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