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(12) Patent Application Publication (10) Pub. No.: US 2016/0046955A1 Wilkerson Et Al

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(12) Patent Application Publication (10) Pub. No.: US 2016/0046955A1 Wilkerson Et Al US 201600.46955A1 (19) United States (12) Patent Application Publication (10) Pub. No.: US 2016/0046955A1 Wilkerson et al. (43) Pub. Date: Feb. 18, 2016 (54) P-COUMAROYL-COA:MONOLIGNOL Related U.S. Application Data TRANSFERASE (60) Provisional application No. 61/576,515, filed on Dec. (71) Applicants: John Sedbrook, Bloomington, IL (US); 16, 2011. BOARD OF TRUSTEES OF MICHIGAN STATE UNIVERSITY, O O East Lansin, MI (US); WISCONSIN Publication Classification ALUMNI RESEARCH FOUNDATION, Madison, WI (US) (51) Int. Cl. CI2N 5/82 (2006.01) (72) Inventors: Curtis Wilkerson, Swartz Creek, MI CI2N IS/II3 (2006.01) (US); John Ralph, Madison, WI (US); CI2N 9/10 (2006.01) Saunia Withers, Durham, NC (US); (52) U.S. Cl. John Sedbrook, Bloomington, IL (US) CPC ........ CI2N 15/8255 (2013.01); C12N 15/8218 (2013.01); C12N 9/1029 (2013.01): CI2N (73) Assignees: Board of Trustees of Michigan State 15/I 137 (2013.01); C12N 23 10/14 (2013.01) University, East Lansing, MI (US); Wisconsin Alumni Research Foundation, Madison, WI (US); The (57) ABSTRACT Board of Trustees of Illinois State University, Normal, IL (US) The invention relates to nucleic acids encoding a p-couma royl-CoA:monolignol transferase and to inhibitory nucleic (21) Appl. No.: 14/365,744 acids adapted to inhibit the expression and/or translation of a p-coumaroyl-CoA:monolignol transferase RNA. Inhibition (22) PCT Filed: Dec. 14, 2012 of p-coumaroyl-CoA:monolignol transferase in plants (86). PCT No.: PCT/US2O12/069902 improves the incorporation of monolignol ferulates into the lignin of plants, giving rise to plant biomass that is more S371 (c)(1), easily processed into useful products such as paper and bio (2) Date: Jun. 16, 2014 fuels. Patent Application Publication Feb. 18, 2016 Sheet 1 of 47 US 2016/0046955 A1 * HC- FIG, Aiii,FIG 1A1 to C OMe O - O OM HO CMe Med C OH OH OMe FIG. IAI Patent Application Publication Feb. 18, 2016 Sheet 2 of 47 US 2016/0046955 A1 HO HO ? OMe MeO HO C OH HO HO OMe hor? C KX-OMe HO HO C (-oiC ( OMe HO C H O CH OH MeO C O OMe HO HC) Y OH S. OH HC C HO CMe O 8- ) v to-1-(O ) MeO OH O OMe (CHO) HO OMe OH FIG, 1A2 Patent Application Publication Feb. 18, 2016 Sheet 3 of 47 US 2016/0046955 A1 CH MO OM OH FIG. IB1 HO FIG, IBT O OH MO HO O MO OM OH HO CMe O OH MC HO O OMe OH HO OH MeO O OH HO G OMe HC O Key-OMe C FIG. IB1 Patent Application Publication Feb. 18, 2016 Sheet 4 of 47 US 2016/0046955 A1 HO OMe OH MeC) OMe O OH OMe HO OH OMe FIG. IB2 Patent Application Publication Feb. 18, 2016 Sheet 5 of 47 US 2016/0046955 A1 s g s g s s OW Patent Application Publication Feb. 18, 2016 Sheet 6 of 47 US 2016/0046955 A1 g s s s s OW Patent Application Publication Feb. 18, 2016 Sheet 7 of 47 US 2016/0046955 A1 [udd] V@'9IH Patent Application Publication Feb. 18, 2016 Sheet 9 of 47 US 2016/0046955 A1 Patent Application Publication Feb. 18, 2016 Sheet 10 of 47 US 2016/0046955A1 s s g s g s s OW Patent Application Publication Feb. 18, 2016 Sheet 11 of 47 US 2016/0046955A1 S N |||s y |||s * S are:23 s SCS n -- esco IOI y TNF | - | l OW Patent Application Publication Feb. 18, 2016 Sheet 12 of 47 US 2016/0046955A1 Patent Application Publication Feb. 18, 2016 Sheet 13 of 47 US 2016/0046955A1 g s g s s Patent Application Publication Feb. 18, 2016 Sheet 14 of 47 US 2016/0046955A1 s Patent Application Publication Feb. 18, 2016 Sheet 15 of 47 US 2016/0046955A1 Patent Application Publication Feb. 18, 2016 Sheet 16 of 47 US 2016/0046955A1 sss --MOM s s y ce 92 s ge - 9 en ca C gd ge ad ad ge Ced es ge ded Mad N. ea ed y ed ed ed Ced e Patent Application Publication Feb. 18, 2016 Sheet 17 of 47 US 2016/0046955A1 Superdex.75 gel filtration fractions 19 20 21 22 23 24 25 26 we an as an arre as we we FIG. 8 Patent Application Publication Feb. 18, 2016 Sheet 18 of 47 US 2016/0046955A1 Y () O. O. 92: d )-o ifC) s d Q sO ed 5 O. O. \, y 3O ON S SA -1 O C O O O S s O : : Patent Application Publication Feb. 18, 2016 Sheet 19 of 47 US 2016/0046955A1 WT1 FMT9 FMT13 WT1 FMT9FMT13 un- un- un- WT1 FMT9 FMT13 WT1 FMT9 FMT13 input input input boundboundbound wash wash wash eluate eluate eluate FIG, 10A AU 0.3 -- : 2: WTPOPLAR : LEAF EXTRACT O 0.3 -- EXPRESSINGPOPLAR LEAF EXTRACT o -----------------ow-or------------------------------------------------------2 4 6 " ) 12 14 MINUTES FIG, 10B Patent Application Publication Feb. 18, 2016 Sheet 20 of 47 US 2016/0046955A1 ARABIDOPSISLEAFRT-PCR Kb PLUS EMPTY VECTOR AtFMT DNA S. X s S. S. S. s 1 --ARABDOPSIS EMPTY WECTOR EXTRACT & we are as K. Exa, ea. A aar ax...'ar as a. as aa: aaa, a -as-as-s-s-s-s-s-ra as- as ar... r * r * 2 : s +ARABIDOPSIS iii. FMTEXTRACT s 3 MINUTES FIG, 11B Patent Application Publication Feb. 18, 2016 Sheet 21 of 47 US 2016/0046955A1 A Hibiscus cannabinus FMT protein purification fraction fraction kDa soluble insoluble 29 3O SO 25 FIG, 12A Patent Application Publication Feb. 18, 2016 Sheet 22 of 47 US 2016/0046955A1 s : s s : s s : s : s s s ; : s : s . S. l,f v.- S. K N as : sh (L) ; : + t ; : ; : : :; :i. on as s a o : : : ; : x: OO retic - E. it: a reiss SS S : - gi'er. d --- saaxa ... " retrix: " -era- . wesi . i ski w'i' * si : ... v. ... it' se- s *'''..., t *w-ana-. : s terra " (N R S s s s s s .-----r ANT- k re-ra. s k -. s r - R s : s s ; : S s : s s ; : s s S s * , s s s K s R s s R ; : S - S s s - s s : : & : s S d Patent Application Publication Feb. 18, 2016 Sheet 25 of 47 US 2016/0046955A1 ACCACCATCACCACCACCTCGAAGGTCTTGAGCTCCATCTCCGGCGACGGCGGCGACGAC GACGACGACGGCGAGGAGGAGCTAGTAGCTAGCTGAGCCAGACAGCATGGGGTTCGCGGT GGTGAGGACGAACCGGGAGTTCGTGCGGCCGAGCGCGGCGACGCCGCCGTCGTCCGGCGA GCTGCTGGAGCTGTCCATCATCGACCGCGTGGTGGGGCTCCGCCACCTGGTGCGGTCGCT GCACATCTTCTCCGCCGCCGCCCCGAGCGGCGGCGACGCCAAGCCGTCGCCGGCGCGGGT GATCAAGGAGGCGCTGGGGAAGGCGCTGGTGGACTACTACCCGTTCGCGGGGAGGTTCGT GGACGGCGGCGGCGGGCCGGGGAGCGCCCGCGTGGAGTGCACCGGCGAGGGCGCCTGGTT CGTGGAGGCCGCCGCCGGCTGCAGCCTCGACGA CGTGAACGGCCTCGACCACCCGCTCAT GATCCCCGAGGACGACCTCCTCCCCGAOGCCGCCCCCGGTGTCCACCCCCTCGACCCCC CCTCATGATGCAGGTATAATACTACCCGTATACGTACGTTTCTACGTACGTAAGTACGTG CTATACTTGCGAGCAGACAAAAACAAATAAAATCGGTAACAACAATTAACCATCCAGTTA TGCTTACAACTAATTCAAATTATCTTAATTAATTAAAACTGTCCGGCTAATTAAGTGATT ATTAAGGGTGTGTTTTTATCACATCTTCCCGACTGGTACTCCCTCATTTTCCACACGGAT GTTTTACAACTGCAAACGGTACGTATTACAGAAAAAAGTTATATATAAAATTGTTTT AAAATCATATTAATCTATTTTTAAGTTTATTTTAGCTAATAGTTAAATAAACACGCGCTA ACGGATCATTATGTTTTGTGTGTGGGGAGAAAGTTTCTAACCTCCACCTCTAAACACA GCATAATTGTTGGTACGTAGGGCCTATTCACTTTAACGCAAAAAAAGAACCTTACCAAGT TGCCAAAATTTTGGTAGGATTTCTTATATAGTTACTAAAATTTGATAG CAAACAAATAT AACCACTTTTTTATAACTTTACCAAAATTTGCTAAGATTGAAAATGGCATCAAAGTGAAC AGGCCCGTATACGTACGGAGAATGCTGACCTCTCCGGATGATACCTTTAATTTTTCACTT GTGTGGATGTGCACACATGTACGAGGACGAACACATTCA AACCCGTGAAGATTTTAATAT GTGGACGAACTCGATCTATGGTATTGTTGCTGACGAATTAATTACAAAAGTGCTCAAGGA GTTATGTAACTATAAGAACAAAACTATATATGTTTGCCCAAGTAGAAATATATACGAACA AAAACACAGACATGAATAGAACCTACGCGTACGTACAATGTGCCATTACATGCATGTAC ACAATCATTAGCTAGTGTCCTGGATTATATTCAGTCAATTATAACTTTCTAGAAATTAG GTACTAATATATGTATGACTCTCAAACTGTAGTCATGCTTGTGTCAAGTTATAATTAAGT ACAATAATCACACCGATTTATTTTACATAAAGTACAGTAGGATTCAAGATAAG ACTGAGC TATATAGTACTAGGCAGGATGATGAGCTAGCTAGAGCTTAGTGCTCAACATAAACTAGTT GGAGCGTG (CACTG CAATTTTCAAAGTAAAATTAGTTAATTTGCACAGGTGAAGTTGATC CTGTCAGGTAGGTAAGCTCACCAA CTCCAAAGATTGGACAGAATGAAGCATCTGTGGAAG TGAAAGCAGTTGCGTTGGCGTAAGACCACACTAACCAGAGAACTCATAATACAAAATACA TAACAGCACACAATTTATATTGTGTATATATATATATATATATATATATATATGATGT ATGTATGTATGTATGTATTCTAACTGTGTTATCCAATTTTTAAGAAATTTCATCTTTTCA AAAGTAGTAGTATTTGAGTGATGCATGTGCACGTTTTTAGATATGTACATATACCTCATC TACTTTAAAAAAAAAAAATTTTATACAGAGTCGGAACACTAAGCTTAACACTGAT ATCTGACGATAGCATGACGGGATGAGCTTGTCATCAATTGCAGCAGGGCAATTAGGCATG TAAACTGGGGCCATTGATTTCTGTCGAGCACACTATGCTTTCCCTGTCTTATTCTGCCTA FIG, 14C1 Patent Application Publication Feb. 18, 2016 Sheet 26 of 47 US 2016/0046955A1 ACTTAACACTAATATTTGACACACTATCAATTGTTAGCTATTGATATGGCAGTTTGACAT CGACCCTGCTCCATCATTATTACTGCATGCCCGCCCATTCGATGATTGACTTGACCAAAC CCACAAGTGCAAATTGGAAAATTAATAATTAATAATTAGCAAGAAAAAATCCATC AGGGATTCAGGATCAGGTCATGGATGTAATCACTCTCAAACATAG CAATCATTGTGCTT ATGGTCCAAGTGATCATTCCCCCTAATCAACAA CTCGCTTGCTAG CAAGACGTCCCTTCG AATGGATTATTTGATAGCTAGAGCATATCACCTTGCACTTCACCACTCCCCTTATGCAGA GTGTACGTATGTCTAACCAGAATCTAGTGGTGAGCGTAAAAGATCAAAGTG CCCTTATCA ATAACAAAATACTCCGTAATACATTTGGTGGATATATAGGTATATAAGTATTAAAGGAAT AAAACTTTCAAATTTGTGGATTCTAATAAAAACTAATATTAATTTTGATAAACCTGAATT GTAGATACTCTAACTTAGGGTAGTAGTTGAAGCATGCAAAGCTCTAAAAATATATATGAA TTTCGGCGTGTTTATATATATTTCTCCGTGGATATAAAAGCTTAAAATTTATAATCATTT TATGATGATCAGGTGACGGAGTTCAGTTGCGGAGGGTTCGTGGTGGGCCTGATCTCGGTG CACACGATGGCGGACGGGCTAGGGGCCGGGCAGTTCATCAACGCGGTGGGCGACTACGCC CGCGGGCTGGACAGGCCGAGGGTGAGCCCGGTCTGGGCCCGCGAGGCCATCCCGAGCCCG CCGAAGCTGCCCCCGGGCCCGCCGCCGGAGCTGAAGATGTTCCAGCTCCGCCACGTCACC GCCGACCTGAGCCTGGACAGCATCAACAAGGCCAAGTCCGCCTACTTCGCCGCCACCGGC CACCGCTGCTCCACCTTCGACGTCGCCATCGCCAAGACGTGGCAGGCGCGCACCCGCGCG CTCCGCCTCCCGGAACCCACCTCCCGCGTCAACCTCTGCTTCTTCGCCAACACCCGCCAC CTCATGGCCGGCGCCGCCGCCTGGCCCGCACCCGCCGCCGGCGGCAATGGCGGCAATGGG TTCACGGCAA CGCTTCACCCGGTGTCGGGGTGGCGGAGAGCGGGGCGGTGGAGGCG GCGGACGTGGCCGGGGTGGTGGGGATGAACGGGAGGCGAAGGCGAGGCTGCCGGCGGAC TTCGCGCGGTGGGCGGTGGCCGACTTCAGGGAGGATCCGTACGAGCTGAGCTTCACGTAC GATTCCCTGTTCGTCTCCGACTGGACGCGGCTGGGGTTCCTGGAGGCGGACTACGGGTGG GGGCCGCCGTCGCACGTCATACCCTTCGCGTACACCCGTTCAGGCCGTCGCCATCAC GGCGCGCCGCCGGTGCCCAAGACCGGCGCCCGGATCATGACGCAGTGCGTCGAGGACGAC CACCTGCCGGCGTTCAAGGAGGAGATCAAGGCCTTCGACAAGTAAAATGCTTGTGAAATG TGAACTTTGTTATTGTTACTACTTCTATGGGCTCGTTGCTCAATGGGCTTTTTTTTGCTT TTGTTTTGTGTGTGTGGGCCGACACGATTGGTCAAAAGGGATTTGGTGGAGGCCCAGTTG TAATAAGATGGTCCACGCATCATGGATTAATCGTTAATTGTAAGGTAGTACTACACGGAT TTGTTAACAAGGAATAAGTTCACTTGGTGACCCAGTGA FIG, 14C2 Patent Application Publication Feb. 18, 2016 Sheet 27 of
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