US010501760B2 United States Patent (10 ) Patent No.: US 10,501,760 B2 Kumaran et al. (45 ) Date of Patent : Dec. 10 , 2019 ( 54 ) METHODS FOR PRODUCTION OF (56 ) References Cited OXYGENATED TERPENES U.S. PATENT DOCUMENTS ( 71) Applicants :GIVAUDAN SA , Vernier ( CH ) ; 5,847,226 A * 12/1998 Muller C07C 29/50 MANUS BIOSYNTHESIS , INC . , 568/346 Cambridge, MA (US ) 6,200,786 B1 3/2001 Huang et al . 6,890,960 B1 5/2005 Henderson et al. 7,211,420 B1 5/2007 Wong et al . ( 72 ) Inventors : Ajikumar Parayil Kumaran , 7,273,735 B2 9/2007 Schalk et al . Cambridge , MA (US ) ; Chin Giaw Lim , 8,097,442 B2 1/2012 Hitchman et al . Allston , MA (US ) ; Liwei Li, Arlington , 8,415,136 B1 4/2013 Gardner et al. 8,512,988 B2 8/2013 Ajikumar et al. MA (US ) ; Souvik Ghosh , Cambridge , 8,927,241 B2 1/2015 Ajikumar et al. MA (US ) ; Christopher Pirie , 9,284,570 B2 3/2016 Stephanopoulos et al. Cambridge, MA (US ) ; Anthony 9,359,624 B2 6/2016 Ajikumar et al. 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(74 ) Attorney, Agent, or Firm Stoel Rives LLP CPC C12P 7/26 (2013.01 ) ; A01N 27/00 (57 ) ABSTRACT ( 2013.01) ; A23L 27/13 ( 2016.08 ) ; C12N 9/0073 (2013.01 ) ; C12P 5/002 (2013.01 ) ; The present disclosure relates to methods for producing C12Y 114/13078 (2013.01 ) ; A23V 2002/00 oxygenated terpenoids, and preparation of compositions and ( 2013.01 ) ; CO7K 2319/03 (2013.01 ) formulations thereof . Polynucleotides, derivative enzymes , (58 ) Field of Classification Search and host cells for use in such methods are also provided . CPC C12P 7/26 ; C12Y 114/13078 ; A23L 27/13 ; A23V 2002/00 13 Claims, 76 Drawing Sheets See application file for complete search history . Specification includes a Sequence Listing . US 10,501,760 B2 Page 2 ( 56 ) References Cited Kumaran , et al. , Office Action dated Mar. 19 , 2019 for U.S. Appl. No. 15 / 505,503 . Kumaran , et al ., Preliminary Amendment dated Sep. 26 , 2017 for OTHER PUBLICATIONS U.S. Appl . No. 15 / 505,503 . 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Depends on Evolutionary Conserved Gene Clusters , The Plant Cell, 2014 , vol. 26 , pp . 3286-3298 . * cited by examiner U.S. Patent Dec. 10 , 2019 Sheet 1 of 76 US 10,501,760 B2 ??? ? - ??????? ????????????????????- FIG.1 4444: U.S. Patent Dec. 10 , 2019 Sheet 2 of 76 US 10,501,760 B2 1.5-2 1.2-1.5 FoldProductivityrelative(toWT) 0.8-1.2 FIG.2 0.5-0.8 0.2-0.5 140 120 100 80 09 40 20 0 No. of Mutants U.S. Patent Dec. 10 , 2019 Sheet 3 of 76 US 10,501,760 B2 > VvVS ( Vitis vinifera ] MSTQVSASSLAQIPQPKNRPVANFHPNIWGDQFITYTPEDKVTRACKEEQIEDLKKEV KRKLTAAAVANPSOLLNFIDAVORLGVAYHFEQEIEEALQHICNSFHDCNDMDGDLYN IALGFRLLRQQGYTISCDIFNKFTDERGRFKEALISDVRGMLGLYEAAHLRVHGEDIL AKALAFTTTHLKAMVESLGYHLAEQVAHALNRPIRKGLERLEARWYISVYQDEAFHDK TLLELAKLDFNLVOSLHKEELSNLARWWKELDFATKLPFARDRLVEGYFWMHGVYFEP QYLRGRRILTKVIAMTSILDDIHDAYGTPEELKLFIEAIERWDINSINOLPEYMKLCY VALLDVYKEIEEEMEKEGNQYRVHYAKEVMKNQVRAYFAEAKWLHEEHVPAFEEYMRV ALASSGYCLLATTSFVGMGEIATKEAFDWVTSDPKIMSSSNFITRLMDDIKSHKFEOK RGHVTSAVECYMKQYGVSEEQVYSEFQKQIENAWLDINQECLKPTAVSMPLLARLLNF TRTMDVIYKEQDSYTHVGKVMRDNIASVFINAVI ( SEQ ID NO : 1 ) ATGGCTACGCAGGTCTCAGCCTCGTCACTGGCACAAATCCCGCAGCCGAAAAATCGTC CGGTGGCGAACTTCCATCCGAATATCTGGGGTGACCAGTTTATCACGTATACCCCGGA AGATAAAGTGACCCGTGCGTGCAAGGAAGAACAAATTGAAGACCTGAAAAAGGAAGTC AAACGCAAGCTGACCGCAGCAGCAGTGGCAAACCCGTCTCAGCTGCTGAATTTTATCG ATGCGGTTCAACGTCTGGGCGTCGCCTATCATTTCGAACAGGAAATTGAAGAAGCACT GCAACATATCTGCAACAGCTTTCACGATTGTAATGATATGGACGGCGATCTGTATAAC ATTGCTCTGGGTTTCCGTCTGCTGCGCCAGCAAGGCTACACGATTTCCTGTGACATCT TTAATAAATTCACCGATGAACGTGGTCGCTTTAAGGAAGCGCTGATCTCAGACGTTCG TGGCATGCTGGGTCTGTATGAAGCTGCGCATCTGCGCGTCCACGGCGAAGATATTCTG GCCAAAGCACTGGCTTTCACCACGACCCACCTGAAGGCGATGGTCGAATCTCTGGGTT ACCATCTGGCAGAACAGGTGGCACACGCCCTGAACCGTCCGATCCGCAAAGGCCTGGA ACGTCTGGAAGCGCGCTGGTATATTAGTGTGTACCAGGACGAAGCATTTCATGATAAA ACCCTGCTGGAACTGGCTAAGCTGGATTTCAACCTGGTTCAATCTCTGCACAAAGAAG AACTGAGTAATCTGGCCCGTTGGTGGAAAGAACTGGACTTTGCGACCAAGCTGCCGTT CGCCCGTGATCGCCTGGTTGAAGGCTATTTTTGGATGCATGGTGTCTATTTCGAACCG CAGTACCTGCGCGGTCGTCGCATTCTGACGAAAGTGATCGCAATGACCTCGATTCTGG ATGACATCCACGACGCTTACGGCACCCCGGAAGAACTGAAACTGTTTATTGAAGCGAT CGAACGTTGGGATATTAACAGCATCAATCAGCTGCCGGAATATATGAAACTGTGCTAC GTGGCCCTGCTGGATGTTTACAAGGAAATCGAAGAAGAAATGGAAAAGGAAGGTAACC AGTATCGTGTTCATTACGCGAAAGAAGTCATGAAGAATCAAGTGCGCGCCTACTTTGC AGAAGCTAAATGGCTGCATGAAGAACACGTGCCGGCGTTCGAAGAATATATGCGCGTT GCGCTGGCCAGCTCTGGCTACTGTCTGCTGGCCACGACCTCTTTTGTGGGCATGGGTG AAATTGCAACGAAAGAAGCGTTTGACTGGGTTACCAGTGATCCGAAGATTATGAGTTC CTCAAACTITATCACCCGTCTGATGGATGACATTAAATCCCATAAGTTCGAACAGAAA CGCGGTCACGTCACCTCAGCCGTGGAATGCTATATGAAACAGTACGGCGTTTCGGAAG AACAAGTCTATAGCGAATTTCAGAAACAAATCGAAAACGCATGGCTGGATATTAATCA GGAATGTCTGAAACCGACGGCAGTCTCCATGCCGCTGCTGGCTCGTCTGCTGAATTTT ACGCGCACGATGGATGTGATCTATAAAGAACAGGATTCGTACACCCATGTGGGCAAGG TTATGCGCGATAACATTGCAAGCGTGTTCATTAATGCTGTTATCTAA ( SEQ ID NO : 2 ) FIG . 3A U.S. Patent Dec. 10 , 2019 Sheet 4 of 76 US 10,501,760 B2 > Vv1M1 | engineered valencene synthase MATOVSASSLAQIPQPKNRPVANFHPNIWGDQFITYTPEDKVTRACKEEQIEDLKKEV