Cloning, functional characterization and evaluating potential in metabolic engineering for lavender ( +)-bornyl diphosphate synthase

We isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) from Lavandula x intermedia. The in planta functions of ( +)-LiBPPS were evaluated in sense and antisense transgenic plants. The monoterpene ( +)-borneol contributes scent and medicinal properties to some plants. It also is the immediate precursor to camphor, another important determinant of aroma and medicinal properties in many plants. ( +)-Borneol is generated through the dephosphorylation of bornyl diphosphate (BPP), which is itself derived from geranyl diphosphate (GPP) by the enzyme ( +)-bornyl diphosphate synthase (( +)-BPPS). In this study we isolated and functionally characterized a novel ( +)-BPPS cDNA from Lavandula x intermedia. The cDNA excluding its transit peptide was expressed in E. coli, and the corresponding recombinant protein was purified with Ni–NTA agarose affinity chromatography. The recombinant ( +)-LiBPPS catalyzed the conversion of GPP to BPP as a major product, and a few minor products. We also investigated the in planta role of ( +)-LiBPPS in terpenoid metabolism through its overexpression in sense and antisense orientations in stably transformed Lavandula latifolia plants. The overexpression of ( +)-LiBPPS in antisense resulted in reduced production of ( +)-borneol and camphor without compromising plant growth and development. As anticipated, the overexpression of the gene led to enhanced production of borneol and camphor, although growth and development were severely impaired in most transgenic lines strongly and ectopically expressing the ( +)-LiBPPS transgene in sense. Our results demonstrate that LiBPPS would be useful in studies aimed at the production of recombinant borneol and camphor in vitro, and in metabolic engineering efforts aimed at lowering borneol and camphor production in plants. However, overexpression in sense may require a targeted expression of the gene in glandular trichomes using a trichome-specific promoter.


Introduction
Several species of the genus Lavandula (Lamiaceae) are cultivated worldwide for the production of essential oils (EOs), cut flowers and other horticultural purposes. Of these, L. angustifolia, L. latifolia and their natural hybrid L. x intermedia are widely grown for EO production (Wells et al. 2018;Erland and Mahmoud 2015;Upson and Andrews 2004).
The quality of lavender EO is mainly determined by its monoterpene constituents, which mainly include linalool, linalyl acetate, ( +)-borneol, camphor and 1,8-cineole (Upson and Andrews 2004). Linalool and linalyl acetate contribute pleasant scents and are desirable in EOs used in perfumery and aromatherapy. Borneol and camphor are odorous, and are typically not found in EOs used in perfumery and aromatherapy; however, they both have medicinal properties and are abundant in oils obtained from L. latifolia plants. These oils have extensive applications in traditional and alternative medicinal preparations (Wells et al. 2018). The EO derived from L. x intermedia species has moderate levels of all monoterpenes found in both parents. However, L. x intermedia plants have a much higher oil yield (up to 10 X) than either parent. Thus, there is substantial interest in developing L. x intermeida plants that produce EOs comparable in composition to those found in L. angustifolia or L. latifolia species. These objectives may be achieved through plant biotechnology once all related genes are cloned, and necessary molecular tools are developed.
Most Lavandula monoterpenes are derived from the head-to-tail condensation of the universal isoprene units isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) generated through the MEP pathway in plastids. IPP and DMAPP are initially condensed to geranyl diphosphate (GPP) -the linear precursor to most monoterpenes -by geranyl diphosphate synthase. GPP is then converted to other prenyl diphosphate and monoterpenes by specific short-chain isoprenyl diphosphate synthase (IDS), and terpene synthase (TPS) enzymes, respectively (Fig. 1). Several Lavandula TPS and IDS genes have been identified and functionally characterized in vitro (Landmann et al. 2007;Woronuk et al. 2011;Demissie et al. 2011Demissie et al. , 2012Sarker et al. 2012;Benabdelkader et al. 2015;Adal et al. 2017Adal et al. , 2019Despinasse et al. 2017;Adal and Mahmoud 2020).  (BPP). Isopentenyl diphosphate (IPP) dimethylallyl diphosphate (DMAPP) are the substrates for the biosynthesis of geranyl diphosphate (GPP). Monoterpene synthases designated as bornyl diphosphate synthases (BPPS) catalyze GPP into BPP, a precursor for borneol, and multiple monoterpenes with different levels in Lami-aceae. These functionally studied monoterpene synthases were previously identified from S. officinalis (SoBPPS) (Wise et al. 1998) and L. angustifolia cv. Diva (LaBPPS) (Despinasse et al. 2017). Products derived by ( +)-LiBPPS shown in a broken circle are from this study. b The subsequent biosynthesis of borneol and camphor from BPP as a substrate. The broken arrow with "?" shows unknown catalyzing enzymes from plants 1 3 The biosynthesis of borneol and camphor in plants begins with the formation of bornyl diphosphate (BPP) from GPP through the reaction catalyzed by BPP synthase (BPPS) (Fig. 1). BPP is then used as a substrate for the synthesis of borneol, which is subsequently oxidized into camphor by borneol dehydrogenase ). To date, five genes encoding BPPS have been reported from Salvia officinalis (Wise et al. 1998), L. angustifolia (Despinasse et al. 2017), Lippia dulcis (Hurd et al. 2017), Cinnamomum burmanni (Ma et al. 2021) and Amomum villosum (Wang et al. 2018). From these, Wise et al (1998) and Ma et al. (2021) specifically identified the BPPSs as ( +)-BPPS type in S. officinalis and C. burmanni, respectively. Most reported plant BPPSs are multi-product enzymes catalyzing GPP to BPP (major) and multiple monoterpenes (minor) (Wise et al. 1998;Despinasse et al. 2017;Wang et al. 2018;Ma et al. 2021). For example, S. officinalis BPPS produces 25% monoterpenes and 75% BPP, while that of L. angustifolia generates 70% monoterpenes and 30% BPP. The only exception so far is the P. dulcis BPPS, which converts GPP to a single product BPP (Hurd et al. 2017). In this study, we identified a unique ( +)-BPPS gene in a previously reported Lavandula transcriptome database (Adal et al. 2019), cloned the gene from L. x intermedia, and functionally characterized the recombinant form of the encoded protein. We also evaluated the effects of overexpressing the gene in sense and antisense on borneol and camphor production in stably transformed L. latifolia plants.

Plant materials and reagents
Sample flower tissues were harvested from plants grown in an outdoor lavender experimental site at the University of British Columbia Okanagan (Kelowna, BC, Canada). L. latifolia seeds purchased from Seed Needs LLC (United States) were used to grow plants for transformation studies. Transformed and wild-type L. latifolia plants were maintained in a growth room at 25 °C, with a photoperiod of 16 h light and 8 h darkness. All sample tissues collected from both L. x intermedia and L. latifolia plants for RNA extraction were immediately flash-frozen in liquid nitrogen and stored at −80 °C until used.

Cloning of ( +)-LiBPPS and LaBPPS
We screened a previously reported L. x intermedia transcriptome database (Adal et al. 2019) for potential BPPS synthase, and identified a candidate with significant homology to previously reported plant BPPS genes. Using genespecific primers, the full-length LiBPPS cDNA (GenBank: MW846855) was cloned into pGEM-T (Promega) and, its sequence was confirmed by Sanger sequencing. The fulllength genomic DNA for ( +)-LiBPPS was amplified from L. x intermedia cv. Grosso, cloned into pGEM-T (Promega), and sequenced by Sanger sequencing.
For comparative studies, we also cloned the L. angustifolia BPPS (LaBPPS) cDNA using previously published cloning primers (Despinasse et al. 2017) from our L. angustifolia EST database (Lane et al. 2010). Briefly, the cDNA encoding LaBPPS was amplified by PCR and cloned into various vectors for confirmation of DNA sequence, and for heterologous expression in bacteria (see below). All primers used in this study are listed in Table S1.

Genome organization analysis
Genomic organization of ( +)-LiBPPS was predicted using the NCBI Spidey genomic DNA-mRNA aligner (Wheelan et al. 2001), targeting intron-exon number and size, as well as intron position in the sequences. The intron-exon structure of ( +)-LiBPPS was then compared with those previously reported TPSs from Lavandula.

Phylogenetic analysis and molecular modeling
Multiple sequence alignments of ( +)-LiBPPS along with previously reported BPPS genes from other plants were made using T-Coffee (http:// tcoff ee. crg. cat/ apps/ tcoff ee/ do: regul ar). The BOXSHADE 3.21 software (www. ch. embnet. org/ softw are/ BOX_ form. html) was used for shading amino acid sequence alignments. To infer the evolutionary history of ( +)-LiBPPS, the full-length amino acid sequences encoded functionally characterized BPPS and lavender TPS genes were aligned using ClustalW, and a maximum likelihood phylogenetic tree was constructed using MEGA7 (Kumar et al. 2016) with default settings. All TPSs clustered into previously defined distinct TPS subfamilies (Bohlmann et al. 1998;Chen et al. 2011).
A homology-based model of LiBPPS protein was produced using the SWISS-MODEL automated mode server (Bordoli et al. 2009;Guex et al. 2009). The crystal structure of S. officinalis bornyl diphosphate synthase (SoBPPS) (PDB code 1N1Z) containing the substrate analog diphosphate (POP) (Whittington et al. 2002) was detected as the top structural homologous template. A docking study with the model LiBPPS protein and GPP was conducted using autodock4 in a molecular docking server (Bikadi and Hazai 2009). The position of substrate GPP from SoBPPS PDB 1N20 was used as a docking positional template, and the result was visualized and analyzed using PyMOL v1.1 (The PyMOL Molecular Graphics System, Schrödinger, LLC). The active site cavities were detected from the model using Swiss-pdbviewer (Guex et al. 2009), in which the binding residues at the active site pockets were predicted.

Bacterial expression and recombinant protein purification
For heterologous expression of the gene in E. coli, the ORF excluding the transit peptide for ( +)-LiBPPS or LaBPPS was amplified by PCR and cloned into the NdeI/EcoRI sites of pET41b( +) by sticky-end cloning (Zeng 1998). The respective construct was then expressed in E. coli Rosetta™ (DE3)plysS cells (EMD Chemicals, Darmstadt, Germany). E. coli cells were grown in LB medium supplemented with 30 mg/l kanamycin and 34 mg/l chloramphenicol at 37 °C to an OD 600 of 0.5-0.6, followed by induction with 0.4 mM IPTG at 20 °C overnight. After harvesting, the pelleted cells were resuspended in lysis buffer (50 mM NaH 2 PO 4 , 300 mM NaCl and 10 mM imidazole, pH 8.0) and disrupted by sonication. The soluble ( +)-LiBPPS/LaBPPS protein was then purified using PerfectPro Ni-NTA Agarose chromatography (5Prime GmbH, Germany), according to the manufacturer's instructional manual. The purified recombinant proteins were then quantified through Bradford assays.

Enzymatic assays and product identification
The activity of the purified recombinant ( +)-LiBPPS (or LaBPPS) protein was assayed with each of GPP, FPP, DMAPP and a combination of IPP and DMAPP as substrates. The assay reactions were prepared in a final volume of 500 µl, containing an enzyme reaction buffer (2 mM dithiothreitol, 12.5% (v/v) glycerol, 1 mM MgCl 2 and 1 mM MnCl 2 ), MOPS (pH 6.5) buffer, purified enzyme and substrate(s), and were overlaid with 400 µl of pentane and incubated at 30 °C for 2 h. Calf intestinal alkaline phosphatase (CIP, 30 U) (NEB Biolabs) was added to the assay, and incubated at 30 °C overnight to hydrolyze the BPP product to borneol. The assays were frozen at -80 °C, and the pentane fraction was removed for product analysis according to previous reports Adal et al. 2017;Adal and Mahmoud 2020). As a control, heat-denatured ( +)-LiBPPS enzyme and purified proteins from E. coli lysate cells harboring the pET41b( +) lacking ( +)-LiBPPS were assayed with substrates under similar conditions. Assay products were identified using gas chromatography-mass spectrometry (GC-MS), following methods described in Adal et al. (2020). Further, borneol enantiomers (( +) and (−)) were analyzed by GC-flame ionization detector (GC-FID) using a 30 m × 0.25 mm fused silica capillary chiral column coated with Chirasil-DEX (cyclodextrin directly bonded to dimethylpolysiloxane) (Agilent Technologies Canada Inc, Mississauga, ON, CAN) according to Adal et al. (2019). Authentic standards of borneol isomers and thymol were also run on GC-FID under similar conditions to help identify the assay products. We used the monoterpene thymol (which is not present in lavender species examined) as an internal standard in all GC runs.

Generation of binary vectors and plant transformation
For expression in L. latifolia, the full-length ORF of ( +)-LiBPPS was cloned in the pCAMBIA1390 vector (which bears a hygromycin resistance gene) between the CaMV 35S promoter and the NOS transcriptional terminator in either sense or antisense orientation. The resulting constructs (35S::LiBPPS-sense and 35S::LiBPPS-antisense) were separately transformed into Agrobacterium tumefaciens strain GV3101. Young leaves (ca. 1 cm in diameter) from tissue culture grown plantlets generated from seeds were transformed by A. tumefaciens carrying 35S::LiBPPS-sense or 35S::LiBPPS-antisense, according to Nebauer et al. (2000), with the following modifications. Infected leaves were cultured on Murashige and Skoog (MS) medium supplemented with plant growth hormones (0.6 µM IAA, 8.8 µM BA) and selection antibiotic (7.5 mg/l hygromycin) for shoot initiation. The newly emerged shoots were then excised and elongated on MS media containing 0.06 µM IAA, 8.9 µM BA and 7.5 mg/l hygromycin. Elongated shoots were then transferred to rooting media (half-strength MS salts supplemented with 2.9 µM IAA as previously reported (Erland and Mahmoud 2014)). Wild-type L. latifolia plants were used as negative controls for antibiotic selection and further analyses. Rooted plants were transferred to pots and placed in a growth room until analyzed.

Relative expression
The spatial expression of ( +)-LiBPPS was examined between leaf and flower tissues of L. x intermedia cv. grosso. The expression of ( +)-LiBPPS was also compared in leaf tissues of L. x intermedia, L. angustifolia and L. latifolia. For transgenic plants, the transcript levels of ( +)-LiBPPS were quantified in transgenic and wild-type L. latifolia leaf tissues. All relative expression studies were performed by qPCR assays using the StepOne Plus Real-Time PCR system (Applied Bioscience). In brief, total RNA was extracted from sample tissues of the aforementioned plants using RNeasy Plant Mini Kit (Qiagen) according to the manufacturer's protocol. RNA from each sample was then converted to cDNA using iScript cDNA synthesis kit (Bio-Rad). β-actin was used as a reference gene to normalize the expression levels. qPCR was performed with a final reaction volume of 10 µl containing 5 µl SYBR premix, 0.6 µM of each primer and 150 ng of cDNA. PCR amplifications were then carried out using the following program; (1) holding stage at 50 °C for 2 min, and 95 °C for 2 min, followed by (2) amplification stage of 50 cycles at 95 °C for 3 s and 60 °C for 30 s, as well as (3) a final melting curve stage at 95 °C for 15 s and 60 °C for 1 min. The generated data were analyzed using the Livak method (Livak and Schmittgen 2001), expressed as 2 −ΔΔCT . The primers used in this study are listed in Table S1.

EO extraction and GC-MS analysis
Young leaves (~ 0.5 g) of transgenic or control L. latifolia plants were harvested and immediately placed in separate 15-ml glass test tubes each containing 5 ml pentane, and 50 μg thymol as an internal standard. This was followed by sonication on ice for 30 min. Pentane, containing EO constituents, from each sample was then treated with activated charcoal and run on a Varian CP 3800 GC coupled to a Saturn 2200 MS/MS according to previously described methods (Adal et al. 2017). Briefly, a 1 μl sample was injected with the following instrument setting. Column flow rate: 1 ml/ min, injector temperature: 40 °C for 10 s, and increased to 100 °C (20 °C /min) for the remaining of the run; column oven temperature: 40 °C for 5 min, rise to 170 °C at a rate of 6 °C/ min, hold for 4 min, then ramp to 230 °C at a rate of 30 °C/ min, and hold for 5 min. The relative percent composition of EO constituents in transgenic and control samples was calculated based on an internal standard (thymol) as previously described (Erland and Mahmoud 2015). Statistical analysis (Student's t-test) was performed using SigmaPlot v 12.5 (Systat Software, Germany).

Cloning of ( +)-LiBPPS
In this study, we identified and cloned the cDNA for ( +)-LiBPPS, a terpene synthase enzyme that can convert GPP to BPP in vitro. The full-length cDNA was amplified by PCR from reverse-transcribed RNA obtained from L. x intermedia, cv. Grosso, flower tissue. The ORF of the cDNA (GenBank: MW846855) encoded a protein of 593 amino acids, bearing a 50 amino acid plastid targeting sequence.
The encoded protein was predicted to have a molecular mass of 69.6 kDa and a pI of 5.08. The deduced amino acid sequence was further characterized for the conserved motifs against known orthologous TPS proteins. As with other plant TPSs, the key motifs that are crucial for prenyl pyrophosphate ionization (RRx8W), and for divalent metal ion binding ((DDxxD) and (N,D)D(L,I,V)x(S,T)x3E) were well-conserved in this enzyme (Fig. S1). ( +)-LiBPPS exhibited a relatively high degree of amino acid identity with S. officinalis BPPS (65.5%), while it was less homologous to other known BPPSs from L. angustifolia (LaBPPS; 51.6%), P. dulcis (PdBPPS; 47.5%), C. burmanni (CbBPPS; 43%) and A. villosum (AvBPPS; 37.5%).

Genomic DNA organization and phylogenetic analysis
The genomic sequence for the ( +)-LiBPPS was amplified from L. x intermedia leaf genomic DNA. The cloned ( +)-LiBPPS genomic DNA is 2487 bp long and contains an intron-exon structure typical of Lavandula and other angiosperm sesquiterpene (TPS-a) and monoterpene (TPS-b) synthase genes; it bears 7 exons ranging in size from 138 bp in exon-5 to 384 bp in exon-3, and 6 introns ranging in size from 68 bp in intron-1 to 226 bp in intron-3 ( Fig. 2; Fig.  S2). Four of the exons (exon-1, exon-4, exon-6 and exon-7) contained the common TPS conserved motifs at the expected positions. All introns and exons, but exons 4-7, were different in length between the two Lavandula BPPSs.

Homology modeling of ( +)-LiBPPS
We determined the 3D structure of ( +)-LiBPPS based on homology to the previously reported structural model for the S. officinalis BPPS (Whittington et al. 2002). Using the information, we predicted the 3D structure of the active site pocket before and after substrate (GPP) docking (Fig. 4). We also identified key amino acid residues positioned within and in close proximity of the active site pocket. Several amino acid residues within the active site are predicted to play important roles in substrate binding and selectivity.

Recombinant ( +)-LiBPPS expression and product identification
We expressed the ( +)-LiBPPS ORF, excluding the N-terminus transit peptide, in E. coli Rosetta™ (DE3) pLysS strain using pET41b( +) expression vector, and partially purified the protein using Ni-NTA-agarose affinity chromatography (Fig. S3). The partially purified recombinant protein was assayed for activity with GPP, FPP, DMAPP as well as a combined IPP and DMAPP as substrate, and the resulting product was treated with calf intestine alkaline phosphatase (CIP) (New England Biolabs) to produce the nearest alcohol. The products of the reactions (before and after alkaline phosphatase treatment) were analyzed along with the authentic standards by GC-MS. Borneol and other monoterpenes (multiple products) were detected from assays of ( +)-LiBPPS with GPP followed by CIP treatment (Table 1 and Fig. 5), whereas only monoterpenes were detected from assays of ( +)-LiBPPS with GPP. ( +)-LiBPPS produced a product mixture that contained ~ 60% BPP and ~ 40% other minor products (Fig. 5). However, the recombinant ( +)-LiBPPS protein was catalytically inactive when assayed with FPP, DMAPP, or IPP and DMAPP (together) (data not shown). The borneol produced in the assay was also analyzed by gas chromatography using a chiral column and identified as ( +)-borneol (Fig. S4). As a control, affinity-purified extracts from E. coli transformed with the empty vector, or heat-denatured purified recombinant ( +)-LiBPPS proteins were assayed. The results revealed that none of the control reactions produced detectable products (data not shown).
To compare the in vitro activity of ( +)-LiBPPS with the previously cloned LaBPPS (Despinasse et al. 2017), we cloned the cDNA for LaBPPS from our L. angustifolia cv. Lady cDNA library (Lane et al. 2010). The cloned LaBPPS shared over 98% nucleotide identities with the previously reported LaBPPS (from cv. Diva). However, the full-length LaBPPS from cv. Lady contains two nucleotide insertions at the C-terminus that cause frame-shift mutations and the introduction of premature termination codons. As a result, we are unable to produce recombinant proteins for LaBPPS in our study.

( +)-LiBPPS expression studies
Using qPCR, we studied transcript levels for ( +)-LiBPPS in L. x intermedia leaf and flower tissues (Fig. 6a). Results indicated that ( +)-LiBPPS transcripts were more abundant in leaf tissue. We also measured ( +)-LiBPPS transcripts in leaves of L. x intermedia parents, L. angustifolia and L. latifolia (Fig. 6b). The ( +)-LiBPPS transcripts accumulated at much higher levels in leaves of L. x intermedia compared to both of its parents.
To evaluate the function of ( +)-LiBPPS in planta, we generated transgenic L. latifolia plants expressing the antisense and sense versions of LiBPPS cDNA placed under the control of CaMV 35S promoter (Fig. 7a). Most plants transformed with the sense construct exhibited stunted growth and did not survive; only nine independent lines expressing ( +)-LiBPPS in sense survived, four of which died soon after early analysis. On the other hand, expression in antisense did not affect plant growth, and over 35 lines expressing the gene in antisense were produced (Fig. 7b). To examine the transcriptional expression of ( +)-LiBPPS, qPCR assays were performed in wild-type (Wt) and representative sense and antisense transgenic lines ( Fig. 7c and d). As anticipated, the ( +)-LiBPPS mRNA was much more abundant in transgenic lines that expressed ( +)-LiBPPS in sense compared to wild-type controls. Similarly, as expected ( +)-LiBPPS transcript levels were much less abundant in plants expressing ( +)-LiBPPS gene in antisense compare to wild-type plants (Fig. 7).
We examined the transgenic lines for the accumulation of key monoterpenes and total oil yield. Borneol (0.87 -2.63%) and camphor (11.6-15.9%) levels were substantially lower in the EO of antisense transgenic plants compared to those in wild-type plants, which contained 4.18 ± 0.23% borneol and 23.5 ± 1.45% camphor (Table 2). Additionally, plants expressing ( +)-LiBPPS in antisense accumulated substantially more all known BPPS genes. The trees were generated using the Phylogeny Analysis MEGA7.0 program (Kumar et al., 2016). The resulting trees were bootstrap analyzed with 1,000 replicates, and bootstrap values > 70% are mentioned on the trees. The scale bar shows the number of amino acid substitutions per site. Accession numbers for all TPSs used in this study are listed in Table S2 1, 8-cineole than Wt plants. However, limonene levels were not affected in antisense plants compared to Wt controls ( Table 2).
As noted, most of the regenerated plants overexpressing ( +)-LiBPPS in the sense did not survive for EO analysis. Of the five that did, three lines (S1, S2 and S5) produced substantially and significantly more borneol and camphor, but accumulated less 1,8-cineole than Wt plants. In the other two sense lines (S3 and S4), the concentrations of all tested monoterpenes were more or less the same as those in Wt plants, although these plants accumulated slightly lower amounts of borneol and camphor. Notably, as seen for antisense plants, limonene levels were not affected in any of the sense plants. However, oil yield was also slightly lower than Wt plants in S3 and S4 lines.

Discussion
Lavandula x intermedia, a high EO yielding natural hybrid of L. angustifolia and L. latifolia, produces an EO constituted of monoterpenes, mainly linalool, linalyl acetate, borneol, camphor and 1,8-cineole. Due to the relatively high levels of borneol and camphor, L. x intermedia EOs typically are used in low-value products such as air fresheners and soaps. The cloning of genes responsible for borneol and camphor production opens several avenues for reducing the production of these compounds, and hence increasing the commercial value of the EO in L. x intermedia plants. In this context, the cloning of BPPS, an enzyme that catalyzes the committed step (conversion of Fig. 4 Homology-based modeling of ( +)-LiBPPS, displaying active site pocket and binding residues. a Three-dimensional protein structure with an active site pocket. b The active site pocket with docked GPP and predicted binding residues. c Key active site residues involved in carbocation stabilization. These amino acid residues within the active site were compared with previously reported BPPS proteins from S. officinalis (Wise et al. 1998) and L. angustifolia (Despinasse et al. 2017) GPP to BPP) in borneol (and camphor) production is of key importance.

Phylogenetic analysis and genome organization of ( +)-LiBPPS
In this study, we identified ( +)-LiBPPS, which encoded 593 amino acids consisting of all a single copy of conserved motifs present in typical plant TPSs, such as RR(× 8)W, DDxxD and (N,D)D(L,I,V)x(S,T)x3E (Wise et al. 1998). The first motif, RR(× 8)W, is located at the very beginning of the N-terminus of the protein and is required in mono-TPSs for cyclization (Williams et al. 1998), or protein stabilization (Hyatt et al. 2007). The DDxxD and ((N,D)D(L,I,V)x(S,T)x3E) motifs presented at C-terminus are responsible for substrate binding and coordination of divalent metal ion cofactors (Degenhardt et al. 2009). Uniquely, a double copy of RR(× 8)W motif was detected from P. dulicis BPPS, in which the first copy inhibited the in vitro protein activity (Hurd et al. 2017). However, alterations of the common residues in these conserved motifs are often observed through deletion and/or substitution without compromising the functional activities. For example, a deletion of an arginine (R) at RR(× 8)W motif of cadinol and germacrene D synthases, and substitution of an aspartate (D) by glutamate (E) at x intermedia BPPS (LiBPPS) from geranyl diphosphate (GPP) as a substrate. Reaction products from ( +)-LiBPPS assay were analyzed by GC-MS before (a) and after (b) treatments of assay products by calf intestinal alkaline phosphatase (CIP). Note that BPP does not show in GC-MS analysis, as it is not volatile. a Chromatography of monoterpene produced from GPP. Peaks 1 -5 represent monoterpenes directly formed from GPP by the recombinant ( +)-LiBPPS. These peaks are α-Pinene (1), Camphene (2), β-Pinene (3), Limonene (4) and Terpinolene (5). b GC-MS analysis of borneol (produced from BPP upon CIP treatment) and other monoterpenes derived from GPP. Assays of recombinant ( +)-LiBPPS were run with GPP (50 µM), followed by overnight hydrolysis using calf intestine alkaline phosphatase (CIP) to generate borneol. c Mass spectra of ( +)-borneol from assay product, and d mass spectrum of the borneol authentic standard from the NIST library 1 3 DDxxD motif of germacrene D synthases were identified in L. angustifolia (Jullien et al. 2014).
Based on analysis of deduced amino acid sequences, ( +)-LiBPPS is highly homologous to the L. angustifolia limonene synthase (LaLimS), L. x intermedia 3-carene synthase (Li3CarS), and Lavandula viridis α-pinene synthase (LvPinS) and fenchol synthase (LvFencS) (Landmann et al. 2007;Benabdelkader et al. 2015;Adal et al. 2017). All these TPSs are clustered together under the angiosperm TPS-b subfamily, a monoterpene synthase subfamily (Bohlmann et al. 1998). Intriguingly, ( +)-LiBPPS is distantly related to most of the known BPPSs from different plants, including that from L. angustifolia, and closes to SoBPPS, with which it shares 65.48% identity at the amino acid level. This indicates that plant mono-TPSs originated from the same ancestor or one has evolved from the other through gene duplication, leading to divergent functional evolution through subfunctionalization and / or neofunctionalization of TPSs (Bohlmann et al. 1998;Chen et al. 2011). It is not also surprising that ( +)-LiBPPS is more homologous to other Lavandula TPSs, as most TPS genes with different functions have more similarity to each other within a given species than TPSs with the same function in a different species. For example, Li3CarS shares 75% amino acid identity with LaLimS (from half-parent species) compared to 29-50% sequence homology with 3-carene synthases of P. abies and S. stenophylla (Adal et al. 2017). While, in Sitka spruce, 3-carene synthase gene had 89-92% nucleotides identity with another mTPS, (-)-sabinene synthase (Roach et al. 2014).
The genomic structure of ( +)-LiBPPS has typical features of the Class III TPSs, which are known by seven exons and six introns (Chen et al. 2011). The common conserved motifs are also located at the same exon positions of most TPSs in several angiosperms.

In vitro activity of the recombinant ( +)-LiBPPS
The genes and encoding enzymes for most major Lavandula monoterpene synthases have been identified and functionally characterized in vitro (Landmann et al. 2007;Demissie et al. 2011Demissie et al. , 2012Adal et al. 2017Adal et al. , 2019. The ( +)-LiBPPS cloned in this study was found to encode a monoterpene synthase that catalyzes the conversion of GPP to ( +)-bornyl diphosphate (BPP), and a few other monoterpenes. The generation of BPP and multiple monoterpenes by ( +)-LiBPPS in vitro was not surprising, as most of the known BPPSs and some other mono-TPSs are multiproduct monoterpenes (Despinasse et al. 2017;Hurd et al. 2017;Wang et al 2018;Ma et al 2021;Wise et al. 1998;Fäldt et al. 2003; (Hurd et al. 2017). A few minor monoterpenes with a total proportion of 39% were also detected from L. x intermedia BPPS activity. Production of a few minor monoterpene products by the ( +)-LiBPPS was not surprising, as most other reported BPPS proteins are multiproduct enzymes.

( +)-LiBPPS is differentially expressed in lavenders
( +)-LiBPPS is constitutively expressed in L. x intermedia leaf and flower tissues, with a strong transcriptional expression observed in leaves. Given that high levels of monoterpenes derived from BPP (i.e., borneol and camphor) are produced in leaves, compared to flowers, in L. x intermedia (Boeckelmann 2008;Hassiotis et al. 2014;Seidler-Lozykowska et al. 2014), this finding was not surprising. As well, ( +)-LiBPPS transcripts were more abundant in L.
x intermedia leaves, compared to those of L. angustifolia and L. latifolia. It is not surprising to observe a lower expression of the gene in L. angustifolia, as this species does not accumulate high levels of camphor or borneol (both of which are derived from BPP). On the other hand, given that L. latifilia is a parent of L. x intermedia, and that it produces large quantities of camphor and borneol, we expected L. latifolia plants to express the same BPPS gene (i.e., ( +)-LiLBPPS) strongly. However, our results indicate that this is not the case, and that another BPPS gene could be responsible for the production of BPP in L. latifolia. Further investigation is needed to resolve this matter.

Expression of ( +)-LiBPPS impacts the biosynthesis of monoterpenes in the transgenic L. latifolia
Metabolic engineering of monoterpenes has been reported in Lamiaceaces, particularly in lavender and mint, (Mahmoud and Croteau 2001;Muñoz-Bertomeu et al. 2006, 2008Lange et al. 2011;Mendoza-Poudereux et al. 2014;Wang et al. 2016;Tsuro et al. 2019;Li et al. 2020). These studies targeted mainly the DXP pathway genes or terpene synthase genes. For example, expression of the Arabidopsis DXS gene increases the concentration of monoterpenes and essential oil yield without impacting primary metabolites, such as chlorophyll and carotenoids in L. latifolia transgenic plant leaves and inflorescences (Muñoz-Bertomeu et al. 2006). A high level of linalool was also produced through the expression of a full-length clarkia linalool synthase in L. latifolia leaves (Mendoza-Poudereux et al. 2014).
In this study, we expressed the full-length ( +)-LiBPPS cDNA in sense or antisense orientation under the control of the CaMV 35S promoter in L. latifolia plants. Plants that most strongly expressed the transgene in sense did not survive to full maturity. The exact nature of this effect is unclear and requires further investigation. However, most likely the unregulated production of large amounts of BPP and monoterpenes derived from BPP (in particular camphor (Fig. 1), a known agent of allelopathy) is lethal to the plant. Given that normally BPPS and other monoterpene synthase genes are expressed in the secretory cells of glandular trichomes, which are specialized to produce and store large quantities of monoterpenes, the above scenario makes sense. To avoid this issue, BPPS should be expressed under the control of a trichome-specific promoter. In addition, two plants expressing ( +)-LiBPPS in sense (S3 and S4) unexpectedly (and contrary to other sense plants) produced less borneol and camphor, and had a lower oil yield compared to Wt plants. While it is difficult to know the exact cause of this phenomenon, these plants may express a truncated and nonfunctional version of the transgene. Such transgene truncations sometimes occur during the Agrobacterium-mediated transformation of plants. The validity of this argument requires further investigation.
Reduced BPPS transcript levels in transgenic plants expressing ( +)-LiBPPS in antisense correlated well with the accumulation of borneol/camphor in these plants. This suggests that ( +)-LiBPPS controls the synthesis of these monoterpenes through the supply of the primary precursor bornyl diphosphate. This outcome was not unexpected as the production of numerous monoterpenes is controlled through the transcriptional regulation of the corresponding terpene synthase gene. For example, the production of 1,8 cineole was recently shown to be controlled via the regulation of the cineol synthase gene in lavender Tsuro et al. 2019).

Conclusions
We have isolated and functionally characterized a new ( +)-bornyl diphosphate synthase (( +)-LiBPPS) cDNA from L. x intermedia. ( +)-LiBPPS, which is clustered under the TPS-b subfamily, is a typical Class III TPS, and has unique active site residues. The recombinant (+)-LiBPPS protein catalyzes the conversion of GPP into BPP and a few other monoterpenes. Ectopic expression of ( +)-LiBPPS impacted the accumulation of borneol and camphor in L. latifolia leaves, prompting decreased levels of these terpenes in antisense transgenic plants. Although overexpression of ( +)-LiBPPS in the sense version resulted in increased production of BPP-derived terpenes in transgenic plants, most overexpressors exhibited severely stunted growth. Therefore, the cloned ( +)-LiBPPS would be useful in metabolic engineering studies, targeting the production of recombinant borneol and camphor in microbial systems and plants. However, overexpression of this gene to enhance borneol or camphor production in plants must be done using trichome-specific promoters.