TOMATONTAUS009994883B2 TULUMLARI TANITI (12 ) United States Patent ( 10 ) Patent No. : US 9 ,994 , 883 B2 Goossens et al. ( 45 ) Date of Patent: Jun . 12 , 2018 ( 54 ) TRITERPENOID SAPOGENIN PRODUCTION ( 56 ) References Cited IN PLANT AND MICROBIAL CULTURES FOREIGN PATENT DOCUMENTS @(71 ) Applicants : VIB VZW , Ghent (BE ) ; Universiteit Gent, Ghent (BE ) EP 2351846 AL 8 / 2011 WO 2013167751 A1 11 /2013 @(72 ) Inventors : Alain Goossens , Lokeren (BE ) ; Tessa Moses , Ghent ( BE ) ; Jacob Pollier , Ghent (BE ) ; Lorena Almagro Romero , OTHER PUBLICATIONS Aljucer ( ES ) Lambert et al ., Modulation of Triterpene Saponin Production : In Vitro Cultures , Elicitation , and Metabolic Engineering , Applied @(73 ) Assignees: VIB VZW , Ghent (BE ) ; Universiteit Biochemistry and Biotechnology , 2011 , pp . 220 - 237 , vol. 164 , Gent, Ghent (BE ) Humana Press Inc . , New York , US . Kirby et al. , Engineering triterpene production in Saccharomyces ( * ) Notice: Subject to any disclaimer, the term of this cerevisiae -beta - amyrin synthase from Artimisia annua , FEBS Jour patent is extended or adjusted under 35 nal, Apr. 10 , 20008 , pp . 1852 - 1859 , vol. 275 , No . 8 . U . S . C . 154 (b ) by 602 days . Kastelic - Shudolc et al ., Involvement of the Cell Wall to Nocardia Restricta on the Bioconversions of Steroid Sapogenins, World ( 21 ) Appl. No .: 14 /400 , 527 Journal of Microbiology and Biotechnology , Jan . 1 , 1991, pp . 22 - 24 , vol . 7 , No. 1 Rapid Communications of Oxford , Oxford , GB . Augustin et al ., Molecular activities , biosynthesis and evolution of ( 22 ) PCT Filed : May 13 , 2013 triterpenoid saponins, Photochemistry , Jan . 11 , 2011, pp . 435 - 457 , vol. 72 , No . 6 , Pergamon Press , GB . ( 86 ) PCT No. : PCT/ EP2013 / 059821 Singer et al ., Microbial Transformations in a Cyclodextrin Medium $ 371 (c ) ( 1 ) , Part 2 , Reduction of Androstenedione to Testosterone by Sac ( 2 ) Date : Nov. 11 , 2014 charomyces -cerevisiae , Applied Microbiology and Biotechnology , 1991 , pp . 731- 737 , vol. 35 , No . 6 . Moses , Tessa , Metabolic engineering for production of triterpenoid (87 ) PCT Pub. No. : W02013 / 167751 saponin building blocks in plants and yeast , Thesis , Ghent Univer PCT Pub . Date : Nov . 14 , 2013 sity , Faculty of Sciences, Belgium , Dec . 1 , 2012 , pp . 1 - 225 . Moses et al ., Bioengineering of plant ( tri )terpenoids : from meta (65 ) Prior Publication Data bolic engineering of plants to synthetic biology in vivo and in vitro , New Phytologist , May 14 , 2013 , retrieved from the Internet : URL : US 2015 / 0141633 A1 May 21, 2015 http : // onlinelibrary .wiley . com / doi/ 10 . 1111 /nph . 12325 /pds [ Retrieved on Aug . 9 , 2013 ] . Related U . S . Application Data Triterpene sapogenins, Phytolab , online catalogue 2012 , pp . 1 - 2 , (60 ) Provisional application No . 61 /645 , 998, filed on May XP002711222 , Retrieved from the Internet : URL : http : // phyproof . 11, 2012 , provisional application No . 61 /732 ,817 , phytolab . de /reference - substances / isoprenoids/ terpenoid - type / filed on Dec . 3 , 2012 . triterpenes / triterpene - sapogenins. html ? _ from _ store = default & _ store = english & dir = asc & limit = 16 & order= cas & p = 3 , retrieved on (51 ) Int. Ci. Aug . 13 , 2013 . C12P 15 / 00 (2006 . 01 ) PCT International Search Report , PCT/ EP2013 /059821 , dated Oct. 28 , 2013 C12P 33 /00 ( 2006 .01 ) Jambhekar et al ., Cyclodextrins in pharmaceutical formulations I : C07C 35 /44 ( 2006 . 01 ) structure and physicochemical properties, formation of complexes , C07C 47746 ( 2006 .01 ) and types of complexes , Feb . 2016 , Drug Discovery Today, vol. 21 , C07C 49 /727 ( 2006 . 01 ) No . 2 , pp . 357 - 362. C07C 62 /32 ( 2006 . 01 ) Li et al ., Triterpenoids of Marine Origin as Anti - Cancer Agents , CO7D 307/ 00 ( 2006 .01 ) Molecules 2013 , 18 , pp . 7886 - 7909 . C12N 9 / 90 ( 2006 . 01 ) C12P 17 / 04 ( 2006 .01 ) Primary Examiner — Kagnew H Gebreyesus (52 ) U .S . CI. ( 74) Attorney , Agent, or Firm — TraskBritt, P . C . CPC .. .. .. .. C12P 15 /00 (2013 .01 ) ; C07C 35 / 44 ( 2013 .01 ) ; C07C 47 / 46 ( 2013 .01 ) ; C07C (57 ) ABSTRACT 49 /727 ( 2013 .01 ) ; C07C 62 /32 ( 2013 .01 ) ; The disclosure relates to a method for enhancing the bio C07D 307700 ( 2013 .01 ) ; C12N 9 / 90 synthesis and / or secretion of sapogenins in the culture (2013 .01 ) ; C12P 17 /04 ( 2013 .01 ) ; C12P 33/ 00 medium of plant and microbial cell cultures . Further , the ( 2013 .01 ) ; C07C 2603 /52 ( 2017 .05 ) ; C12Y disclosure also relates to the identification of novel genes 504 / 99 ( 2013 .01 ) involved in the biosynthesis of sapogenin intermediates , as (58 ) Field of Classification Search well as to novel sapogenin compounds . CPC . .. C12P 15 /00 ; C12P 17 /04 ; C07C 62 /32 ; CO7D 307/ 00 , C12Y 114 / 13134 11 Claims, 33 Drawing Sheets See application file for complete search history . ( 15 of 33 Drawing Sheet ( s ) Filed in Color ) atent Jun . 12 , 2018 Sheet 1 of 33 US 9 , 994 , 883 B2 STEROLS man ** * * * * * GINSENOSIDE RE- 1 ???D?? SQUALENE MOX YCLOARTENOL OAMMARANEDIOLL mun LUPEOLO Hitta VOXIDOSQUALENESQUALENG SAIKOSAPONINA SEUUNICACO CYCP480, GT Sort !1111 ** *# **** **Pildis" OLEANOUCACID "*O CERO.GT peppe optare- MOON r OYIP450 GLYCYRRHIZIN 26000& PODPREDP GT,SPESE AT , NY PAPASAPANp BARDOXOLONE - 0 ON METHYL i personne. AVENACINAAVENACINA E by 0 . * 25-21 * W * FIG . 1 U . S . 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