SEQUENCE LISTING

<110> CSIR
<120> A process for producing tirucalladienol for synthesis of limonoids
<130> NCLI-INV-2018-070
<140> PCT/IN----/0-----
<141> PCT filing date:
<150> Indian Patent Application No.
<151> Indian Filing date
<160> 36

<210> SEQ ID NO:1
<211> 2283
<212> DNA 
<213> Artificial sequence 
<220>
<223> cDNA encoding tirucalladienol synthase cloned from Azadirachta indica
<400> 1
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac    60 
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt   120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct   180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct   240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag   300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat   360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt   420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag   480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt   540 
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc   600 
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg   660 
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc   720 
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc   780 
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc   840 
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac   900 
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta   960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca  1020 
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa  1080 
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct  1140 
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat  1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat  1260 
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca  1320 
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat  1380 
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat  1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg  1500 
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat  1560 
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc  1620 
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat  1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac  1740 
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa  1800 
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga  1860 
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc  1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag  1980 
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg  2040 
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca  2100 
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc  2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac  2220 
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat  2280
taa                                                                2283


<210> SEQ ID NO: 2
<211> 760
<212> PRT	
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase encoded by SEQ ID NO: 1
<400> 2
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn

<210> SEQ ID NO: 3
<211> 1593
<212> DNA 
<213> Artificial sequence 
<220>
<223> cDNA encoding squalene epoxidase cloned from Azadirachta indica
<400>   3
atggcggctg tgattgatca atacattgtt tggactttct gtgcctctct cattgggttt    60
cttcttctct tcattttgcg actcaatgat aagaacgagt caaaggagga taccggaaac   120
tgcaatatcc aaaatgacat tgttaagagc tcttctaacg atggagattc ctcgccggag   180
catggctccc gcactgacgt catcattgtt ggcgccggcg ttgctggtac tgccttggct   240
cacacacttg gcaaggatgg aaggcgagtt catgtgatag aaagagactt gacagagcct   300
gacagaattg ttggtgaact gcttcagcct ggggaatacc tgaaactagt tgagttgggc   360
cttgaggact gtgtggaaga aattgatgcc cagcaagtac ttggttatgc tcttttcaa    420
gatggaaaaa atactagact gtcatatccc ttggagaaat ttcatgcgga cgtctctggg   480
aggagcttcc acaatgggcg tttcattcag cggatgagag aaaaagcagc tactcttccc   540
aatgtacagc tagatcaagg aactgtaaca tccctacttg aagaaaatgg gactataaaa   600
ggggttcagt acaaaactaa ggatggtcaa gaacttcaat cttttgcccc tcttacaatt   660
gtgtgtgatg gttgcttctc aaatctgcgc cgttctcttt gcaaaccaaa ggttgatgtg   720
ccctcttgtt ttgtgggttt agtcttagag aactgccagc ttccatttgc gaaccacggg   780
catgtcattc tggcagatcc ttcccccatc ttattttatc caatcagtag cacagagatt   840
cgatgcttgg ttgatgttcc tggtcagaag ataccaccaa tagctaatgg tgaaatggcc   900
aattatttga agactgtggt ggcaccgcag gttccccctg agcttcatga tgccttcatt   960
tctgcagttg agaaaggaaa tattagaact atgccaaata gaagcatgcc agctgatccc  1020
cagcctactc ctggagccct tctgatgggt gatgctttca acatgcgtca tcctttaact  1080
ggcggcggaa tgactgtggc attatctgat attgttgtac tgcggaatct ccttaagcca  1140
ctcaatgact ttcatgattc agcttccctt accacatatc ttgaatcctt ttatacctta  1200
cgcaagccag ttgcatcgac aataaatact ctggcaggtg ctctttacaa ggtgttttcc  1260
tcttcccctg atcaagcaag aaaggaaatg cgccaagcat gttttgacta tttaagtctt  1320
ggaggtgtct tttcatccgg accaattgct ttactctctg gtctaaaccc tcgcccctta  1380
agtttagttc tgcatttctt tgctgtggca atttatggtg ttggtcgttt gctacttcca  1440
tttccatcgc ttaagaggat gtggattggg gctaggctaa tatcgagtgc atcaggtatt  1500
atttttccca tcatcaaggc agaaggagtt aggcaaatgt tcttccctgc cactattcct  1560
gcaatataca gagctcctcc tgttgacgat tga                               1593

<210> SEQ ID NO: 4
<211> 530
<212> PRT
<213> Artificial sequence 
<220>
<223> Squalene epoxidase encoded by SEQ ID NO: 3
<400> 4
Met Ala Ala Val Ile Asp Gln Tyr Ile Val Trp Thr Phe Cys Ala Ser 
Leu Ile Gly Phe Leu Leu Leu Phe Ile Leu Arg Leu Asn Asp Lys Asn 
Glu Ser Lys Glu Asp Thr Gly Asn Cys Asn Ile Gln Asn Asp Ile Val 
Lys Ser Ser Ser Asn Asp Gly Asp Ser Ser Pro Glu His Gly Ser Arg 
Thr Asp Val Ile Ile Val Gly Ala Gly Val Ala Gly Thr Ala Leu Ala
His Thr Leu Gly Lys Asp Gly Arg Arg Val His Val Ile Glu Arg Asp 
Leu Thr Glu Pro Asp Arg Ile Val Gly Glu Leu Leu Gln Pro Gly Glu 
Tyr Leu Lys Leu Val Glu Leu Gly Leu Glu Asp Cys Val Glu Glu Ile 
Asp Ala Gln Gln Val Leu Gly Tyr Ala Leu Phe Lys Asp Gly Lys Asn 
Thr Arg Leu Ser Tyr Pro Leu Glu Lys Phe His Ala Asp Val Ser Gly
Arg Ser Phe His Asn Gly Arg Phe Ile Gln Arg Met Arg Glu Lys Ala 
Ala Thr Leu Pro Asn Val Gln Leu Asp Gln Gly Thr Val Thr Ser Leu 
Leu Glu Glu Asn Gly Thr Ile Lys Gly Val Gln Tyr Lys Thr Lys Asp 
Gly Gln Glu Leu Gln Ser Phe Ala Pro Leu Thr Ile Val Cys Asp Gly 
Cys Phe Ser Asn Leu Arg Arg Ser Leu Cys Lys Pro Lys Val Asp Val 
Pro Ser Cys Phe Val Gly Leu Val Leu Glu Asn Cys Gln Leu Pro Phe 
Ala Asn His Gly His Val Ile Leu Ala Asp Pro Ser Pro Ile Leu Phe 
Tyr Pro Ile Ser Ser Thr Glu Ile Arg Cys Leu Val Asp Val Pro Gly 
Gln Lys Ile Pro Pro Ile Ala Asn Gly Glu Met Ala Asn Tyr Leu Lys 
Thr Val Val Ala Pro Gln Val Pro Pro Glu Leu His Asp Ala Phe Ile
Ser Ala Val Glu Lys Gly Asn Ile Arg Thr Met Pro Asn Arg Ser Met 
Pro Ala Asp Pro Gln Pro Thr Pro Gly Ala Leu Leu Met Gly Asp Ala 
Phe Asn Met Arg His Pro Leu Thr Gly Gly Gly Met Thr Val Ala Leu 
Ser Asp Ile Val Val Leu Arg Asn Leu Leu Lys Pro Leu Asn Asp Phe 
His Asp Ser Ala Ser Leu Thr Thr Tyr Leu Glu Ser Phe Tyr Thr Leu
Arg Lys Pro Val Ala Ser Thr Ile Asn Thr Leu Ala Gly Ala Leu Tyr 
Lys Val Phe Ser Ser Ser Pro Asp Gln Ala Arg Lys Glu Met Arg Gln 
Ala Cys Phe Asp Tyr Leu Ser Leu Gly Gly Val Phe Ser Ser Gly Pro 
Ile Ala Leu Leu Ser Gly Leu Asn Pro Arg Pro Leu Ser Leu Val Leu 
His Phe Phe Ala Val Ala Ile Tyr Gly Val Gly Arg Leu Leu Leu Pro 
Phe Pro Ser Leu Lys Arg Met Trp Ile Gly Ala Arg Leu Ile Ser Ser 
Ala Ser Gly Ile Ile Phe Pro Ile Ile Lys Ala Glu Gly Val Arg Gln 
Met Phe Phe Pro Ala Thr Ile Pro Ala Ile Tyr Arg Ala Pro Pro Val 
Asp Asp

<210> SEQ ID NO: 5
<211> 43
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 1 synthesis
<400> 5
gatcacgaat tcaacacaat gtctatgtgg aagctgaaga ttg  			43
?
<210> SEQ ID NO: 6
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 1 synthesis
<400> 6
catgcagcgg ccgcattagg caatggaact tttc   				34

<210> SEQ ID NO: 7
<211> 30
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 3 synthesis
<400> 7
acacaggatc cgatggcggc tgtgattgat 					30

<210> SEQ ID NO: 8
<211> 51
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 3 synthesis
<400> 8
atattcaagc tttcaatggt gatggtgatg atgatcgtca acaggaggag c	   	51

<210> SEQ ID NO: 9 
<211> 2283
<212> DNA
<213> Artificial Sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution Y125F
<400> 9
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180 
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtttttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa      							  2283

<210> SEQ ID NO: 10 
<211> 27
<212> DNA
<213> Artificial Sequence
<220> 
<223> DNA primer sequence for SEQ ID NO: 9 synthesis 
<400> 10
tctggcccta tgtttttcct tcctcca  					  	27
<210> SEQ ID NO: 11 
<211> 27
<212> DNA
<213> Artificial Sequence
<220> 
<223> DNA primer sequence for SEQ ID NO: 9 synthesis
<400> 11
tggaggaagg aaaaacatag ggccaga  						27

<210> SEQ ID NO: 12 
<211> 760
<212> PRT
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase with Y125F mutation encoded by SEQ ID NO: 9
<400> 12
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Phe Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln 
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp 
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His 
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His 
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala 
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe 
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn


<210> SEQ ID NO: 13 
<211> 2283
<212> DNA
<213> Artificial sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution F260Y
<400> 13
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240 
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgctac 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa      							  2283

<210> SEQ ID NO: 14
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 13 synthesis 
<400> 14
caaatgtggt gctactgccg gctggtt 						   27


<210> SEQ ID NO: 15 
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 13 synthesis
<400> 15
aaccagccgg cagtagcacc acatttg 						   27

<210> SEQ ID NO: 16
<211> 760
<212> PRT
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase encoded by SEQ ID NO:13
<400> 16
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln
Met Trp Cys Tyr Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His 
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn


<210> SEQ ID NO:17 
<211> 2283
<212> DNA
<213> Artificial Sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution T413S
<400> 17
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagtcat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa      							  2283


<210> SEQ ID NO: 18
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 17 synthesis
<400> 18
gaatgaaagt tcagtcattt ggcagtcaaa c  					   31


<210> SEQ ID NO: 19
<211> 31
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 13 synthesis
<400> 19
gtttgactgc caaatgactg aactttcatt c  					   31


<210> SEQ ID NO: 20 
<211> 760
<212> PRT 
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase encoded by SEQ ID NO:17
<400> 20
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln 
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Ser Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His 
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn


<210> SEQ ID NO: 21 
<211> 2283
<212> DNA
<213> Artificial Sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution V484L
<400> 21
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1220
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaac tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa   								  2283


<210> SEQ ID NO: 22
<211> 34
<212> DNA
<213> 
<223> 
<223> DNA primer sequence for SEQ ID NO: 21 synthesis
<400> 22
gatcatggtt ggcaactttc agattgtact gcag					   34

<210> SEQ ID NO: 23
<211> 34
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 21 synthesis
<400> 23
ctgcagtaca atctgaaagt tgccaaccat gatc					   34


<210> SEQ ID NO: 24
<211> 760
<212> PRT 
<213> Artificial sequence 
<220>
<223> tirucalladienol synthase encoded by SEQ ID NO:21
<400> 24
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His
Gly Trp Gln Leu Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His 
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn


<210> SEQ ID NO: 25 
<211> 2283
<212> DNA
<213> Artificial Sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution V534A
<400> 25
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag catgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa                                                               2283


<210> SEQ ID NO: 26
<211> 28
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 25 synthesis
<400> 26
ggtggaatag cagcatggga gaaagctg   					28


<210> SEQ ID NO: 27 
<211> 28
<212> DNA
<213> Artificial Sequence
<223> DNA primer sequence for SEQ ID NO: 25 synthesis
<400> 27
cagctttctc ccatgctgct attccacc   					28


<210> SEQ ID NO: 28 
<211> 760
<212> PRT 
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase encoded by SEQ ID NO:25
<400> 28
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln 
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val 
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp 
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His 
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Ala Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Leu Glu Asp Leu Ile Val Glu His 
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe 
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn     
 

<210> SEQ ID NO:29 
<211> 2283
<212> DNA
<213> Artificial Sequence
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution L553F
<400> 29 
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctgta gagtttttcg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa    								  2283


<210> SEQ ID NO: 30 
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 29 synthesis
<400> 30
cctgtagagt ttttcgagga ccttatt						   27
?

<210> SEQ ID NO: 31
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 29 synthesis
<400> 31  
aataaggtcc tcgaaaaact ctacagg				 		   27


<210> SEQ ID NO: 32
<211> 760
<212> PRT 
<213> Artificial sequence 
<220>
<223> Tirucalladienol synthase encoded by SEQ ID NO:29
<400> 32
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile 
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser 
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln 
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu 
Glu Trp Leu Asn Pro Val Glu Phe Phe Glu Asp Leu Ile Val Glu His
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn


<210> SEQ ID NO: 33 
<211> 2283
<212> DNA
<210> Artificial sequence 
<220>
<223> cDNA encoding mutant tirucalladienol synthase with substitution V550T  
<400> 33
atgtggaagc tgaagattgc agagggtgac aaaaatagcc catatatttc tacaacaaac 60
aatttcgttg gaaggcaaat atgggaattt gatccgaacg ccggaactgc tgaagagctt 120
gctgaagttg aagaagctcg tcagaatttc tacaagaatc gccatcaagt caaacctgct 180
agtgatctta tttttcgtct tcagtttctt agagagaaaa acttcaagca aacgattcct 240
caagtgaagg ttgaagatgg ggaggagatc acatatgaca ctgccacagc agcaatgaag 300
agggctgctc actacttctc agcaattcag gctagcgatg gccattggcc tgctgaaaat 360
tctggcccta tgtatttcct tcctccattt gtattctgct tgtacattac aggacatctt 420
gatactgtat ttacagctgc tcatcgcaga gaagtccttc gttacttata caatcatcag 480
catgaagatg gaggatgggg aatacacata gaagcgccaa gcagtatgtt tggtacagtt 540
tacagttatc ttacaatgcg tttgctaggg ttaggaccca acgatggtga aaacaatgcc 600
tgcgccagag ctagaaaatg gattcgtgat aatggtggtg tcacttacat tccctcttgg 660
ggaaagaatt ggctttcgat tcttggtttg tttgaatggg ctggaacaca cccaatgccc 720
ccagagttct ggatgcttcc ttctcatttt ccacttcatc cagcccaaat gtggtgcttc 780
tgccggctgg tttacatgcc cttgtgttat ttatacggca aaagatttgt tggtccaatc 840
actccactta tcaaacaact gagagaagaa cttcatacag agccttacga taaaatcaac 900
tggaggaaag ttcgtcatca atgtgcaaag actgatctct actaccccca tccattcgta 960
caagaagttc tatgggatac tctatacttt gctacagagc ctctgcttac tcgttggcca 1020
ttgaacaagt atgtcagaga gaaggctttg aaacaaacga tgaagatcat tcattatgaa 1080
gaccaaagca gtcgatatat tactattgga tgcgtcgaga agccgctgtg tatgcttgct 1140
tgttgggtgg aggatcctga aggggttgct ttcaagaagc atcttgagag aattgctgat 1200
tttatttgga ttggagaaga tggaatgaaa gttcagacat ttggcagtca aacatgggat 1260
actgctcttg gacttcaagc tttgcttgct tgcaatatcg ttgatgaaat tggacctgca 1320
cttgctaaag gacacgacta cttgaagaaa gctcaggtga gggataatcc agtgggtgat 1380
tatacaagca atttccgaca cttttccaaa ggagcatgga ctttctctga tcaagatcat 1440
ggttggcaag tttcagattg tactgcagaa agtttgaagt gctgcctgca tttctcaatg 1500
ctgcctccag aaattgttgg agagaaacat gatcctgaga gattatatga agctgtcaat 1560
ttcatactct ctcttcagga taaaaatggt ggaatagcag tttgggagaa agctggtgcc 1620
tctttgatgt tagagtggct caatcctaca gagtttctgg aggaccttat tgttgagcat 1680
acttacgtgg aatgcactgc ttcagcaatc gaggcatttg ttatgttcaa gaaattatac 1740
ccacatcatc gcaagaagga gattgaaaat ttcctcgtaa aagctgtaca gtacattgaa 1800
aatgaacaaa ctgctgatgg ttcatggtat ggaaactggg gagtttgctt cttatatgga 1860
acatgttttg cacttggagg tttacatgct gctggaaaga cttacaacaa ttgtcttgcc 1920
attcgtagag cagttgagtt tctgcttcaa gcacagagtg atgatggtgg ttggggagag 1980
agctacaaat cttgccctag taagatatac gtacctcttg atgggaaaag atcaactgtg 2040
gtacacactg cattggctat tcttggttta atccatgctg ggcaggctga aagagaccca 2100
acccctattc atcgtggtgt aaaattgctg atcaactctc aattggagaa tggagacttc 2160
cctcaacagg aaattatggg agtttttatg agaaactgta tgttacacta tgctcaatac 2220
aggaatattt ttcctttgtg ggctttagct gaatatagaa gaaaagttcc attgcctaat 2280
taa                                                               2283


<210> SEQ ID NO:34
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 33 synthesis
<400> 
tggctcaatc ctacagagtt tctggag  						   27


<210> SEQ ID NO:35
<211> 27
<212> DNA
<213> Artificial Sequence
<220>
<223> DNA primer sequence for SEQ ID NO: 33 synthesis
<400> 29
ctccagaaac tctgtaggat tgagcca						   27


<210> SEQ ID NO:36
<211> 760
<212> PRT 
<213> Artificial sequence 
<220>
<223> tirucalladienol synthase encoded by SEQ ID NO:33.
<400> 36
Met Trp Lys Leu Lys Ile Ala Glu Gly Asp Lys Asn Ser Pro Tyr Ile
Ser Thr Thr Asn Asn Phe Val Gly Arg Gln Ile Trp Glu Phe Asp Pro 
Asn Ala Gly Thr Ala Glu Glu Leu Ala Glu Val Glu Glu Ala Arg Gln 
Asn Phe Tyr Lys Asn Arg His Gln Val Lys Pro Ala Ser Asp Leu Ile 
Phe Arg Leu Gln Phe Leu Arg Glu Lys Asn Phe Lys Gln Thr Ile Pro
Gln Val Lys Val Glu Asp Gly Glu Glu Ile Thr Tyr Asp Thr Ala Thr 
Ala Ala Met Lys Arg Ala Ala His Tyr Phe Ser Ala Ile Gln Ala Ser
Asp Gly His Trp Pro Ala Glu Asn Ser Gly Pro Met Tyr Phe Leu Pro 
Pro Phe Val Phe Cys Leu Tyr Ile Thr Gly His Leu Asp Thr Val Phe 
Thr Ala Ala His Arg Arg Glu Val Leu Arg Tyr Leu Tyr Asn His Gln
His Glu Asp Gly Gly Trp Gly Ile His Ile Glu Ala Pro Ser Ser Met 
Phe Gly Thr Val Tyr Ser Tyr Leu Thr Met Arg Leu Leu Gly Leu Gly 
Pro Asn Asp Gly Glu Asn Asn Ala Cys Ala Arg Ala Arg Lys Trp Ile 
Arg Asp Asn Gly Gly Val Thr Tyr Ile Pro Ser Trp Gly Lys Asn Trp 
Leu Ser Ile Leu Gly Leu Phe Glu Trp Ala Gly Thr His Pro Met Pro
Pro Glu Phe Trp Met Leu Pro Ser His Phe Pro Leu His Pro Ala Gln 
Met Trp Cys Phe Cys Arg Leu Val Tyr Met Pro Leu Cys Tyr Leu Tyr 
Gly Lys Arg Phe Val Gly Pro Ile Thr Pro Leu Ile Lys Gln Leu Arg 
Glu Glu Leu His Thr Glu Pro Tyr Asp Lys Ile Asn Trp Arg Lys Val 
Arg His Gln Cys Ala Lys Thr Asp Leu Tyr Tyr Pro His Pro Phe Val
Gln Glu Val Leu Trp Asp Thr Leu Tyr Phe Ala Thr Glu Pro Leu Leu 
Thr Arg Trp Pro Leu Asn Lys Tyr Val Arg Glu Lys Ala Leu Lys Gln 
Thr Met Lys Ile Ile His Tyr Glu Asp Gln Ser Ser Arg Tyr Ile Thr 
Ile Gly Cys Val Glu Lys Pro Leu Cys Met Leu Ala Cys Trp Val Glu 
Asp Pro Glu Gly Val Ala Phe Lys Lys His Leu Glu Arg Ile Ala Asp 
Phe Ile Trp Ile Gly Glu Asp Gly Met Lys Val Gln Thr Phe Gly Ser 
Gln Thr Trp Asp Thr Ala Leu Gly Leu Gln Ala Leu Leu Ala Cys Asn 
Ile Val Asp Glu Ile Gly Pro Ala Leu Ala Lys Gly His Asp Tyr Leu 
Lys Lys Ala Gln Val Arg Asp Asn Pro Val Gly Asp Tyr Thr Ser Asn 
Phe Arg His Phe Ser Lys Gly Ala Trp Thr Phe Ser Asp Gln Asp His
Gly Trp Gln Val Ser Asp Cys Thr Ala Glu Ser Leu Lys Cys Cys Leu 
His Phe Ser Met Leu Pro Pro Glu Ile Val Gly Glu Lys His Asp Pro 
Glu Arg Leu Tyr Glu Ala Val Asn Phe Ile Leu Ser Leu Gln Asp Lys 
Asn Gly Gly Ile Ala Val Trp Glu Lys Ala Gly Ala Ser Leu Met Leu
Glu Trp Leu Asn Pro Thr Glu Phe Leu Glu Asp Leu Ile Val Glu His
Thr Tyr Val Glu Cys Thr Ala Ser Ala Ile Glu Ala Phe Val Met Phe 
Lys Lys Leu Tyr Pro His His Arg Lys Lys Glu Ile Glu Asn Phe Leu 
Val Lys Ala Val Gln Tyr Ile Glu Asn Glu Gln Thr Ala Asp Gly Ser 
Trp Tyr Gly Asn Trp Gly Val Cys Phe Leu Tyr Gly Thr Cys Phe Ala 
Leu Gly Gly Leu His Ala Ala Gly Lys Thr Tyr Asn Asn Cys Leu Ala
Ile Arg Arg Ala Val Glu Phe Leu Leu Gln Ala Gln Ser Asp Asp Gly 
Gly Trp Gly Glu Ser Tyr Lys Ser Cys Pro Ser Lys Ile Tyr Val Pro 
Leu Asp Gly Lys Arg Ser Thr Val Val His Thr Ala Leu Ala Ile Leu 
Gly Leu Ile His Ala Gly Gln Ala Glu Arg Asp Pro Thr Pro Ile His 
Arg Gly Val Lys Leu Leu Ile Asn Ser Gln Leu Glu Asn Gly Asp Phe 
Pro Gln Gln Glu Ile Met Gly Val Phe Met Arg Asn Cys Met Leu His 
Tyr Ala Gln Tyr Arg Asn Ile Phe Pro Leu Trp Ala Leu Ala Glu Tyr 
Arg Arg Lys Val Pro Leu Pro Asn



