Integrated platform for structural and functional analysis of terpene synthases of Cannabis sativa

Cloning of CsTPS

Expression constructs encoding three full-length sesqui-TPS (CsTPS9FN, CsTPS16CC and CsTPS20CT), two mono/sesqui-TPS (CsTPS5FN and CsTPS19BL), and five mono-TPS sequences (CsTPS12PK, CsTPS13PK, CsTPS3FN, CsTPS1SK and CsTPS37FN), which had their plastidial-targeting sequence motif truncated (Table 1), were synthesised (Twist Bioscience) as codon-harmonised (Codon Wizard) and codon-optimised for Escherichia coli. The genes were cloned into a pET28a⁺ expression vector (Luna-Vargas et al., 2011), using NdeI and XhoI restriction sites.

Protein expression and purification

Recombinant TPS enzymes were expressed in E. coli BL21(DE3) T7 Express lysY/Iq competent cells (New England Biolabs, Ipswich, MA, USA) using the NEB high-efficiency transformation protocol. Briefly, 25 µl aliquots of competent cells were mixed with 2 µl of plasmid DNA by heat shock and incubated in Luria-Bertani (LB) medium (1.0% (w/v) tryptone, 0.5% (w/v) yeast extract, 1.0% (w/v) NaCl) at 37 °C for 1 h with shaking at 200 rpm. Transformed cells were plated on LB-agar containing 50 µg ml⁻¹ kanamycin and incubated overnight at 37 °C.

A single colony was used to inoculate a 10 mL starter culture in Terrific Broth (TB; 1.2% (w/v) tryptone, 2.4% (w/v) yeast extract, 0.4% (v/v) glycerol, 17 mM KH₂PO₄, 72 mM K₂HPO₄) with 50 µg ml⁻¹ kanamycin and grown overnight at 37 °C with shaking at 200 rpm. The starter culture (1:50 dilution) was used to inoculate 400 mL TB medium supplemented with kanamycin (50 µg ml⁻¹). Cultures were grown at 37 °C for 16 h, induced with 1 mM isopropyl β-D-1-thiogalactopyranoside (IPTG), and incubated at 16 °C for an additional 16 h. Cells were harvested by centrifugation at 10,330 × g for 30 min at 4 °C.

Cell pellets were resuspended in 30 mL lysis buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10 mM imidazole) and lysed by sonication (Ultrasonics, Brookfield, CT, USA) at 40% amplitude for 30-s bursts with 30-s rest intervals repeated three times, on ice. Lysates were clarified by centrifugation at 10,330 x g at 4 °C for 20 min. The process was repeated to ensure maximal removal of cell debris. The clarified lysate was syringe-filtered (0.22 µm), prior to purification.

Cleared lysates were loaded onto a gravity column containing 2 mL of 50% (v/v) of Ni-NTA resin slurry (Thermo Fisher Scientific, Waltham, MA, USA), pre-equilibrated with wash buffer (20 mM Tris-HCl (pH 7.5), 500 mM NaCl, 10 mM imidazole). The column was washed twice with 30 mL wash buffer, and TPS proteins were eluted with 5 mL elution buffer (50 mM Tris-HCl (pH 7.5), 500 mM NaCl and 500 mM imidazole) in 1 mL fractions. Each step of the purification process was analysed by sodium dodecyl sulphate−polyacrylamide gel electrophoresis (SDS−PAGE) and Coomassie blue staining.

Ni-affinity eluted fractions containing the proteins of interest were pooled and further purified by size-exclusion chromatography (SEC) using a HiPrep 16/60 Superdex 200 column (GE Healthcare Life Sciences, Marlborough, MA, USA) on a ÄKTA Basic Fast Protein Liquid Chromatography (FPLC) system. The gel filtration buffer comprised 25 mM 4-(2-Hydroxyethyl) piperazine-1-ethane-sulfonic acid (HEPES; pH 7.0), 10 mM MgCl₂, 100 mM KCl and 1 mM Dithiothreitol (DTT) and all steps were performed at a flow rate of 1 mL min⁻¹. Peak fractions containing the CsTPS enzymes (>95% pure as judged by SDS−PAGE) were combined and concentrated to 10 mg mL⁻¹ using Amicon centrifugal filters (10 kDa molecular weight cut off, Millipore). Protein concentrations were measured using Bradford’s method (Bradford, 1976) and stored at 4 °C.

Enzyme activity assay

Enzymatic activity assays were performed as described by Allen et al. (2019) with minor modifications. TPS activities were assayed in triplicate in a final volume of 500 µL assay buffer (20 mM HEPES (pH 7.5), 100 mM KCl, 5 mM MgCl₂, 1 mM dithiothreitol (DTT), 10% (v/v) glycerol), 100 µM geranyl diphosphate (GPP), geranyl-geranyl diphosphate (GGPP) or farnesyl diphosphate (FPP) (Merck, Sigma Inc., St. Louis, MO, USA) and purified protein. 500 µL of hexane (Sigma-Aldrich) containing 2.5 µM isobutylbenzene as an internal standard was overlaid to trap the volatile products. Reactions were incubated at 30 °C for 16 h and vortexed for 30 s. Volatile products were extracted by centrifugation at 1,000 x g for 30 min at 4 °C. Boiled enzyme controls were included to determine the background noise of the assay.

Product identification by gas-chromatography mass spectrometry

Gas-chromatography mass spectrometry (GC-MS) analysis was conducted using a Thermo Scientific system equipped with a Trace 1310 GC interfaced with a triple quadrupole MS TSQ 8000 Evo and a TriPlus Robotic Sample Handling autosampler (Thermo Fisher Scientific, Waltham, MA, USA). The column was a Thermo Scientific TG-5SILMS capillary column (60 m × 0.25 mm ID, 1.0 µm film thickness), and carrier gas was He at a constant flow rate of 1.5 mL min⁻¹. The inlet temperature was 280 °C with a split ratio of 5:1, and the injection volume was 1 μL. The initial oven temperature was set at 50 °C with 5 min hold time, then increased to 300 °C at a rate of 5 °C min⁻¹, and held at 300 °C for 5 min. The MS was set in full scan mode with a mass range of 35–400 amu, delay time of 10.5 min, and ionisation by electron impact with ionisation energy of 70 eV. The ion source temperature was 230 °C, and the MS transfer line temperature was 280 °C. Gerstel Maestro software v1.5 controlled the autosampler, and data were acquired using Thermo Scientific XCalibur Software v4.7. Data was processed using Thermo Scientific XCalibur Qual Browser v4.7 or FreeStyle v1.8 (Thermo Fisher Scientific, Waltham, MA, USA).

To confirm the presence of most terpenes, authentic standards (Sigma, St. Louis, MO, USA) were used during the analysis. However, the sesquiterpenes δ-elemene, β-elemene, γ-elemene, epi-β-caryophyllene, alloaromadendrene, elemol, germacrene-D, guaiol, globulol, γ-eudesmol, and α-eudesmol were identified tentatively through comparisons with the NIST library due to the lack of available internal standards (Fig. S4). Similarly, several monoterpenes, including β-phellandrene, allo-ocimene, fenchol, β-terpineol, pinene hydrate, and geranyl methyl ether, were also putatively identified via NIST library comparisons. While geranyl methyl ether and pinene hydrate were detected as chromatographic peaks and labelled accordingly, these compounds are neither monoterpenes nor sesquiterpenes—the primary focus of this study—and were therefore excluded from the final quantification percentages.

Malachite green assay for kinetic measurements

Kinetic parameters were determined using the malachite green assay (Vardakou et al., 2014). Reactions were performed in triplicate in 96-well flat-bottomed plates (Greiner Bio-One, Kremsmünster, Austria). Standard curves were established for both monophosphate (Pi) and pyrophosphate (PPi) using serial 2-fold dilutions ranging from 0.01 to 50 µM. For kcat determination, reactions (50 µL) contained malachite green assay buffer (25 mM 2-(N-morpholino) ethanesulfonic acid (MES), 25 mM 3-(Cyclohexylamino)-1-propanesulfonic acid (CAPS), 50 mM Tris, 5 mM MgCl2, pH 7.5), 25 mU of inorganic pyrop/hosphatase from Saccharomyces cerevisiae (Sigma, St. Louis, MO, USA), 100 µM substrate (FPP or GPP), and serial 2-fold dilutions of protein (0.003–0.2 µM). Reactions were incubated at 37 °C for 30 min prior to being terminated by the addition of the malachite green development solution (prepared according to Pegan et al. (2009)) and further incubated for 15 min before measuring absorbance at 623 nm using a plate reader (Bio-Rad, Hercules, CA, USA). Steady-state kinetic measurements were also conducted in triplicate in a 50 µL reaction volume containing malachite green assay buffer, a fixed concentration of 0.014 µM of the protein of interest, and serial 2-fold dilutions of the pyrophosphate substrate (FPP or GPP) ranging from 0.02–100 µM. Reactions were also incubated at 37 °C for 30 min before termination and absorbance measurement at 623 nm, following the same procedure described above The kinetic parameters (V_max, K_M and k_cat) were obtained from non-linear regression analysis of the data using the Michaelis–Menten model in GraphPad Prism.

Thermofluor protein stability assay

For downstream applications, purified CsTPS enzymes were concentrated to 10 mg ml⁻¹. To optimise storage conditions an 80-condition buffer screen (Table S1) was developed, based on commonly used buffers for TPS enzymes. The temperature at which CsTPS unfolding occurred for each of the conditions (T_m values) was compared with the original storage buffer, and changes in unfolding temperature (∆T_m) were calculated. Thermal denaturation was monitored using SYPRO Orange dye (Sigma) detected on a real-time PCR machine. Melting temperatures (T_m) were calculated as described previously (Ericsson et al., 2006). Briefly, solutions of 7.5 µL of 300 x SYPRO Orange (Merck Sigma, St. Louis, MO, USA), 12.5 µL of test buffer conditions (Table S1), and 5 µL of 2.5 mg mL⁻¹ CsTPS proteins were added to the wells of a 384-well thin wall PCR plate (Bio-Rad, Hercules, CA, USA). Water was added instead of a buffer in the control samples. The plates were sealed with Optical-Quality Sealing Tape (Bio-Rad, Hercules, CA, USA) and heated in an ABI QuantStudio 5Dx Real-Time qPCR System from 20 to 95°C in increments of 1 °C. Fluorescence changes in the wells of the late were monitored simultaneously with a charge-coupled device (CCD) camera. The wavelengths for excitation and emission were 490 and 575 nm, respectively.

Creation of the TPS-crystallisation screen

A 48-condition screen specifically directed towards the crystallisation of TPSs was developed. To create this screen, the Protein Data Bank (PDB; Berman et al., 2000), was searched to identify the crystallisation conditions of TPSs and related proteins. Crystallisation conditions for 120 deposited structures of proteins were returned. Individual searching of each entry led to the removal of 16 entries that had not published crystallisation conditions, leaving the final count of crystallisation conditions at 104. Various conditions from these entries were analysed, such as pH, buffer, precipitant, and salt to develop a TPS-specific sparse matrix screen containing 48 different conditions (Table S2).

Crystallisation and optimisation of TPS crystals

After creating the TPS-crystallisation screen, crystallisation experiments were performed. Each of the 10 CsTPS proteins were screened through the 48 conditions of the newly created TPS-crystallisation screen. Crystallisation was performed using the hanging-drop vapour diffusion method at 293 K. Briefly, a 1 μL drop of protein solution (10 mg mL⁻¹ CsTPS) was mixed with a 1 μL drop of precipitant solution (Table S2) and equilibrated against a 500 μL reservoir of the precipitant solution.

Development of a pipeline for the characterisation of CsTPS enzymes

Existing protocols for TPSs overexpression in bacterial systems often suffer from challenges such as protein misfolding, formation of inclusion bodies, low yield and stability issues (Raman et al., 2014; Wiles et al., 2022). To overcome these limitations, we established a robust pipeline for the overexpression, functional and structural characterisation of TPS’s (Fig. 1). Currently, 55 different TPS have been identified in the Cannabis genome (Allen et al., 2019). We selected 10 CsTPS to act as candidates in our structural and functional pipeline (Table 1). The selection criteria included representing different TPS types (mono-TPS and sesqui-TPS), including TPSs that can utilise multiple substrates or produce multiple products, and encompassing enzymes from both drug and fibre-type cannabis from a broad selection of different cultivars.

Recombinant expression and purification of CsTPS enzymes

The ten candidate CsTPS enzymes were successfully expressed recombinantly in E. coli and purified using immobilised metal-ion affinity chromatography (IMAC). SDS-PAGE analysis confirmed the presence of a single protein band with the expected molecular mass of 66.32–73.75 kDa for each enzyme, indicating that all ten CsTPS proteins were expressed and purified in their soluble forms (Fig. S1). After initial ion-affinity purification, yields of each CsTPS ranged from 2 to 25 mg L⁻¹. To further purify and characterise all CsTPS were subjected to size-exclusion chromatography (SEC). A typical trace chromatogram for each of the CsTPS (Fig. S2) revealed a single peak corresponding to a molecular weight of approximately 66.32–73.75 kDa, suggesting that all CsTPS are monomers in solution.

Impact of optimal buffer conditions for CsTPS stability

The purified CsTPS enzymes were concentrated for structural studies. However, different levels of protein precipitation occurred after overnight storage. To overcome this problem, a thermofluor assay was used to determine the optimal storage buffer. Relatively large variations in stability could be observed when varying the buffers (Fig. 2), and a few buffers appear to be generally more favourable for protein stabilisation (Fig. 2B; Table 2). Tris pH 8.0 and 8.5, and HEPES pH 7.5 were the most stabilising buffers, whereas MES pH 5.5, Bis-Tris pH 6.0 and CAPS pH 9.0 significantly destabilised several of the CsTPS proteins (Table 2, Fig. 2B). Several of the 56 buffer conditions investigated in the buffer screen gave no measurable transitions in combination with one or a few of the proteins, possibly caused by destabilisation or partial unfolding and potential aggregation of the proteins. For example, a clear thermal transition could be detected with only 6 of the 10 proteins in combination with CAPS pH 9.0. However, for some of the buffers, among them the overall most stabilising buffers, a measurable transition could be recorded together with all the proteins. The three most stabilising buffers were determined for each CsTPS protein, and Tris pH 8.0 was consistently found to improve the stability of the CsTPS enzymes. This contrasts with both the literature, where buffers with a pH of 7–7.5 are generally used for TPS proteins. While a pH of 8.0 is above the theoretical isoelectric point (PI), of approximately 5–6, which generally helps maintain solubility by increasing the net negative charge on the protein, it is notable that a slightly more alkaline conditions provided greater stability than the commonly used neutral to mildly acidic buffers. Given that the median ∆Tm for Tris pH 8.0 with the addition of the salt additives 100 and 200 mM NaCl were found to be more than 4 °C, these buffer conditions were considered to improve the stability of the CsTPS proteins significantly and were deemed suitable buffers for continuing functional characterisation of the enzymes.

Product profile analysis of CsTPS

To determine if the recombinant CsTPS were enzymatically active, all recombinant CsTPS were assayed as purified proteins with GGPP, GPP and FPP, and the reaction products were analysed by GC-MS. No products were seen for any CsTPS when assayed with GGPP, indicating that none of the CsTPS enzymes analysed form diterpenes.

Monoterpene synthases (GPP assay)

Five of the ten chosen CsTPS had previously been identified as proposed monoTPSs, while two were suggested to have dual activity with both GPP and FPP as substrates (Table 1). The five monoTPSs (CsTPS1SK, CsTPS3FN, CsTPS12PK, CsTPS13PK, and CsTPS37FN) exhibited enzymatic activity, producing a diverse array of monoterpenes (Fig. 3, Table 3). CsTPS1SK predominantly produced limonene (74.72%), in line with previous studies (Günnewich et al., 2007), but we also detected the minor products β-pinene (5.18%), α-terpineol (4.83%), and β-terpineol (2.30%) (Fig. 3; Table 3). Interestingly, we also identified camphene (0.98%) and fenchol (3.55%), which were not previously reported for this enzyme (Fig. 3, Table 3). CsTPS3FN exhibited strict specificity for β-myrcene, producing this monoterpene at 100% when incubated with GPP, consistent with previous reports (Fig. 3, Table 3; Booth, Page & Bohlmann, 2017). CsTPS5FN also produced myrcene as its most abundant monoterpene product (37.09%) (Fig. 3, Table 3). In addition to β-myrcene, several other monoterpenes were produced, including (-)-α-pinene (23.45%), (-)-limonene (17.33%), sabinene (15.26%), and (-)-β-pinene (8.87%), consistent with previous reports (Fig. 3, Table 3; Booth, Page & Bohlmann, 2017). CsTPS12PK predominantly produced α-terpinene (68.42%), a result consistent with previous studies (Booth, Page & Bohlmann, 2017), while also generating minor amounts of γ-terpinene (15.29%) and α-phellandrene (4.21%) (Fig. 3, Table 3). Interestingly, our study also identified trace amounts of sabinene (3.87%), which had not been previously reported for this enzyme (Fig. 3, Table 3). CsTPS13PK was highly specific for (Z)-β-ocimene, producing 94.11% of this product, which aligns with prior findings for this enzyme (Fig. 3, Table 3; (Booth, Page & Bohlmann, 2017)). Small amounts of α-ocimene (3.49%) were also detected, though this was not previously noted (Fig. 3, Table 3). For CsTPS37, the dominant product was terpinolene (81.62%), in agreement with past studies (Livingston et al., 2020). Additionally, minor amounts of limonene (6.38%) and β-myrcene (3.89%) were observed, adding new insights to the product profile of this enzyme (Fig. 3, Table 3). The TPS CsTPS19BL, when incubated with GPP, produced a mixture of monoterpenes, with the major products being (+)-linalool (54.12%) and (-)-linalool (40.87%) (Fig. 3, Table 3). These results align with the previous studies that have suggested this enzyme’s strong preference for linalool production when GPP is present in the reaction (Zager et al., 2019). The TPS CsTPS20CT was previously characterised as a sesquiterpene synthase, with its primary product being hedycaryol when incubated with FPP (Zager et al., 2019). However, when incubated with GPP in this study, CsTPS20CT displayed an unexpected monoterpene production profile, forming geraniol as the dominant product (87.64%) (Fig. 3, Table 3).

Table 3:

Terpene products formed by recombinant Cannabis sativa terpene synthase (CsTPS) enzymes.

TPS enzyme	Substrate	Terpenes produced	Percent total (%)
Monoterpene synthases
CsTPS3FN	GPP	β-myrcene	100.00
CsTPS1SK	GPP	α-pinene	2.98
		camphene	0.98
		β-myrcene	2.25
		β-pinene	5.18
		limonene	74.72
		terpinolene	1.53
		fenchol*	3.55
		β-terpineol*	2.30
		α-terpineol	4.83
		geraniol	1.69
CsTPS12PK	GPP	α-terpinene	29.50
		limonene	33.36
		γ-terpinene	24.89
		β-myrcene	12.25
CsTPS13PK	GPP	(E)-β-ocimene	79.52
		allo-ocimene	1.80
		(Z)-β-ocimene	18.68
CsTPS37FN	GPP	α-pinene	3.03
		β-phellandrene*	2.17
		β-myrcene	2.16
		β-pinene	4.77
		delta-3-Carene	3.67
		α-terpinene	2.86
		limonene	1.96
		y-terpinene	1.13
		terpinolene	70.69
		linalool	2.95
		geraniol	4.61
Sesquiterpene synthases
CsTP9FN	FPP	β-caryophyllene	2.76
		humulene	4.49
		epi-β-caryophyllene*	67.30
		germacrene D*	15.26
		globulol*	10.19
CsTPS16CC	FPP	β-elemene*	1.05
		γ-elemene*	3.82
		germacrene B*	92.73
		alloaromadendrene*	1.06
Mono/Sesquiterpene synthases
CsTPS19BL	GPP	linalool	100.00
	FPP	nerolidol	100.00
CsTPS5FN	GPP	α-pinene	23.00
		β-myrcene	37.00
		β-pinene	8.00
		limonene	17.00
		sabinene*	15.00
	FPP	farnesol*	100.00
CsTPS20CT	GPP	β-myrcene	7.89
		limonene	12.72
		(Z)-β-ocimene	2.64
		terpinolene	7.74
		α-terpineol	26.06
		geraniol	42.95
	FPP	hedycaryol (elemol*)	31.42
		guaiol	19.96
		γ-eudesmol*	20.51
		α-eudesmol*	28.11

DOI: 10.7717/peerj.19723/table-3

Note:

* No reference standard available, putative identification of compound using National Institute of Standards and Technology (NIST) library.

Sesquiterpene synthases (FPP assay)

All previously proposed sesquiTPSs analysed in this study were found to be active with FPP. CsTPS9FN produced a diverse range of sesquiterpenes, with epi-β-caryophyllene being the predominant product (67.30%) with β-caryophyllene (2.76%), humulene (4.49%) found to be present in low abundance (Fig. 4, Table 3). Additionally, germacrene D (15.26%) and globulol (10.19%) were also detected, both of which were not previously reported for this enzyme (Fig. 4, Table 3; Booth, Page & Bohlmann, 2017). This contrasts previous studies where β-caryophyllene and α-humulene were the only products reported. Consistent with previous studies, when incubated with FPP, CsTPS20CT primarily produced hedycaryol (which was detected as elemol due to GC-MS thermal degradation) (31.42%) (Fig. 4, Table 3; (Zager et al., 2019)). Other significant sesquiterpene products included γ-eudesmol (20.51%), α-eudesmol (28.11%), and guaiol (19.96%). This broad range of products indicates that CsTPS20CT is a multi-product sesquiterpene synthase, capable of producing a diverse array of eudesmol-type sesquiterpenes. The TPS CsTPS16CC produced γ-elemene (92.73%) as the dominant sesquiterpene when incubated with FPP, consistent with the enzyme’s sesquiterpene synthase activity (Fig. 3, Table 3). Other minor products included β-elemene (3.82%) and germacrene B (1.05%), further supporting its activity toward forming elemene-type sesquiterpenes. Interestingly, alloaromadendrene (1.06%) was also detected, which had not been previously associated with this enzyme. CsTPS5FN, when incubated with FPP, resulted in the exclusive production of farnesol (100%), a linear sesquiterpene alcohol (Fig. 4, Table 3). Farnesol had previously been reported as the single sesquiterpene product of this enzyme, highlighting the dual activity of CsTPS5FN with GPP and FPP as substrates (Booth, Page & Bohlmann, 2017). This enzyme’s ability to produce both monoterpenes and sesquiterpenes makes it a versatile target for biotechnological applications. Finally, the TPS CsTPS19BL produced 100% nerolidol when incubated with FPP, consistent with prior studies (Fig. 4, Table 3; (Zager et al., 2019)). These findings demonstrate that each TPS exhibits a distinct product profile, with some producing a diverse array of sesquiterpenes and others generating a single, consistent product, highlighting the enzyme-specific nature of terpene biosynthesis.

Kinetic properties of CsTPS enzymes

To better inform protein engineering strategies for TPSs, there is a need to understand their catalytic activity. We chose a high-throughput enzyme assay that can be performed without specialised equipment which is based on phosphate being produced. The kinetic profiles of the CsTPS are shown in Fig. 5. Substrate concentrations (FPP, GPP) ranged from 0 to 100 µM. The calculated k_{cat ap} for each CsTPS was found to be between 0.0011 and 0.0204 s⁻¹ (Table 4).

The kinetic analysis revealed a broad range of affinities and turnover rates across the different CsTPS variants, reflecting their diverse roles in terpene biosynthesis. For instance, CsTPS1SK exhibited a relatively low K_m for GPP (7.809 ± 0.678 µM) paired with a high V_max (0.2038 ± 0.0053 µM⁻¹ s⁻¹) and k_cat (0.0204 s⁻¹), indicating a strong substrate affinity and rapid catalysis, which suggests its efficiency in monoterpene production. On the other hand, CsTPS13PK which also utilizes GPP, displayed a moderately higher K_m (12.96 ± 1.23 µM) with a lower V_max (0.0918 ± 0.0029 µM⁻¹ s⁻¹), signifying a slightly reduced affinity for GPP compared to CsTPS1SK but still an efficient monoterpene producer.

For enzymes using FPP as a substrate, CsTPS9FN exhibited the highest K_m (41.7 ± 3.73 µM), indicating a lower affinity for FPP compared to CsTPS20CT, which had a significantly lower K_m (16.86 ± 6.42 µM). However, CsTPS9FN also showed a much higher V_max (0.1127 ± 0.0047 µM⁻¹ s⁻¹), than CsTPS20CT (0.0144 ± 0.0022 µM⁻¹ s⁻¹), suggesting distinct catalytic efficiencies and roles in sesquiterpene biosynthesis. This variability highlights the challenge of predicting enzyme function based solely on sequence similarity and the need for functional and kinetic characterisation. By elucidating these kinetic properties, our study provides a foundation to understand which CsTPS enzymes efficiently produce specific terpenes. This can be used to inform engineering of this class of enzymes for targeted terpene production.

Development of a targeted TPS crystallisation screen

Currently, many macromolecular crystallisation screens are commercially available; however, relatively few are specifically designed for particular protein families. We created a 48-condition screen, called the TPS screen, with conditions specifically directed towards the crystallisation of the TPS protein family by analysing the crystallisation conditions for all TPS and TPS-related proteins identified in the PDB. Most TPS crystals were obtained between pH 6.0 and 6.9 (34.4%), with the next highest range from pH 7.0 to 7.9 (23.4%). The majority of TPS crystallised in Bis-Tris buffer (Figs. 6A, 6B), with 24.6% of conditions ranging widely in pH from 3.8 to 10 (Figs. 6A, 6B). Tris was the next most common buffer used to obtain TPS crystals (19.7%). Combining this data with the pH data resulted in the selection of six buffer/pH combinations for use in the TPS-directed screen. These buffers are Bis-Tris (pH 6.0), Bis-Tris (pH 6.5), Bis-Tris (pH 7.0), Tris (pH 7.5), Tris (pH 8.0) and Tris (pH 8.5). Of the 104 crystallisation conditions identified for TPS crystal production, 50 conditions reported the inclusion of salts to crystallise the molecule (Fig. 6C). Although 22 different salts were used in these crystallisation conditions, a large majority (26%) of these conditions contained MgCl₂. This is consistent with TPS functioning as metalloenzymes, where Mg²⁺ or Mn²⁺ plays a crucial role in stabilising the PP_i substrates leaving group and facilitating catalysis. NaCl was the next most common salt used and was seen in 12% of crystallisation conditions. This data led to the selection of both magnesium chloride and sodium chloride as counter ions in the TPS-directed screen.

The final components of the analysed crystallisation conditions were precipitants, which were provided for 62 of the 104 conditions. Of the 16 different precipitants used, various molecular weights of polyethylene glycol (PEG) comprised 83.9% of the conditions (Fig. 6D (Inset)). The next most used precipitant was ammonium sulphate, which was used in 8% of the conditions, indicating a strong preference for PEG in the crystallisation of TPS proteins. When examining the 52 conditions containing PEG in more depth (Fig. 6D), PEG-3350 and PEG-8000 were most often used (30.6% and 19.4% of conditions, respectively). As a result, PEG-3350 and PEG-8000 were chosen to be included in the TPS-directed screen.

To create the final TPS screen, four concentrations (5, 15, 25 and 35%) of each molecular weight PEG (3,350 and 8,000) were chosen, along with 100 mM of each of the buffers (Bis-Tris pH 6.0, Bis-Tris pH 6.5, Bis-Tris pH 7.0, Tris pH 7.5, Tris pH 8.0 and Tris pH 8.5). Each condition additionally contains 200 mM MgCl₂ and 200 mM NaCl (Table S2). This systematic approach to screen design provides a balanced matrix of variables aimed at identifying conditions for TPS crystal formation. By incorporating a diverse yet targeted range of parameters, this screen facilitates the formation of crystals, addressing the challenges of obtaining high-quality crystals essential for structural characterisation and subsequent protein engineering efforts.

Crystallisation and optimisation of CsTPS crystals

Each of the 10 CsTPS proteins were screened against the 48 conditions of the TPS-screen, as well as several other commercially available screens, all at room temperature. Initial crystal formation was observed as early as 24 h post-screen setup, in many conditions of the TPS-screen. All 10 proteins demonstrated varying degrees of crystallisation, with at least some form of precipitate forming in every case. Well-defined crystals were observed for CsTPS37FN, CsTPS1Sk, CsTPS3FN, CsTPS13PK, and CsTP12PK, while several of the other proteins produced spherical aggregates. Interestingly, with the exception of CsTPS1SK, all CsTPS formed crystals with either star or cubic morphologies. CsTPS1SK, on the other hand, consistently produced rod-shaped crystals (Fig. 7). The best crystals in terms of size and morphology were found in condition 16 of the TPS-screen (0.1 M Bis-Tris (pH 7.0), 25% PEG-3350, 0.2 M NaCl, 0.2 M MgCl₂), using a 2:1 ratio of CsTPS protein (10 mg ml⁻¹) to reservoir solution.