C-19
22 5 19
EST RT-PCRol i vetol synthase
50%
In thi s study, I cloned a gene encodi ng ol i vetol synthase, the
pol yketide synthase that synthesi zes the phenol i c moi ety of cannabi noi ds i n , by means of EST anal ysi s and RT-PCR, and then characteri zed the bacteri al l y
expressed recombi nant ol i vetol synthase to cl ari fy the structural and functi onal properties i n detai l . In addi ti on, I attempted to obtai n a gene f or the prenyl transferase that synthesi ze the carbon skel eton of cannabi noi ds, resul ti ng i n cl oning of two candi date genes f or the prenyl transf erase. Interesti ngl y, I conf i rmed that these putati ve prenyl transferases show more that 50 % identi ty wi th the prenyl transferase i nvolved i n secondary metabol i sm i n hop, a cl ose rel ati ve of .
2008 2, 600, 000 780, 000 3, 380, 000 2009 800, 000 240, 000 1, 004, 000
B
EST
Cloning and application of cannabinoid biosynthetic genes based on
EST analysis
TAURA FUTOSHI
3,400,000 1,020,000
1,040,000
4,420,000
t et rahydrocannabi nol ( THC)
THC
tetrahydrocannabi nol i c aci d THCATHCA synthase cDNA
279, 39767-39774, 2004 THCA synthase
cannabi gerol i c aci d CBGATHCA
361, 675-680, 2007 THCATHC
THC
CBGA
CBGATHCA
synthase
CBGA
ol ivetol i c aci d
CBGA
RT-PCR ESTExpressed Sequence Tag
RT- PCR EST
THCA synthaseTHCA
THC
1
mRNA cDNA
PCR
5’ RACE 3’ RACE
RT-PCR
2
mRNA cDNA
cDNA
5’
EST
3
1pET24a
BL21(DE3)
acyl CoAHPLC
41
pPICZa
1
RT-PCR CsPKScDNA
CsPKS
chal cone synthase 65%
chal cone synthase
CsPKS pET24a
Hi s-bi nd resi n
ol i vetol i c aci dhexanoyl -CoA mal onyl -CoA
HPLCol i vetol i c acid
NMRCsPKS derai l ment producttri keti de pyrone
tetraketide pyrone al dol
ol i vetol i c aci dol i vetol
CsPKS hexanoyl -CoA ol i vetoltetraketi de pyrone 3. 46 mi n-11. 06 mi n-1 cat
ol i vetolol ivetol synthase OLS
Ol i vetolal kyl resorci nol PKS
OLSPKS
-coumaroyl -CoAresveratrol sti l bene synthase
sti l bene synthasehexanoyl -CoA pyrone
CoA
hexanoyl -CoACoA butyryl -CoAi soval eryl -CoA octanoyl -CoA
butyryl -CoAcat: 0. 25 mi n-1 resorci nol
tri keti de pyronebenzoyl -CoA -coumaroyl - CoA
CoAC4
C8 CoAhexanoyl -CoA
OLShexanoyl -CoA
OLS OLS
western bl otti ng
ol i vetolOLS
OLS ol l i vetol i c aci d
hexanoyl -CoAal dol
ol i vetol i c aci d
2
ubi A
ubi A
PCR CsPTCsPT2
5’ RACE 3’ RACEcDNA
CsPT1 404 CsPT2 384PSORT
transi t peptide
NQxxDxxxD
f lavonoi d prenyl transferase
bi tter aci d50%
CsPT12
cDNA
EST
Marks( 60, 3715-3726,
2009)
4 C. Taguchi , F. Taura
, T. Tamada, Y. Shoyama, Y. Shoyama, H. Tanaka, R. Kuroki ,
S. Morimoto, Crystal l i zati on and prel i mi nary X-ray di f f racti on studies of
pol yketide synthase-1 (PKS- 1) f rom . ,
, , 64, 2008, 217-220
F. Taura
, Molecul ar characteri zati on of the bi osynthetic enzyme for the
bi otechnol ogical producti on of
tetrahydrocannabinol , the active consti tuent
of mari j uana,
, , 2, Supplement, 2008, 59-59
F. Taura
, S. Tanaka, C. Taguchi , T. Fukami zu, H. Tanaka, Y. Shoyama, and S. Mori moto Characteri zation of ol i vetol synthase, a pol yketi de synthase putati vel y i nvol ved in cannabi noi d bi osyntheti c pathway, FEBS Letters,
, 583, 2009, 2061-2066 F. Taura
, Studi es on tetrahydrocannabi nol i c aci d synthase that produces the acidi c precursor of tetrahydrocannabi nol , the pharmacol ogi cal l y active cannabi noi d i n mari j uana,
, , 3, 2009, 83-87
2
129 21 327 F. Taura
Characteri zati on of ol i vetol synthase, a pol yketi de synthase putati vel y i nvol ved in cannabi noi d bi osyntheti c pathway Asi an Symposi um for Pharmaceuti cal Sci ences i n JSPS Asi an Core Program i n Fukuoka 21 11 6
1 F. Taura
, S. Si rikantaramas, Y. Shoyama, Y. Shoyama and S. Mori moto, Phytocannabi noi ds i n Cannabis sati va: Recent studi es on bi osyntheti c enzymes in: D. M. Lambert (Ed. ) , Cannabi noi d i n Nature and Medi cine, Springer Verl ag, 2009, 51-65
http: / / sei gyo. phar. kyushu-u. ac. j p/
( 1) TAURA FUTOSHI
00301341
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