Plasmid pGB7/PMA1 was constructed by integrating a 700 pb PMA1 promoter in the NotI-SacI digested plasmid pGB7 [14]. The pSM5 plasmid was constructed through integration of O. basilicum geraniol synthase (GES), by means of recombination in the pGB7/PMA1 plasmid, in fusion with the green fluorescent protein (GFP) from pGB7. The pSM5 plasmid was then used for site-directed mutations of GES. The plasmids carrying the mutations were created using the QuikChange SiteDirected Mutagenesis Kit from Stratagene, in accordance with the manufacturer’s instructions. The complementary Forward and Reverse primers were designed as described in Table 1. All of the plasmids were verified by sequencing.

The haploid G strain (Mata, his3D, leu2∆0, ura3-, trp1∆63, YJL167W::kanMX4 [pFL44erg20K197G]) [13] was transformed with a pSM5 plasmid series expressing wild type and mutated GES. A 1.5 mL aliquot of G cells, grown to the stationary phase YNB medium supplemented with the appropriate auxotrophic amino acids, was centrifuged (1 min, 10,000 g) and 10 mL of mutated plasmid pSM5 (125 ng) was added to the yeast pellet. Transformed cells were selected for uracil prototrophy and G418 resistance on a minimal medium.

The cells from a stationary phase culture were harvested by centrifugation (5000 g for 5 min). Octanol (4 mg) and Ethylheptanoate (4 mg) were added as an internal standard to 20 mL culture medium supernatant. Monoterpenoids were extracted using a stir bar (Twister; Gerstel, Mülheim a/d Ruhr; Germany) sorptive extraction liquid desorption gas chromatograhy mass spectrometry (SBSELD-GC-MS) [15] with the following modifications: acetonitrile as solvent and a 1 µL injection volume.

Monoterpenol extracts were analyzed by GC-MS using an Agilent 6890N gas chromatograph equipped with a Gerstel MPS2 sampler, coupled to an Agilent 5975B inert MSD (Agilent Technologies). The gas chromatograph was fitted with a DB-Wax capillary column (60 m × 0.32 mm i.d. × 0.50 µm film thickness, J&W Scientific) and helium was used as the carrier gas (1 mL×min⁻¹ constant flow). The GC oven temperature was programmed to increase from 45˚C to 82˚C at a rate of 20˚C×min⁻¹, and then to increase to 235˚C at a rate of 2.7˚C×min⁻¹ (hold for 15 min). The injector was set to 250˚C and used in pulsed splitless mode (25 psi for 0.50 min). The temperatures of the interface, MS ion source and quadrupole were 270˚C, 230˚C and 150˚C, respectively. The mass spectrometer was operated in the electron impact ionization mode (EI, 70 eV), and the masses were scanned over a m/z range of 29 - 300 amu. Agilent MSD ChemStation software (G1701DA, Rev D.03.00) was used for instrument control and data processing. The mass spectra were compared with the Wiley’s library reference spectral bank. Total amounts of geraniol, linalool, citronellol, nerol and a-terpineol were determined using linear calibration curves, leading to a value of 0.98 for R², over the range of concentrations from 0 to 200 mg/mL.

Homology modeling was implemented using Modeller [16] with Mentha spicata (4S)-limonene synthase as template [17]. The O. basilicum GES sequence was aligned with Mentha spicata (4S)-limonene using ClustalX

Table 1. Specificities of forward oligonucleotides used to modify the O. Basilicum GES. Mutated codons are underlined.

[18]. Visual analysis of the model and the illustrations were made using PyMOL [19].

Although the product specificities of monoterpenol synthases have been extensively studied using heterologous expression, the manner in which their amino acidic sequence relates to the specific enzymatic function still remains largely unknown, with the notable exception of the sabinene and 1,8 cineole synthases from S. fruticosa [11]. Although classical sequence comparisons led to the identification of seven subfamilies [20], further analysis of their enzymatic activity was not possible, due to the history of this protein’s family. The original plant terpenoid synthase was a di-terpene synthase, and it is thought that monoterpenoid synthases originated via the loss of the domain of approximately 200 amino-acids [21]. This loss did not take place in a single ancestral event, but occurred several times during plant evolution, leading to an ongoing process of mono and sesqui-TPS generation [22]. In the present study, to enable catalytic site analysis, we positioned each residue differing from O. basilicum and P. frutescens GES and LIS within the predicted catalytic site, and used an in vivo characterization approach to investigate the functional consequences of amino-acidic substitutions at the putative catalytic site of O. basilicum GES. We discovered that 3 mutations resulted in an abolished function of GES and one mutation resulted in a partial loss of function, whereas 5 mutations did not affect the original function.  

Expression of GES in the erg20K197G yeast strain resulted in the biosynthesis of a number of monoterpenols (geraniol, linalool, citronellol, nerol) in an YNB medium (Table 2), whereas almost no production of a-terpineol was detected. Untransformed control erg20K197G cells exhibited a much lower production of monoterpenols. The production level of geraniol was 16 times higher arising from expression of GES. An increase in linalool, citronellol and nerol was also detected, but their overall amounts represented only 4% - 12% of the geraniol detected. Expression of 4 mutated GES K197G DM1, K197G DM2, K197G DM5 and K197G DM7 resulted in a monoterpenol profile very similar to that observed with the untransformed control. The expression of 5 other GES mutants (K197G DM3 – K197G DM4 – K197G DM6 – K197G DM8 – K197G DM9) resulted in a monoterpenol profile, very similar to that observed with erg20K197G yeast cells transformed with wild type GES. Only one strain (K197G DM10) exhibited an intermediate monoterpenol profile, between that of erg20K197G transformed with wild type GES and that of the untransformed control.

It is of interest to compare our results with previous structure function studies of O. basilicum GES. Using a GES-(R)-LIS protein chimeric approach, the function was evaluated by Iijima et al. [10]. Replacement of the first 360 aa on GES by their (R)-LIS homologues resulted in a loss of function. Our catalytic site modeling indicates that only one area in the first 360 aa on GES could potentially be involved in the catalytic site, suggesting a significant role for this region in the proposed GES function (Figures 2(A) and (B)). The site-directed modification of this area corresponds to DM1, and the resulting K197G DM1 strain indeed exhibits a dramatic loss of GES activity (Table 2). This result prompted us to test another mutation, DM2, replacing the 2 amino-acids

Table 2. Monoterpenol content (mg×L⁻¹) of FPPS mutated yeast strains, untransformed or transformed by control or mutated GES plasmid. Terpenoids were extracted from a minimal medium at their stationary growth phase. The results are the mean of 3 experiments ± standard deviation.

ND: Not Detected.

corresponding to the difference between P. frutescens GES and (R)-LIS in O. basilicum GES. GES activity in the K197G DM2 strain is also suppressed. It is also noteworthy that P. frutescens belongs to the same family (Lamiaceae) as sweet basil. From this comparison it appears that there is a good correlation between DM1, DM2 and the GES-(R)-LIS inactive chimeric construct. One can infer that the loss of GES function obtained by Iijima et al. [10] was due to a modification of this area, defined by DM1 and DM2. The same area also appeared to be of interest in the sabinene and 1,8 cineole synthases analysis of S. fruticosa made by Kampranis et al. [11], since it corresponds to their Region 1. Kampranis et al. [11] suggested that these residues, located on helix a 14 and aligned along the pore of the catalytic centre, could be involved in the deprotonation of the water molecule, facilitating the attack on the intermediate a-terpinyl carbocation. The multiple changes they introduced in Region 1 resulted in a progressive shift from a sabinene or 1,8 cineole synthase activity to a wider range of detected products. On the basis of our results and those reported by Iijima et al. [10], we infer that any modification of size (Y) or charge (T) of the amino-acids in this region is likely to substantially alter the GES enzymatic function on the intermediate geranyl carbocation, whereas a simple change from a GES→LIS signature in this area may not be sufficient to affect the original GES enzymatic specificity.

Following replacement of the aa after position 360 on the GES, by their (R)-LIS homologues, Iijima et al. [10] found a dual geraniol/linalool synthase function. As monoterpenol biosynthesis proceeds via an intermediate carbocation [9], one could therefore infer that they affected the intermediate geranyl carbocation, thereby shifting the charges towards an intermediate potential geranyl or linalyl carbocation. One could therefore consider that mutations in the second half of the GES protein may either inactivate the enzyme or shift the specificity of GES towards mixed geraniol and linalool production. A comparison of O. basilicum GES, (R)-LIS, P. frutescens GES and (R)-LIS (Figure 1), combined with O. basilicum GES modeling (Figure 2), suggested 6 O. basilicum GES modeling (Figure 2), suggested 6 areas of potential interest.

DM3 and DM4 are in the middle of the catalytic site, and in the area where the 3 Mg²⁺ are thought to be located [17]. DM3 was intended to modify this area, making it more similar to O. basilicum (R)-LIS, and DM4 was used to induce a F→Y change since both O. basilicum and P. frutescens GES have an F at this position, whereas their (R)-LIS have a Y. The DM3 and DM4 modifications did not affect GES activity or specificity, suggesting that this area has only a minor in vivo role in directing the enzymatic function.

DM5 and DM7 are located in the region, which is predicted to interact with the first and middle parts of GPP, respectively [17]. Although both the V and F residues, introduced respectively into DM5 and DM7, are larger than the original A and L amino-acids, they remain hydrophobic. DM7 is targeted to the motif in order to achieve metal dependent ionization of the prenyl diphosphate substrates (N,D)DXX(S,T)XXXE. The modification of these two GPP-interacting residues tested in our experiments gave similar results, leading to a loss of GES function. These results indicate that conformational changes in the processing of the GPP substrate are needed for GES function. It is likely that any mutation of these residues affects the conformation, and either induces the release of GPP out of the catalytic region before it is transformed into a monoterpenol, or even prevents its access to the catalytic region. These observations are corroborated by the fact that any disturbance of the substrate stability in the catalytic pocket (i.e. GPP from yeast FPPS) [13,23] results in a partial or total loss of enzymatic function. One could thus infer that the loss of GES specificity by GPP destabilization would not result in a shift towards a geraniol/linalool monoterpenol synthase function, but rather in a loss of activity.

DM6 is located at the very bottom of the pocket, and affects an amino-acid pointing to a region complementtary to the area tested in DM1 and DM2. This drastic M→P change observed in P. frutescens (R)-LIS, and recreated in DM6, had the potential to affect the nearby helix and thus to produce constraints on the pocket. However, it resulted in a GES activity very similar to that observed with the wild type GES protein (Table 2). This area is therefore likely to play no, or only a minor, role in the GES activity and specificity.

DM8 is on a loop at the very bottom of the pocket site, and is therefore located outside the helix parts of the catalytic site. It is likely that this residue forms part of the entrance of the GES pore. Amino acids located on a loop are not considered to be critical in monoterpene synthase activity, but they have been shown to be important in diterpene synthases [24]. DM8 was tested because the L→M modification could destabilize the neighboring metallic cations from the catalytic pocket. This might affect the enzymatic reaction via substrate wobbling at the main catalytic site, and release different reaction products. However, quite expectedly, this minor modification did not affect GES efficiency or specificity, indicating that its presence in O. basilicum (R)-LIS may be fortuitous.

DM9 corresponds to a single amino-acidic difference observed between O. basilicum GES and LIS, at the end of the protein sequence, but within the catalytic site. DM9 had no effect on GES protein activity. This was very surprising, since changing the negatively charged D into a positively charged H at this position, facing SARM and in contact with Mg²⁺, was expected to substantially affect the enzyme activity. This outcome, together with the results observed with DM3, DM4 and DM8, suggests that these residues, which are involved in the coordination of the divalent cations, act strongly and that the link is very difficult to disturb with a single directed mutagenesis.

DM10 is also at the end of the protein sequence and corresponds to a single amino-acid difference, observed between P. frutescens GES and LIS. Albeit being next to the amino-acidic position described in DM9, in P. frutescens this H→N mutation acts on the carbonate part of the substrate. In DM10, this area was modified to make this amino acid and the previous one look similar to the amino acid observed in P. frutescens. Strain K197G DM10 exhibited a monoterpenol profile, intermediate between the profile observed with the untransformed erg20K197G yeast strain, and that corresponding to the same strain expressing the wild-type GES (Table 2). This area therefore participates in the formation of the catalytic site. More interestingly, the enzyme with DM10 was altered in its efficiency in behaving as GES, but maintained its entire specificity, with no shift of production towards other terpenes. This type of behavior is clearly different to that observed by Kampranis et al. [11] for S. fruticosa sabinene and 1,8 cineole synthases.