Molecular cloning and functional expression of geranylgeranyl pyrophosphate synthase from Coleus forskohliiBriq
© Engprasert et al; licensee BioMed Central Ltd. 2004
Received: 20 August 2004
Accepted: 18 November 2004
Published: 18 November 2004
Isopentenyl diphosphate (IPP), a common biosynthetic precursor to the labdane diterpene forskolin, has been biosynthesised via a non-mevalonate pathway. Geranylgeranyl diphosphate (GGPP) 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 359 amino acids, in which 1,077 nucleotides long with calculated molecular mass of 39.3 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.
Forskolin, a labdane diterpene, is a major active compound isolated from tuberous roots of Coleus forskohlii Briq. (Lamiaceae) . 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 [3, 4].
Isoprenoids are essential for the normal growth and development processes in all living organisms. Isopentenyl diphosphate (IPP; C5) is a common metabolic precursor of all isoprenoids. Recently, several groups have demonstrated that two distinct pathways synthesise IPP in plants. The mevalonate (MVA) pathway occurs in the cytoplasm, and an alternative mevalonate-independent (2C-methyl-D-erythritol 4-phosphate; MEP) pathway occurs in plastids [5–7].
Geranylgeranyl diphosphate (GGPP) 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 [9, 10], 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 (FARM) and the second aspartate-rich motif (SARM) . 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 [13, 18].
Carotenoids arise from the coupling of two molecules of GGPP. The carotenoid biosynthetic gene cluster (crt genes) 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 (phytoene synthase), crtE (GGPP synthase), crtI (phytoene desaturase), crtX (zeaxanthin β-glucosidase), crtY (lycopene cyclase) and crtZ (β-carotene hydroxylase). 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 and discussion
cDNA cloning and sequencing of C. forskohlii GGPP synthasegene
Transient expression of putative localization signal of C. forskohliiGGPP synthase in tobacco cells
Heterologous expression and activity of C. forskohliiGGPP synthase
Expression of GGPP synthase gene in organs of C. forskohlii
Forskolin production via 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.
Plant materials and reagents
C. forskohlii plantlets were cultured in hormone-free MS (Murashige and Skoog) medium at 25°C under a 16 hours light cycle. The light intensity was 3000 lux and the relative humidity was 60%. Shoot cuttings (10 mm in length) propagated by shoot tip culture were successively cultivated in vermiculite. BY-2 tobacco single cell suspension  was cultured in liquid modified LS (Leinsmaier and Skoog) medium supplemented with 0.2 mg l-1 of 2,4-D (2,4-dichlorophonoxy acetic acid) under dark conditions at 25°C on an orbital incubator. Restriction enzymes, ligase, and PCR-polymerase were purchased from Takara Shuzo Co., Ltd. (Tokyo, Japan) and Toyobo Co., Ltd. (Tokyo, Japan). Fosmidomycin (FR-3154) was purchased from Molecular Probes (Oregon, USA). Chemical reagents were purchased from Sigma Chemical Company (St. Louis, USA) and Nacalai Tesque Inc. (Tokyo, Japan)
Bacterial strains and plasmids
E. coli TOP10F' and E. coli DH10B carrying the plasmid pACCAR25ΔcrtE were used in the present investigation. The pUC119 vector was used for cDNA cloning and sequencing. The pBluescript II KS- vector was used as a GGPP synthase expression plasmid. The 35SΩ-sGFP(S65T) plasmid was used as a green fluorescent protein (GFP) reporter plasmid. The pBI121 plant vector and Agrobacterium tumefaciens LBA4404 were used for transformation of GFP and GFP-fusion genes to plant cells.
cDNA cloning and sequencing of C. forskohlii GGPP synthasegene
Primers for cDNA cloning of GGPP synthase.
Sequences (5' to 3')
conserved sequence (LCIAACE)
conserved sequence (VEMIHTM)
conserved sequence (DILDVTK)
conserved sequence (KTAGKDL)
oligo-dT adapter primer
degenerated PCR product (KPTNHK)
degenerated PCR product (VYGENVA)
degenerated PCR product (AILGGAD)
degenerated PCR product (DEAVEKL)
N-terminal sequence (MSLIAS)
N-terminal sequence (EKAERVN)
All amplified cDNA fragments were purified and digested with restriction enzymes at sites introduced via the PCR primers, and cloned into the vector pUC119. After transformation to E. coli TOP10F', clones harboring inserts were sequenced using a Model 310 Genetic Analyzer (PE Biosystems) using a BigDye Terminator Cycle Sequencing Kit.
The amino acid sequence deduced from the nucleotide sequence was compared with sequence databases in the Genome Net WWW server using the FASTA program. Multiple amino acid sequence alignment was performed using the CLUSTALW Multiple Sequence Alignment in the GenomeNet CLUSTALW Server.
Construction and expression of putative localization signal of C. forskohliiGGPP synthase
A 240 bp fragment of the N-terminal region of C. forskohlii GGPP synthase was PCR-amplified using primers P and Q and the PCR product was digested and cloned into the SalI-NcoI site of the 35SΩ-sGFP(S65T) plasmid. 35SΩ-pt-sGFP(S65T) was used as the positive control for chloroplast targeting [29, 30]. GFP, GGPP synthase-GFP fusion and pt-GFP fusion with CaMV35SΩ promoter and NOS3' terminator [35SΩ-sGFP (S65T), 35SΩ-GGPP synthase-sGFP (S65T) and 35SΩ-pt-sGFP (S65T), respectively] were subcloned into the HindIII-EcoRI site of the pBI121 vector and then transformed into A. tumefaciens LBA4404. The transformants were cultured at 28°C for two days in YEB liquid medium containing 25 μg/ml of kanamycin and 25 μg/ml of rifampicin. The transformants were washed twice and re-suspended in YEB medium. Agrobacterium transformants (108 cells) were applied to four ml of five-day-old BY-2 suspension culture. The culture was incubated at 28°C for two days under dark conditions. GFP and GFP fusion protein were analysed by fluorescence microscopy using Nikon Eclipse TE2000-U model. Cells were observed at a 400 × magnification.
Construction of plasmid for C. forskohliiGGPP synthase expression
The coding region of a cDNA of C. forskohlii GGPP synthase was amplified by PCR using specific primers M and O. A PCR product was digested; purified and cloned into the KpnI-SalI site of pBluescript II KS- vector, namely pGGPPS. This plasmid was transformed into E. coli XL1-Blue MRF' for over-expression. The transformants were cultured in LB liquid medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol. The culture was induced with 1 mM isopropyl-1-thio-β-D-galactoside (IPTG) and incubated for six hours at 37°C. The cells were harvested and washed with 50 mM Tris-HCl pH 8.0 by centrifugation. The pellet was re-suspended, lysozyme was added and the mixture was incubated for 30 minutes. The mixture was then sonicated for four cycles of 15 seconds at one minute intervals. The soluble fraction was obtained after centrifugation at 10,000 × g for 10 minutes. SDS-PAGE was conducted in order to detect the proteins .
Genetic complementation expression
The pACCAR25ΔcrtE plasmid contains the gene cluster crtB, crtI, crtX, crtY and crtZ encoding carotenoid biosynthetic enzymes with the exception of crtE (encoding GGPP synthase). The plasmid pBAA containing mouse GGPP synthase (positive control plasmid) and E. coli DH10B carrying the plasmid pACCAR25ΔcrtE was provided by Dr. M. Kawamukai, Shimane University, Japan . pBluescript II KS- vector, pBS, was used as negative control. pGGPPS, pBAA and pBS were transformed into E. coli DH10B carrying the plasmid pACCAR25ΔcrtE. All transformants were plated on LB agar medium containing 50 μg/ml of ampicillin and 25 μg/ml of chloramphenicol and then incubated for two to three days at 25°C.
Reverse transcriptase-PCR (RT-PCR)
An eight-month-old C. forskohlii was analysed in twelve separate parts; leaf (L1–L4), stem (S1–S5) and root (R1–R3). The numbering is based on the maturation of organs. Total RNA was extracted from each part of plant. One microgram of total RNA was used as the template for the synthesis of the first strand cDNA (using SuperScript First-Strand Synthesis System for RT-PCR, Invitrogen). Primers M and O, the first strand cDNA and KOD-polymerase were used for the amplification of C. forskohlii GGPP synthase with the condition of denaturation, 98°C, 15 seconds; annealing, 60°C, 2 seconds and extension, 74°C, 5 seconds. The 18S rRNA fragment used as an internal control was amplified using primers R and S under the same conditions of C. forskohlii GGPP synthase amplification. The amplified PCR products were analysed by 1.0% agarose gel electrophoresis.
Analysis of forskolin production
C. forskohlii plantlets were treated with various concentrations of fosmidomycin and then investigated for forskolin content using the HPLC method as previously described . Forskolin was detected by comparison with the retention time of a forskolin standard (Sigma) detected by UV absorption at 202 nm.
List of abbreviations
- crt :
first aspartate-rich motif
green fluorescent protein
second aspatate-rich motif
35SΩ-sGFP(S65T) plasmid was generously provided by Dr. Yasuo Niwa, University of Shizuoka, Japan.
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