BACKGROUND
forskolin, a labdane diterpene, is a major active compound isolated from tuberous roots of coleus forskohlii briq.
<cit> .
c. forskohlii has been used as an important folk medicine in india.
futher, forskolin has been found to be a potent activator of adenylate cyclase  <cit> , leading to an increase in levels of c-amp, which affects heart action, blood and intraocular pressure.
recently, forskolin has become commercially available as a drug for treating heart disease in japan.
forskolin is not available by chemical synthesis due to its complicated structure.
however, two groups have reported successful total synthesis of forskolin  <cit> .
isoprenoids are essential for the normal growth and development processes in all living organisms.
isopentenyl diphosphate  is a common metabolic precursor of all isoprenoids.
recently, several groups have demonstrated that two distinct pathways synthesise ipp in plants.
the mevalonate  pathway occurs in the cytoplasm, and an alternative mevalonate-independent  pathway occurs in plastids  <cit> .
geranylgeranyl diphosphate  synthase catalyses the consecutive condensation of an allylic diphosphate with three molecules of ipp to produce ggpp, an essential linear precursor for biosynthesis of diterpenes, carotenoid, retinoids and side chain of chlorophyll  <cit> .
ggpp synthase is an important branch point prenyltransferase enzyme in terpenoid biosynthesis.
ggpp synthase genes have been cloned in a number of organisms including; arabidopsis thaliana  <cit> , taxus canadensis  <cit> , helianthus annuus  <cit> , scoparia dulcis and croton sublyratus  <cit> , sulfolobus acidocaldarius  <cit> , neurospora crassa  <cit> , and mouse and human  <cit> .
amino acid sequence comparison has shown that ggpp synthases contain several domains of conserved amino acid residues including the first aspartate-rich motifs  and the second aspartate-rich motif   <cit> .
futhermore, recent studies suggested that two amino acids at the four and five positions before farm in the sequence, as well as an insertion in farm of plant ggpp synthases play important roles in product length determination  <cit> .
carotenoids arise from the coupling of two molecules of ggpp.
the carotenoid biosynthetic gene cluster  of erwinia uredovora was elucidated  <cit> , and is currently used to investigate the function of carotenoid related genes in a heterologous system.
this crt gene cluster is composed of six genes; crtb , crte , crti , crtx , crty  and crtz .
consequently, the production of carotenoids using e. coli harbouring the crt gene cluster can be used for the determination of ggpp synthase activity.
ggpp synthase is suggested to be a key enzyme in the biosynthesis of forskolin.
herein, we report the cdna encoding c. forskohlii ggpp synthase and its heterologous expression in e. coli.
RESULTS
cdna cloning and sequencing of c. forskohlii ggpp synthase gene
the open reading frame  for full-length ggpp synthase gene encodes a protein of  <dig> amino acids,  <dig>  nucleotides long, with a calculated molecular mass of  <dig>  kda.
the amino acid sequence of c. forkohlii ggpp synthase revealed high homology throughout the entire coding region of catharanthus roseus , arabidopsis thaliana , sinapis alba , croton sublyratus , scoparia dulcis  and mentha piperita  .
however, comparison of the amino acid sequence with that of prokaryotic ggpp synthases showed a low level of homology .
highly conserved residues were designated as domains i-vii.
two conserved aspartate-rich motifs, ddxxd, were identifed.
farm and sarm have been shown to be important in substrate binding and catalysis  <cit> .
transient expression of putative localization signal of c. forskohlii ggpp synthase in tobacco cells
sequence alignment of plant ggpp synthases showed that the n-terminal region has a low level of homology.
it is reasonable to assume that these ggpp synthases have localization signals in their n-terminal regions to target them into specific subcellular compartments.
the n-terminal region of c. forskohlii ggpp synthase was predicted to be localized in chloroplasts by the chlorop  <dig>  prediction server.
in an effort to determine the localization of c. forskohlii ggpp synthase, the sequence coding for the  <dig> amino acid sequence at the n-terminus of c. forskohlii ggpp synthase was fused to the n-terminus of the gfp reporter gene and transformed into by- <dig> tobacco cells.
the pattern of putative localization signal of c. forskohlii ggpp synthase was identical to the positive chloroplast targeting signal  .
the n-terminal region of c. forskohlii ggpp synthase was determined to contain a chloroplast localization signal.
recently, plant ggpp synthases have been determined to be translocated into plastids, mitochondria and cytosol  <cit> .
heterologous expression and activity of c. forskohlii ggpp synthase
in order to express c. forskohlii ggpp synthase, the gene was constructed and cloned into the plasmid pbluescript ii ks-.
the fusion protein of ggpp synthase with lacz had a calculated molecular mass of  <dig>  kda, was observed in the soluble fraction of e. coli carrying pggpps after iptg induction .
functional activity of expressed ggpp synthase was investigated by genetic complementation with the carotenogenic crt gene cluster.
carotenoids are produced in e. coli harbouring a crt cluster gene from e. uredovora.
replacements of a crt gene with an unknown gene with the same activity, can be used to determine the function of the gene  <cit> .
herein, the c. forskohlii ggpp synthase gene was cloned into pbluescript ii ks- vector  in order to produce a lacz fusion protein.
pggpps was then transformed into e. coli dh10b carrying the plasmid paccar25Δcrte in which the crte encoding ggpp synthase had been deleted.
the yellow color of carotenoid was observed in the transformant, indicating that pggpps carried the gene substituting the function of the crte gene .
carotenoid production of the transformants was compared with that of e. coli transformant carrying plasmid paccar25Δcrte and pbaa encoding mouse ggpp synthase   <cit> , and with transformant carrying plasmid paccar25Δcrte and a pbluescript ii ks-  vector .
this result suggested that the coding region of a cdna of c. forskohlii ggpp synthase encodes a functional ggpp synthase.
expression of ggpp synthase gene in organs of c. forskohlii
the expression of ggpp synthase gene was investigated by rt-pcr in different organs of c. forskohlii.
total rna extracted from the roots, stems and leaves of an eight-month-old plant were analysed.
the c. forskohlii ggpp synthase gene was strongly expressed in the leaves, whereas expression was decreased in stems and barely expressed in roots .
therefore, the leaves are thought to be the primary location for forskolin synthesis.
we previously reported the forskolin concentration in clonally propagated plant organs of c. forskohlii  <cit> .
tuberous roots and the stem base were determined to contain a higher concentration of forskolin than the organs.
moreover, the stem base, parts of the epidermis and cortex, the vascular bundle, and the pith were analysed separately.
the highest concentration of forskolin was identified in the vascular bundle tissue.
from these data, we proposed that ggpp synthase involved in biosynthesis of forskolin, is mainly synthesised in leaves, subsequently distributed to stems and finally accumulated in stem bases and roots.
forskolin production via non-mevalonate pathway
in an effort to investigate the forskolin biosynthesis pathway by a non-mevalonate pathway, various concentrations of fosmidomycin, the specific inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase  enzyme in the non-mevalonate pathway were applied to the c. forskohlii culture and the forskolin content of roots was determined .
treatment led to a decrease in forskolin, whereas  <dig> μm fosmidomycin had no effect on forskolin production.
at higher concentrations a dose-dependent inhibitory effect was observed.
at  <dig> μm fosmidomycin, the forskolin content was decreased by up to fifty percent in comparison to the control tissue without inhibitor treatment.
thus, forskolin was thought to be synthesised via a non-mevalonate pathway.
a recent 13c-glucose feeding experiment using 13c-nmr analytical methodology suggested the biosynthetic pathway of forskolin via a non-mevalonate pathway  <cit> .
in addition, the dxr gene regarding the specific enzyme in the first step of the non-mevalonate pathway was cloned from c. forskohlii  <cit> .
CONCLUSIONS
c. forskohlii ggpp synthase was cloned and its subcellular localization was determined.
the n-terminal region contained a signal which was localized in chloroplasts.
functional expression of ggpp synthase was investigated by genetic complementation with the carotenogenic crt gene cluster.
carotenoids were produced when the crte gene was replaced with c. forskohlii ggpp synthase.
ggpp synthase is thought to be involved in biosynthesis of forskolin, which is primary synthesised in the leaves, subsequently distributed to stems and finally accumulated in stem bases and roots.

 forskolin, a labdane diterpene, is a major active compound isolated from tuberous roots of coleus forskohlii briq.. c. forskohlii has been used as an important folk medicine in india. futher, forskolin has been found to be a potent activator of adenylate cyclase, leading to an increase in levels of c-amp, which affects heart action, blood and intraocular pressure. recently, forskolin has become commercially available as a drug for treating heart disease in japan. forskolin is not available by chemical synthesis due to its complicated structure. however, two groups have reported successful total synthesis of forskolin. isoprenoids are essential for the normal growth and development processes in all living organisms. isopentenyl diphosphate is a common metabolic precursor of all isoprenoids. recently, several groups have demonstrated that two distinct pathways synthesise ipp in plants. the mevalonate pathway occurs in the cytoplasm, and an alternative mevalonate-independent pathway occurs in plastids. geranylgeranyl diphosphate synthase catalyses the consecutive condensation of an allylic diphosphate with three molecules of ipp to produce ggpp, an essential linear precursor for biosynthesis of diterpenes, carotenoid, retinoids and side chain of chlorophyll. ggpp synthase is an important branch point prenyltransferase enzyme in terpenoid biosynthesis. ggpp synthase genes have been cloned in a number of organisms including; arabidopsis thaliana, taxus canadensis, helianthus annuus, scoparia dulcis and croton sublyratus, sulfolobus acidocaldarius, neurospora crassa, and mouse and human. amino acid sequence comparison has shown that ggpp synthases contain several domains of conserved amino acid residues including the first aspartate-rich motifs and the second aspartate-rich motif. futhermore, recent studies suggested that two amino acids at the four and five positions before farm in the sequence, as well as an insertion in farm of plant ggpp synthases play important roles in product length determination. carotenoids arise from the coupling of two molecules of ggpp. the carotenoid biosynthetic gene cluster of erwinia uredovora was elucidated, and is currently used to investigate the function of carotenoid related genes in a heterologous system. this crt gene cluster is composed of six genes; crtb, crte, crti, crtx, crty and crtz. consequently, the production of carotenoids using e. coli harbouring the crt gene cluster can be used for the determination of ggpp synthase activity. ggpp synthase is suggested to be a key enzyme in the biosynthesis of forskolin. herein, we report the cdna encoding c. forskohlii ggpp synthase and its heterologous expression in e. coli.  cdna cloning and sequencing of c. forskohlii ggpp synthase gene the open reading frame for full-length ggpp synthase gene encodes a protein of amino acids, nucleotides long, with a calculated molecular mass of kda. the amino acid sequence of c. forkohlii ggpp synthase revealed high homology throughout the entire coding region of catharanthus roseus, arabidopsis thaliana, sinapis alba, croton sublyratus, scoparia dulcis and mentha piperita. however, comparison of the amino acid sequence with that of prokaryotic ggpp synthases showed a low level of homology. highly conserved residues were designated as domains i-vii. two conserved aspartate-rich motifs, ddxxd, were identifed. farm and sarm have been shown to be important in substrate binding and catalysis. transient expression of putative localization signal of c. forskohlii ggpp synthase in tobacco cells sequence alignment of plant ggpp synthases showed that the n-terminal region has a low level of homology. it is reasonable to assume that these ggpp synthases have localization signals in their n-terminal regions to target them into specific subcellular compartments. the n-terminal region of c. forskohlii ggpp synthase was predicted to be localized in chloroplasts by the chlorop prediction server. in an effort to determine the localization of c. forskohlii ggpp synthase, the sequence coding for the amino acid sequence at the n-terminus of c. forskohlii ggpp synthase was fused to the n-terminus of the gfp reporter gene and transformed into by- tobacco cells. the pattern of putative localization signal of c. forskohlii ggpp synthase was identical to the positive chloroplast targeting signal. the n-terminal region of c. forskohlii ggpp synthase was determined to contain a chloroplast localization signal. recently, plant ggpp synthases have been determined to be translocated into plastids, mitochondria and cytosol. heterologous expression and activity of c. forskohlii ggpp synthase in order to express c. forskohlii ggpp synthase, the gene was constructed and cloned into the plasmid pbluescript ii ks-. the fusion protein of ggpp synthase with lacz had a calculated molecular mass of kda, was observed in the soluble fraction of e. coli carrying pggpps after iptg induction. functional activity of expressed ggpp synthase was investigated by genetic complementation with the carotenogenic crt gene cluster. carotenoids are produced in e. coli harbouring a crt cluster gene from e. uredovora. replacements of a crt gene with an unknown gene with the same activity, can be used to determine the function of the gene. herein, the c. forskohlii ggpp synthase gene was cloned into pbluescript ii ks- vector in order to produce a lacz fusion protein. pggpps was then transformed into e. coli dh10b carrying the plasmid paccar25Δcrte in which the crte encoding ggpp synthase had been deleted. the yellow color of carotenoid was observed in the transformant, indicating that pggpps carried the gene substituting the function of the crte gene. carotenoid production of the transformants was compared with that of e. coli transformant carrying plasmid paccar25Δcrte and pbaa encoding mouse ggpp synthase, and with transformant carrying plasmid paccar25Δcrte and a pbluescript ii ks- vector. this result suggested that the coding region of a cdna of c. forskohlii ggpp synthase encodes a functional ggpp synthase. expression of ggpp synthase gene in organs of c. forskohlii the expression of ggpp synthase gene was investigated by rt-pcr in different organs of c. forskohlii. total rna extracted from the roots, stems and leaves of an eight-month-old plant were analysed. the c. forskohlii ggpp synthase gene was strongly expressed in the leaves, whereas expression was decreased in stems and barely expressed in roots. therefore, the leaves are thought to be the primary location for forskolin synthesis. we previously reported the forskolin concentration in clonally propagated plant organs of c. forskohlii. tuberous roots and the stem base were determined to contain a higher concentration of forskolin than the organs. moreover, the stem base, parts of the epidermis and cortex, the vascular bundle, and the pith were analysed separately. the highest concentration of forskolin was identified in the vascular bundle tissue. from these data, we proposed that ggpp synthase involved in biosynthesis of forskolin, is mainly synthesised in leaves, subsequently distributed to stems and finally accumulated in stem bases and roots. forskolin production via non-mevalonate pathway in an effort to investigate the forskolin biosynthesis pathway by a non-mevalonate pathway, various concentrations of fosmidomycin, the specific inhibitor of 1-deoxy-d-xylulose-5-phosphate reductoisomerase enzyme in the non-mevalonate pathway were applied to the c. forskohlii culture and the forskolin content of roots was determined. treatment led to a decrease in forskolin, whereas μm fosmidomycin had no effect on forskolin production. at higher concentrations a dose-dependent inhibitory effect was observed. at μm fosmidomycin, the forskolin content was decreased by up to fifty percent in comparison to the control tissue without inhibitor treatment. thus, forskolin was thought to be synthesised via a non-mevalonate pathway. a recent 13c-glucose feeding experiment using 13c-nmr analytical methodology suggested the biosynthetic pathway of forskolin via a non-mevalonate pathway. in addition, the dxr gene regarding the specific enzyme in the first step of the non-mevalonate pathway was cloned from c. forskohlii.  c. forskohlii ggpp synthase was cloned and its subcellular localization was determined. the n-terminal region contained a signal which was localized in chloroplasts. functional expression of ggpp synthase was investigated by genetic complementation with the carotenogenic crt gene cluster. carotenoids were produced when the crte gene was replaced with c. forskohlii ggpp synthase. ggpp synthase is thought to be involved in biosynthesis of forskolin, which is primary synthesised in the leaves, subsequently distributed to stems and finally accumulated in stem bases and roots. 
therefore, ggpp synthase is thought to be a key enzyme in biosynthesis of forskolin. herein we report the first confirmation of the ggpp synthase gene in coleus forskohlii briq. the open reading frame for full-length ggpp synthase encodes a protein of amino acids, in which nucleotides long with calculated molecular mass of kda. alignments of c. forskohlii ggpp synthase amino acid sequences revealed high homologies with other plant ggpp synthases. transient expression of the n-terminal region of c. forskohlii ggpp synthase-gfp fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. carotenoid production was observed in escherichia coli harboring paccar25Δcrte from erwinia uredovora and plasmid carrying c. forskohlii ggpp synthase. these results suggested that cdna encoded functional ggpp synthase. furthermore, c. forskohlii ggpp synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots. ggpp synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. several highly conserved regions, including two aspartate-rich motifs were identified. geranylgeranyl diphosphate synthase is an important branch point enzyme in terpenoid biosynthesis. isopentenyl diphosphate, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. this investigation proposed that forskolin was synthesised via a non-mevalonate pathway.
 isopentenyl diphosphate, a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. geranylgeranyl diphosphate synthase is an important branch point enzyme in terpenoid biosynthesis. therefore, ggpp synthase is thought to be a key enzyme in biosynthesis of forskolin. herein we report the first confirmation of the ggpp synthase gene in coleus forskohlii briq.  the open reading frame for full-length ggpp synthase encodes a protein of amino acids, in which nucleotides long with calculated molecular mass of kda. alignments of c. forskohlii ggpp synthase amino acid sequences revealed high homologies with other plant ggpp synthases. several highly conserved regions, including two aspartate-rich motifs were identified. transient expression of the n-terminal region of c. forskohlii ggpp synthase-gfp fusion protein in tobacco cells demonstrated subcellular localization in the chloroplast. carotenoid production was observed in escherichia coli harboring paccar25Δcrte from erwinia uredovora and plasmid carrying c. forskohlii ggpp synthase. these results suggested that cdna encoded functional ggpp synthase. furthermore, c. forskohlii ggpp synthase expression was strong in leaves, decreased in stems and very little expression was observed in roots.  this investigation proposed that forskolin was synthesised via a non-mevalonate pathway. ggpp synthase is thought to be involved in the biosynthesis of forskolin, which is primarily synthesised in the leaves and subsequently accumulates in the stems and roots. 