WITHIN CONUSUN TOTAL20170283844A1 UITDI A NTARA PARA A TORI ( 19) United States (12 ) Patent Application Publication (10 ) Pub. No. : US 2017/ 0283844 A1 ITKIN et al. (43 ) Pub . Date : Oct . 5 , 2017 ( 54 ) METHODS OF PRODUCING MOGROSIDES Publication Classification AND COMPOSITIONS COMPRISING SAME (51 ) Int . ci. AND USES THEREOF C12P 33 / 12 (2006 . 01 ) C12N 9 /02 (2006 . 01 ) (71 ) Applicant: The State of Israel , Ministry of A23L 2730 ( 2006 . 01 ) Agriculture & Rural Development, C12N 9 /88 ( 2006 . 01 ) Argricultural Research Organiza , A23L 2760 ( 2006 .01 ) Rishon LeZion (IL ) C07 ) 17 / 00 ( 2006 . 01) C12N 9 / 90 (2006 .01 ) (72 ) Inventors : Maxim ITKIN , Kibbutz HaOgen ( IL ) ; (52 ) U . S . CI. Rachel DAVIDOVICH -RIKANATI , ??? C12P 33 / 12 ( 2013 .01 ) ; C07 ) 17/ 005 Alonei Abba ( IL ) ; Shahar COHEN , (2013 .01 ) ; C12N 9 /0083 ( 2013 .01 ) ; C12N Rishon LeZion ( IL ) ; Vitaly 9 / 90 ( 2013 .01 ) ; C12N 9 /88 ( 2013 .01 ) ; C12N PORTNOY , Nesher ( IL ) ; Adi 9 /0071 (2013 .01 ) ; C12Y 114 /99007 (2013 . 01 ) ; DORON - FAIGENBOIM , C12Y 504 /99033 ( 2013 . 01 ) ; C12Y 402 /01 Ramat- HaSharon ( IL ); Marina ( 2013 .01 ) ; A23L 2 /60 ( 2013 . 01 ) ; A23L 27 36 PETREIKOV , Rishon LeZion ( IL ) ; ( 2016 .08 ) ; A23V 2002 /00 (2013 .01 ) Shmuel SHEN , Moshav Beit - Elazari ( IL ) ; Yaakov TADMOR , Timrat ( IL ) ; (57 ) ABSTRACT Yosef BURGER , Haifa (IL ) ; Efraim Isolated mogroside and mogrol biosynthetic pathway LEWINSOHN , Timrat ( IL ) ; Nurit enzyme polypeptides useful in mogroside biosynthesis are KATZIR , Kiryat - Tivon ( IL ) ; Arthur A . provided . Mogroside biosynthetic pathway enzymes of the SCHAFFER , Hashmonaim ( IL ) ; Elad invention include squalene epoxidase (SE ) , expoxy OREN , Beit Shearim ( IL ) hydratase (EH ), cytochrome p450 (Cyp ), cucurbitadienol synthase (CDS ) and udp - glucosyl- transferase (UGT ) . Also (21 ) Appl. No. : 15 /510 ,708 provided are methods of producing a mogroside using the isolated mogroside and mogrol biosynthetic enzyme poly ( 22 ) PCT Filed : Sep . 10 , 2015 peptides , the methods comprising contacting a mogrol and / or a glycosylated mogrol (mogroside ) with at least one UDP ( 86 ) PCT No. : PCT/ IL2015 / 050933 glucose glucosyl transferase (UGT ) enzyme polypeptide of the invention catalyzing glucosylation of the mogrol and / or $ 371 ( c )( 1 ) , the glucosylated mogrol to produce a mogroside with an ( 2 ) Date : Mar . 13 , 2017 additional glucosyl moietie ( s ) , thereby producing the mogroside . Alternatively or additionally provided is a method of synthesizing a mogrol, the method comprising Related U . S . Application Data contacting a mogrol precursor substrate with one or more (60 ) Provisional application No . 62/ 048 ,924 , filed on Sep . mogrol biosynthetic pathway enzyme polypeptides as 11 , 2014 , provisional application No . 62 /089 , 929 , described herein catalyzing mogrol synthesis from the mog filed on Dec . 10 , 2014 . rol precursor substrate , thereby synthesizing the mogrol. o AACT poHM33MUS HMG hik + •HOC SConcoc MVA pathway MEP away PMK 10.OP DXROXR .0 MCIOH GA OPP* .COP COPLCROP O PP- CDP PassTOP OH OH = OH OHMICS HCC. OPP OH OH NIVD ID ! A nop - - GPS SOE dentender os til den her + + stering HO - krcon pattway OH pulative pathway HOV IH KO CYP450 A VIV CYP450 H?, HYOH HOV open to UOPE HOX ogro ! Chomson OR gecroside R R maç ceide LLE GC HOH 04 megroeide 10 GCGC 100 m noraide I' s Gce mogrede Gloc R.OX mogrosides mogrzeide vil - Get GHS Patent Application Publication Oct. 5 , 2017 Sheet 1 of 26 US 2017 / 0283844 A1 FIG . 1 HMGSHMGS OH OHMGR O H O MK ??? SCOAAACT M , SCOASCOA SCOA "?? “ ???? , MVA pathway MEP pathway ??? HO ' Pova L DXR RICHOF MCT OH OPP. CRpCDP CMK 4, 0P OFOP DXR ?? ?? OHO OPPOPP.COM. CDP ??? OH OH OP M? HOOCC |Oto opp BSST MVD GPS untut opp pps den opp ' dropp CAS 3 deretour - steriod known pathway putative pathway HOLTHY HO, YOH OH 20 CYP450 Cyp450 HO, Hint TOH CS HOY mogrol UDPG // / CYP450 Chanel OR , mogrosides R R or OH mogroside NEE -GicGIC GIC HOHOL T L oHOH mogroside II1 -GIC GIC GicGLC- GICGC Hopoco mogroside IV -GC - GiC GLC- GLC 111 mogroside V - GIO- GIC - GIC C ROROY mogrosides mogroside Vi -Ge - GC -GLC - Gie Patent Application Publication Oct. 5 , 2017 Sheet 2 of 26 US 2017 / 0283844 A1 FIG . 2 Acetyl COA Mevalonate pathway Squalene Squalene epoxidase 2 , 3 -oxidosqualene SqualeneAm epoxidase 3 ; 22 , 23 - diepoxysqualene Cucurbitajieno synthase w 24 , 25 epoxycucurbitadienol Epoxide hydrolase HOVO * 24 , 25 dihydroxycucurbitadienol CYP 102801 POH mogrol Gedepan Glu Glimmm Glu Multiple UGTS * *** * Glum o Mogroside V Patent Application Publication Oct. 5 , 2017 Sheet 3 of 26 US 2017 / 0283844 A1 FIG . 3 Me Me Me * 484444 *** 14 *14 *** onal *+ + + + + + + + + + + + + ++ + **** * * * * ** * * *** **17779 **** 444 ** * 03 Me Me Me Hac 25 CHA ????? vui HO H?C °CH , Patent Application Publication _ 0ct . 5 , 2017 _ Sheet 4 of 26 _ US 2017/ 028384441 ?? ?? ? ? ? ?? ? ?? ? ? ?? ? ? ? ?? ?? ?? ? ? ? ?? ? ? ?? ? ? ?? ?? ? ? ? ? ? ? ? ? ? ?? ?? ?? ? ? ? ? ? ? ? ?? ?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ?? ? ? ? ? ?? ', -' , , ; , , , , ', - . s ," ; ? ? ? ?? ? ?? ? ?? ? ?? ? ?? ? ?? ? ? ?? ? ? ?? ? ?? ? ? ? ? ? ? ? -: - . ;- . - ? ???? ???? ?? ? ?? ? ? ? ? ? ?? ? ? ? ? ?? ? ?? ?? ?? ? ? ? ? ?? ? ? ? ? ? ? ? ? ? ?? ? ? ? ?? ? ? ? ?? ? ??????? ??? . ?- ? ? ? ? ? . ? ?? ?? ? ? ?? ?? ? ????????? ??w5 ? ?? ? ? ? ? ? ? ?? ? ? ? ?? ? ? ? ,- : : ???? ? ?? ? ? ? ? ? ? ? ? ? ? ? ? ? . ???????? ? ??? ? ? .. .. ? ? ?? ? ? ?? ?? ? ? ? ? ? ? ???????????????? ????????????????????? ? ?? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? 3503 3000 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? Ffs. 48 2500 ? ?? 8: 31 ? ? 848 200 ; ?? ? ? ? ? ? ? ? ? ? ? ? ? ?? ? ?? ? ? ? ? ?? ° ? f/ f3 ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? ? _f /f ?????? sia 1500 ? ? _fwf5 1003 ? ? ? ????????????????????? 500 ?????? ' ?? ? ? ?? _ 0 _ 20 _ 40 60 _ 80 _ 200 _ 220 Patent Application Publication Oct. 5 , 2017 Sheet 5 of 26 US 2017 / 0283844 A1 FIG . 5A Fruit Goroscon 15 0* * 20* * * * * * * * 40 60 80 100 FIG . 5B Tume days ) Patent Application Publication Oct. 5 , 2017 Sheet 6 of 26 US 2017 / 0283844 A1 OOOOOOOOOOOOOOOOOOO O O O oooooooooooooooooooooooOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOOO 1.*71 control . : . FIG . 6A . .,Vtirir . Moon . nosso . si. 11. om SgCDS * . .* 4. FIG . 6B %* loncount(407-462Da) &onoogen . 3 %* * . : : - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - - . - iiiiiiiiiii .: * 1 * *. standards . 4+ FIG . 6C * . E .I . + ?. *+ N . 11.' 4+ ". + *. .IT 4+ ". VOLVO wwwwwwwwww w wwwwwwwwww 4 5 6 7 8 epoxycucurbitadienol diepoxysqualene9 10 11 12oxidosqualene cucurbitadienol13 14 15 16 Patent Application Publication Oct. 5 , 2017 Sheet 7 of 26 US 2017 / 0283844 A1 Expression 0.2 1.0 0. -1.0 -2.0 EPH_contig86123EPHcontig102640 EPH_contig28382 EPH_contig73966 EPH_contig22474 EPHcontig102581 EPHcontig102175 EPHcontig101438 113DAA 77DAA GEStudyData1 #666666666666 ? { 5 34DAA FIG.7 15DAA EPH_contig86123EPHcontig102640 EPH_contig28382 EPH_contig73966 EPH_contig22474 EPHconti?102581 EPHcontig102175 EPHcontig101438 te 983 . 70 Patent Application Publication Oct. 5 , 2017 Sheet 8 of 26 US 2017 / 0283844 A1 FIG . 8A G177 + SgCDS SgEPH1 namemenonton record | 1 . dihydroxycucurbitadienol 23 . epoxydocucurbitadienolC30H5203 G1177 + 4 . dioxydosqualene SgCDS * 5 . oxidosqualene SgEPH2 * 6 . cucurbitadienol * loncount(407-462Da) * * * * Gil77 + SgCDS 1 2 4 STEPH3 * * * * ** Gi177 + oneormorepeople SgCDS ? ? Ï Š 10 11 12 13 14 15 Retention time (min ) FIG . 8B 25000000 20000000 peakarea 15000000 10000000 dihydroxycucurbitadienol 5000000 * epoxycucurbitadienol U SDCDSEll_SDCDSEPH1 _SDCDS EPH2 EPH39 )SOCDS = Patent Application Publication Oct. 5 , 2017 _ Sheet 9 of 26 US 2017 / 0283844 Al 34 35 39 39 7238 63 64 3775|100 63}34. |716937 61 72 10075| 373937100 626 60 72 100757273 8 75 87.751007271 6062 60 Seqid#3????contig102581contig101438EPH286123p49EPH173966pEPH3102640EP+428382pcontig102175contig22474 . 596060 . 7271 . *. *. '-.,* . '. 996561616061 98 27 . .: . 60100 . 9859 7660 ,"'. 60 contig10258199100656061 7264contig102175637369- ,'." 6510060 6859 6063 - 63 , |1009965 .? Contig22474343538 . *.: ,61 *.:, *. 62 EPH428382p61 - - *.: EPH286123p.61 - - Contig10143865 - 100Secial38 - EPH173966p60 - - EPH330264062 - Senid#4061 - FIG.9 segid#38 Contig101438 Seeidt40 102840EPH3 Patent Application Publication 0ct. 5 , 2017 _ Sheet 10 of 26 US 2017 / 0283844 Al FIG . 10 GE Study Data 1 0 152A} 7g14 . 2 7g14. 2 3 : 15 . 1 – 696 . ? 8015? & 9 . 21 ~ Y83584921 189584321 . Expression - £18? ! ??321? . 1 ?– 883 . 883 893 . 2 ?????? . 2 $ 350 $ 211 . 3 82113 2. 0 868 1 868. 1 178 s17g8 10 399 ? 3g4Y 529 529 811. 2 81. 2 19g $ 190 . 036 . 2 ???????14 ? 14, 1 – 8266 . ?266?? . + 8562hG3. ? c??A®9??????Ec????? ? -8B9 . 1 - ??4. 3 { 983 1 9745 . 3 - ? ????wgpc? - *tob? -? ?????MAM??-?????????????? -? - 758 . 2 -92 . 1 324 7397 2 ? . 7886 . -??? . 12 62A???? bQE?? @g???-????? ?????????????? 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