Chemical diversity in angiosperms - monoterpene synthases control complex reactions that provide the precursors for ecologically and commercially important monoterpenoids.

Monoterpene synthases (MTSs) catalyze the first committed step in the biosynthesis of monoterpenoids, a class of specialized metabolites with particularly high chemical diversity in angiosperms. In addition to accomplishing a rate enhancement, these enzymes manage the formation and turnover of highly reactive carbocation intermediates formed from a prenyl diphosphate substrate. At each step along the reaction path, a cationic intermediate can be subject to cyclization, migration of a proton, hydride, or alkyl group, or quenching to terminate the sequence. However, enzymatic control of ligand folding, stabilization of specific intermediates, and defined quenching chemistry can maintain the specificity for forming a signature product. This review article will discuss our current understanding of how angiosperm MTSs control the reaction environment. Such knowledge allows inferences about the origin and regulation of chemical diversity, which is pertinent for appreciating the role of monoterpenoids in plant ecology but also for aiding commercial efforts that harness the accumulation of these specialized metabolites for the food, cosmetic, and pharmaceutical industries.

Creation of new germplasm resources, development of SSR markers, and screening of monoterpene synthases in thyme.

BACKGROUND: Thyme derived essential oil and its components have numerous applications in pharmaceutical, food, and cosmetic industries, owing to their antibacterial, antifungal, and antiviral properties. To obtain thyme essential oil with different terpene composition, we developed new germplasm resources using the conventional hybridization approach. RESULTS: Phenotypic characteristics, including essential oil yield and composition, glandular trichome density, plant type, and fertility, of three wild Chinese and seven European thyme species were evaluated. Male-sterile and male-fertile thyme species were crossed in different combinations, and two F1 populations derived from Thymus longicaulis (Tl) × T. vulgaris 'Fragrantissimus' (Tvf) and T. vulgaris 'Elsbeth' (Tve) × T. quinquecostatus (Tq) crosses were selected, with essential oil yield and terpene content as the main breeding goals. Simultaneously, simple sequence repeat (SSR) primers were developed based on the whole-genome sequence of T. quinquecostatus to authenticate the F1 hybrids. A total of 300 primer pairs were selected, and polymerase chain reaction (PCR) was carried out on the parents of the two hybrid populations (Tl, Tvf, Tve, and Tq). Based on the chemotype of the parents and their F1 progenies, we examined the expression of genes encoding two γ-terpinene synthases, one α-terpineol synthase, and maybe one geraniol synthase in all genotypes by quantitative real-time PCR (qRT-PCR). CONCLUSION: We used hybridization to create new germplasm resources of thyme, developed SSR markers based on the whole-genome sequence of T. quinquecostatus, and screened the expression of monoterpene synthase genes in thyme. The results of this study provide a strong foundation for the creation of new germplasm resources, construction of the genetic linkage maps, and identification of quantitative trait loci (QTLs), and help gain insight into the mechanism of monoterpenoids biosynthesis in thyme.

Functional characterization of four mono-terpene synthases (TPSs) provided insight into the biosynthesis of volatile monoterpenes in the medicinal herb Blumea balsamifera.

Blumea balsamifera, a wooden plant belonging to the family Asteraceae, is a medicinal herb with anticancer, antiviral, and multiple pharmacological effects, which are believed to be caused by its essential oil. The essential oil from B. balsamifera is comprised of mono- and sesqui-terpenes as the majority. Unfortunately, this plant has been facing the challenge of resource shortage, which could be effectively alleviated by biological engineering. Therefore, the identification of key elements involved in the biosynthesis of active ingredients becomes an indispensable prerequisite. In this study, candidate genes encoding monoterpene synthase were screened by transcriptome sequencing combined with metabolomics profiling in the roots, stems, and leaves of B. balsamifera. Then, these candidates were successfully cloned and verified by heterologous expression and in vitro enzyme activity assays. As a result, six candidate BbTPS genes were isolated from B. balsamifera, of which three encoded single-product monoterpene synthases and one encoded a multi-product monoterpene synthase. Among them, BbTPS1, BbTPS3, and BbTPS4 could catalyze the formation of D-limonene, α-phellandrene, and L-borneol, respectively. Meanwhile, BbTPS5 functioned in catalyzing GPP into terpinol, ß-phellandrene, ß-myrcene, D-limonene, and 2-carene in vitro. In general, our results provided important elements for the synthetic biology of volatile terpenes in B. balsamifera, which laid a foundation for subsequent heterologous production of these terpenoids through metabolic engineering and increasing their yield, as well as promoting sustainable development and utilization of B. balsamifera. Supplementary Information: The online version contains supplementary material available at 10.1007/s12298-023-01306-8.

The santalene synthase from Cinnamomum camphora: Reconstruction of a sesquiterpene synthase from a monoterpene synthase.

Plant terpene synthases (TPSs) can mediate formation of a large variety of terpenes, and their diversification contributes to the specific chemical profiles of different plant species and chemotypes. Plant genomes often encode a number of related terpene synthases, which can produce very different terpenes. The relationship between TPS sequence and resulting terpene product is not completely understood. In this work we describe two TPSs from the Camphor tree Cinnamomum camphora (L.) Presl. One of these, CiCaMS, acts as a monoterpene synthase (monoTPS), and mediates the production of myrcene, while the other, CiCaSSy, acts as a sesquiterpene synthase (sesquiTPS), and catalyses the production of α-santalene, ß-santalene and trans-α-bergamotene. Interestingly, these enzymes share 97% DNA sequence identity and differ only in 22 amino acid residues out of 553. To understand which residues are essential for the catalysis of monoterpenes resp. sesquiterpenes, a number of hybrid synthases were prepared, and supplemented by a set of single-residue variants. These were tested for their ability to produce monoterpenes and sesquiterpenes by in vivo production of sesquiterpenes in E. coli, and by in vitro enzyme assays. This analysis pinpointed three residues in the sequence which could mediate the change in product specificity from a monoterpene synthase to a sesquiterpene synthase. Another set of three residues defined the sesquiterpene product profile, including the ratios between sesquiterpene products.

[Cloning and characterization of a monoterpene synthase gene from Tripterygium wilfordii].

Tripterygium wilfordii is a medicinal plant commonly used in the treatment of rheumatoid arthritis,and with pharmacological activities in anti-tumor and obesity treatment. The known active ingredients in T. wilfordii are mainly terpenoids,but with very low content. Therefore,the analysis of the biosynthesis pathway of terpenoids in T. wilfordii has become a research hotspot to solve the problem of its resources. Terpenoid synthase( TPS) is a key enzyme that catalyzes the formation of a wide variety of terpenoid skeletons. In this study,a gene fragment with an ORF of 1 785 bp was cloned from T. wilfordii. Bioinformatics analysis was performed using NCBI's BLASTP,ProtParam and Interpro online tools and MEGA 6.0 software. The response of this gene to methyl jasmonate was also detected by real-time fluorescent quantitative PCR,and its catalytic function was verified by prokaryotic expression and in vitro enzymatic assay. Bioinformatics analysis indicated that the amino acid sequence encoded by this gene had both N-terminal domain and C-terminal domain of TPS,as well as the DDxx D conserved domain of the class I of TPS family. And Tw MTS gathered together with TPS-b subfamily in the Neighbor-Joining Tree constructed with known homologous TPSs. The results of RT-PCR showed that 50 µmol·L-1 MeJA 12 h could increase the expression of Tw MTS to 735 times in the control group at 12 h,and 1 644 times at 24 h. In addition,in vitro enzymatic reaction results showed that Tw MTS can catalyze the production of ß-citronellol with GPP as substrate,indicating that Tw MTS was a monoterpene synthase. The above results provided a new element for the synthetic biology database of T. wilfordii terpenoids,and laid the foundation for future biosynthesis research.

Functional analysis of (4S)-limonene synthase mutants reveals determinants of catalytic outcome in a model monoterpene synthase.

Crystal structural data for (4S)-limonene synthase [(4S)-LS] of spearmint (Mentha spicata L.) were used to infer which amino acid residues are in close proximity to the substrate and carbocation intermediates of the enzymatic reaction. Alanine-scanning mutagenesis of 48 amino acids combined with enzyme fidelity analysis [percentage of (-)-limonene produced] indicated which residues are most likely to constitute the active site. Mutation of residues W324 and H579 caused a significant drop in enzyme activity and formation of products (myrcene, linalool, and terpineol) characteristic of a premature termination of the reaction. A double mutant (W324A/H579A) had no detectable enzyme activity, indicating that either substrate binding or the terminating reaction was impaired. Exchanges to other aromatic residues (W324H, W324F, W324Y, H579F, H579Y, and H579W) resulted in enzyme catalysts with significantly reduced activity. Sequence comparisons across the angiosperm lineage provided evidence that W324 is a conserved residue, whereas the position equivalent to H579 is occupied by aromatic residues (H, F, or Y). These results are consistent with a critical role of W324 and H579 in the stabilization of carbocation intermediates. The potential of these residues to serve as the catalytic base facilitating the terminal deprotonation reaction is discussed.

Isolation and functional characterization of a methyl jasmonate-responsive 3-carene synthase from Lavandula x intermedia.

KEY MESSAGE: A methyl jasmonate responsive 3-carene synthase (Li3CARS) gene was isolated from Lavandula x intermedia and functionally characterized in vitro. Lavenders produce essential oils consisting mainly of monoterpenes, including the potent antimicrobial and insecticidal monoterpene 3-carene. In this study we isolated and functionally characterized a leaf-specific, methyl jasmonate (MeJA)-responsive monoterpene synthase (Li3CARS) from Lavandula x intermedia. The ORF excluding transit peptides encoded a 64.9 kDa protein that was expressed in E. coli, and purified with Ni-NTA agarose affinity chromatography. The recombinant Li3CARS converted GPP into 3-carene as the major product, with K m and k cat of 3.69 ± 1.17 µM and 2.01 s-1 respectively. Li3CARS also accepted NPP as a substrate to produce multiple products including a small amount of 3-carene. The catalytic efficiency of Li3CARS to produce 3-carene was over ten fold higher for GPP (k cat /K m = 0.56 µM-1s-1) than NPP (k cat /K m = 0.044 µM-1s-1). Production of distinct end product profiles from different substrates (GPP versus NPP) by Li3CARS indicates that monoterpene metabolism may be controlled in part through substrate availability. Li3CARS transcripts were found to be highly abundant in leaves (16-fold) as compared to flower tissues. The transcriptional activity of Li3CARS correlated with 3-carene production, and was up-regulated (1.18- to 3.8-fold) with MeJA 8-72 h post-treatment. The results suggest that Li3CARS may have a defensive role in Lavandula.

Functional identification of a Lippia dulcis bornyl diphosphate synthase that contains a duplicated, inhibitory arginine-rich motif.

Lippia dulcis (Aztec sweet herb) contains the potent natural sweetener hernandulcin, a sesquiterpene ketone found in the leaves and flowers. Utilizing the leaves for agricultural application is challenging due to the presence of the bitter-tasting and toxic monoterpene, camphor. To unlock the commercial potential of L. dulcis leaves, the first step of camphor biosynthesis by a bornyl diphosphate synthase needs to be elucidated. Two putative monoterpene synthases (LdTPS3 and LdTPS9) were isolated from L. dulcis leaf cDNA. To elucidate their catalytic functions, E. coli-produced recombinant enzymes with truncations of their chloroplast transit peptides were assayed with geranyl diphosphate (GPP). In vitro enzyme assays showed that LdTPS3 encodes bornyl diphosphate synthase (thus named LdBPPS) while LdTPS9 encodes linalool synthase. Interestingly, the N-terminus of LdBPPS possesses two arginine-rich (RRX8W) motifs, and enzyme assays showed that the presence of both RRX8W motifs completely inhibits the catalytic activity of LdBPPS. Only after the removal of the putative chloroplast transit peptide and the first RRX8W, LdBPPS could react with GPP to produce bornyl diphosphate. LdBPPS is distantly related to the known bornyl diphosphate synthase from sage in a phylogenetic analysis, indicating a converged evolution of camphor biosynthesis in sage and L. dulcis. The discovery of LdBPPS opens up the possibility of engineering L. dulcis to remove the undesirable product, camphor.

Differences in Monoterpene Biosynthesis and Accumulation in Pistacia palaestina Leaves and Aphid-Induced Galls.

Certain insect species can induce gall formation on numerous plants species. Although the mechanism of gall development is largely unknown, it is clear that insects manipulate their hosts' anatomy, physiology, and chemistry for their own benefit. It is well known that insect-induced galls often contain vast amounts of plant defensive compounds as compared to non-colonized tissues, but it is not clear if defensive compounds can be produced in situ in the galled tissues. To answer this question, we analyzed terpene accumulation patterns and possible independent biosynthetic potential of galls induced by the aphid Baizongia pistaciae L. on the terminal buds of Pistacia palaestina Boiss. We compared monoterpene levels and monoterpene synthase enzyme activity in galls and healthy leaves from individual trees growing in a natural setting. At all developmental stages, monoterpene content and monoterpene synthase activity were consistently (up to 10 fold on a fresh weight basis) higher in galls than in intact non-colonized leaves. A remarkable tree to tree variation in the products produced in vitro from the substrate geranyl diphosphate by soluble protein extracts derived from individual trees was observed. Furthermore, galls and leaves from the same trees displayed enhanced and often distinct biosynthetic capabilities. Our results clearly indicate that galls possess independent metabolic capacities to produce and accumulate monoterpenes as compared to leaves. Our study indicates that galling aphids manipulate the enzymatic machinery of their host plant, intensifying their own defenses against natural enemies.

Susceptibility to Verticillium longisporum is linked to monoterpene production by TPS23/27 in Arabidopsis.

The fungus Verticillium longisporum is a soil-borne plant pathogen of increasing economic importance, and information on plant responses to it is limited. To identify the genes and components involved in the early stages of infection, transcripts in roots of V. longisporum-challenged Arabidopsis Col-0 and the susceptible NON-RACE SPECIFIC DISEASE RESISTANCE 1 (ndr1-1) mutant were compared using ATH1 gene chips. The analysis revealed altered transcript levels of several terpene biosynthesis genes, including the monoterpene synthase TPS23/27. When transgenic 35S:TPS23/27 and TPS23/27-amiRNA plants were monitored the over-expresser line showed enhanced fungal colonization whereas the silenced genotype was indistinguishable from Col-0. Transcript analysis of terpene biosynthesis genes suggested that only the TPS23/27 pathway is affected in the two transgenic genotypes. To confirm changes in monoterpene production, emitted volatiles were determined using solid-phase microextraction and gas chromatography-mass spectrometry. Levels of all identified TPS23/27 monoterpene products were significantly altered in the transgenic plants. A stimulatory effect on conidial germination and hyphal growth of V. longisporum was also seen in co-cultivation with 35S:TPS23/27 plants and upon exposure to 1,8-cineole, the main product of TPS23/27. Methyl jasmonate treatments of myc2-1 and myc2-2 mutants and analysis of TPS23/27:uidA in the myc2-2 background suggested a dependence on jasmonic acid mediated by the transcription factor MYC2. Taken together, our results show that TPS23/27-produced monoterpenes stimulate germination and subsequent invasion of V. longisporum in Arabidopsis roots.

T-shaped trichome-specific expression of monoterpene synthase ADH2 using promoter-ß-GUS fusion in transgenic Artemisia annua L.

Artemisinin, a sesquiterpene lactone isolated from Artemisia annua L. (sweet wormwood), is extensively used in the treatment of malaria. In order to better understand the metabolism of terpenes in A. annua and the influence of terpene synthases on artemisinin yield, the expression pattern of a monoterpene alcohol dehydrogenase (ADH2) has been studied using transgenic plants expressing promoter-ß-glucuronidase (GUS) fusion. ADH2 played a major role in monoterpenoid biosynthesis including carveol, borneol, and artemisia ketone through in vitro biochemical analysis. In this study, the ADH2 promoter was cloned by the genome walking method. A number of putative cis-acting elements were predicted in promoter region, suggesting that the ADH2 is driven by a complex regulation mechanism. ADH2 gene was highly expressed in old leaves, whereas the artemisinin biosynthetic genes were mainly expressed in bud and young leaves. The expression of ADH2 gene increased quickly during leaf development, revealed by qRT-PCR. GUS expression analysis in different tissues of transgenic A. annua demonstrates that ADH2 expression is exclusively located to T-shaped trichome, not glandular secretory trichome.

Characterization of a monoterpene synthase from Paeonia lactiflora producing α-pinene as its single product.

OBJECTIVES: To identify a terpene synthase that catalyzes the conversion of geranyl pyrophosphate (GPP) to α-pinene and is involved in the biosynthesis of paeoniflorin. RESULTS: Two new terpene synthase genes were isolated from the transcriptome data of Peaonia lactiflora. Phylogenetic analysis and sequence characterization revealed that one gene, named PlPIN, encoded a monoterpene synthase that might be involved in the biosynthesis of paeoniflorin. In vitro enzyme assay showed that, in contrast to most monoterpene synthases, PlPIN encoded an α-pinene synthase which converted GPP into α-pinene as a single product. CONCLUSIONS: This newly identified α-pinene synthase could be used for improving paeoniflorin accumulation by metabolic engineering or for producing α-pinene via synthetic biology.

Identification and Characterization of a Fungal Monoterpene Synthase Responsible for the Biosynthesis of Geraniol.

Geraniol, an acyclic monoterpenoid of substantial value extracted from the essential oils of various aromatic plants, holds significant commercial and industrial importance in the realms of food, cosmetics, medicine, and bioenergy. Geraniol synthase, which is responsible for geraniol production, has been identified in only several plant species to date. Here, we present the first cloning and characterization of a geraniol synthase (PgfTPS) from Penicillium griseofulvum. This enzyme demonstrates pronounced specificity in catalyzing the conversion of geranyl diphosphate into geraniol. Moreover, through protein modeling and site-directed mutagenesis, we have identified key active-site residues crucial for the catalytic function of PgfTPS. Finally, we utilized engineered Saccharomyces cerevisiae as a host for PgfTPS expression to facilitate geraniol production. Our findings not only advance the development of efficient biocatalysts for geraniol generation but also establish a fundamental basis for further exploration into fungal monoterpene biosynthesis.

Identification and Characterization of a Nerol Synthase in Fungi.

Nerol, a linear monoterpenoid, is naturally found in essential oils of various plants and is widely used in the fragrance, food, and cosmetic industries. Nerol synthase, essential for nerol biosynthesis, has previously been identified only in plants that use NPP as the precursor. In this study, a novel fungal nerol synthase, named PgfB, was cloned and characterized from Penicillium griseofulvum. In vitro enzymatic assays showed that PgfB could directly convert the substrate GPP into nerol. Furthermore, the successful expression of PgfB and its homologous protein in Saccharomyces cerevisiae resulted in the heterologous production of nerol. Finally, crucial amino acid residues for PgfB's catalytic activity were identified through site-directed mutagenesis. This research broadens our understanding of fungal monoterpene synthases and presents precious gene resources for the industrial production of nerol.

Biochemical basis for the formation of organ-specific volatile blends in mint.

Above-ground material of members of the mint family is commercially distilled to extract essential oils, which are then formulated into a myriad of consumer products. Most of the research aimed at characterizing the processes involved in the formation of terpenoid oil constituents has focused on leaves. We now demonstrate, by investigating three mint species, peppermint (Mentha Ë£ piperita L.), spearmint (Mentha spicata L.) and horsemint (Mentha longifolia (L.) Huds.; accessions CMEN 585 and CMEN 584), that other organs - namely stems, rhizomes and roots - also emit volatiles and that the terpenoid volatile composition of these organs can vary substantially from that of leaves, supporting the notion that substantial, currently underappreciated, chemical diversity exists. Differences in volatile quantities released by plants whose roots had been dipped in a Verticillium dahliae-spore suspension (experimental) or dipped in water (controls) were evident: increases of some volatiles in the root headspace of mint species that are susceptible to Verticillium wilt disease (peppermint and M. longifolia CMEN 584) were detected, while the quantities of certain volatiles decreased in rhizomes of species that show resistance to the disease (spearmint and M. longifolia CMEN 585). To address the genetic and biochemical basis underlying chemical diversity, we took advantage of the newly sequenced M. longifolia CMEN 585 genome to identify candidate genes putatively coding for monoterpene synthases (MTSs), the enzymes that catalyze the first committed step in the biosynthesis of monoterpenoid volatiles. The functions of these genes were established by heterologous expression in Escherichia coli, purification of the corresponding recombinant proteins, and enzyme assays, thereby establishing the existence of MTSs with activities to convert a common substrate, geranyl diphosphate, to (+)-α-terpineol, 1,8-cineole, γ-terpinene, and (-)-bornyl diphosphate, but were not active with other potential substrates. In conjunction with previously described MTSs that catalyze the formation of (-)-ß-pinene and (-)-limonene, the product profiles of the MTSs identified here can explain the generation of all major monoterpene skeletons represented in the volatiles released by different mint organs.

Elucidation of the essential oil biosynthetic pathways in Cinnamomum burmannii through identification of six terpene synthases.

Cinnamomum burmannii is a traditional plant that has long been used as a spice, food preservative, and food flavoring. Essential oils in C. burmannii, which mainly consist of mono- and sesquiterpenes such borneol, linalool, and caryophyllene, have impressive pharmaceutical properties. Although the transcriptome-based discovery of (+)-bornyl diphosphate synthase (CbTPS1) from C. burmannii was reported in our previous study, the remaining terpene synthases (TPSs) corresponding to various terpene biosynthesis pathways remain unidentified. In this study, we report the results of RNA-sequencing of a borneol type plant and functional characterization of six additional full-length candidate TPS genes (named CbTPS2-7). Phylogenetic analysis revealed that CbTPS2 and CbTPS3 together with the previously identified CbTPS1 protein belong to the TPS-b subfamily, and enzyme assays using geranyl diphosphate (GPP) and farnesyl diphosphate (FPP) as substrates revealed that CbTPS1, CbTPS2 and CbTPS3 catalyze the formation of monoterpenes. CbTPS4, CbTPS5, and CbTPS6, which belong to the TPS-a clade, generated monoterpenes and sesquiterpenes. CbTPS7, which belongs to the TPS-g clade, showed linalool/nerolidol synthase activity. These CbTPSs identified in C. burmannii produced a total of 10 monoterpenes and 14 sesquiterpenes in an in vitro assay. These findings clarify the biosynthesis pathways of 13 monoterpenoids and 12 sesquiterpenoids in the leaf essential oil of C. burmannii and shed light on terpene biosynthesis in Cinnamomum.

Monoterpene Synthase Genes and Monoterpene Profiles in Pinus nigra subsp. laricio.

In the present study, we carried out a quantitative analysis of the monoterpenes composition in different tissues of the non-model conifer Pinus nigra J.F. Arnold subsp. laricio Palib. ex Maire (P. laricio, in short). All the P. laricio tissues examined showed the presence of the same fourteen monoterpenes, among which the most abundant were ß-phellandrene, α-pinene, and ß-pinene, whose distribution was markedly tissue-specific. In parallel, from the same plant tissues, we isolated seven full-length cDNA transcripts coding for as many monoterpene synthases, each of which was found to be attributable to one of the seven phylogenetic groups in which the d1-clade of the canonical classification of plants' terpene synthases can be subdivided. The amino acid sequences deduced from the above cDNA transcripts allowed to predict their putative involvement in the biosynthesis of five of the monoterpenes identified. Transcripts profiling revealed a differential gene expression across the different tissues examined, and was found to be consistent with the corresponding metabolites profiles. The genomic organization of the seven isolated monoterpene synthase genes was also determined.

Plasticity engineering of plant monoterpene synthases and application for microbial production of monoterpenoids.

Plant monoterpenoids with structural diversities have extensive applications in food, cosmetics, pharmaceuticals, and biofuels. Due to the strong dependence on the geographical locations and seasonal annual growth of plants, agricultural production for monoterpenoids is less effective. Chemical synthesis is also uneconomic because of its high cost and pollution. Recently, emerging synthetic biology enables engineered microbes to possess great potential for the production of plant monoterpenoids. Both acyclic and cyclic monoterpenoids have been synthesized from fermentative sugars through heterologously reconstructing monoterpenoid biosynthetic pathways in microbes. Acting as catalytic templates, plant monoterpene synthases (MTPSs) take elaborate control of the monoterpenoids production. Most plant MTPSs have broad substrate or product properties, and show functional plasticity. Thus, the substrate selectivity, product outcomes, or enzymatic activities can be achieved by the active site mutations and domain swapping of plant MTPSs. This makes plasticity engineering a promising way to engineer MTPSs for efficient production of natural and non-natural monoterpenoids in microbial cell factories. Here, this review summarizes the key advances in plasticity engineering of plant MTPSs, including the fundamental aspects of functional plasticity, the utilization of natural and non-natural substrates, and the outcomes from product isomers to complexity-divergent monoterpenoids. Furthermore, the applications of plasticity engineering for improving monoterpenoids production in microbes are addressed.

Experiment and Simulation Reveal How Mutations in Functional Plasticity Regions Guide Plant Monoterpene Synthase Product Outcome.

Monoterpenes (C10 isoprenoids) are a structurally diverse group of natural compounds that are attractive to industry as flavours and fragrances. Monoterpenes are produced from a single linear substrate, geranyl diphosphate, by a group of enzymes called the monoterpene cyclases/synthases (mTC/Ss) that catalyse high-energy cyclisation reactions involving unstable carbocation intermediates. Efforts towards producing monoterpenes via biocatalysis or metabolic engineering often result in the formation of multiple products due to the nature of the highly branched reaction mechanism of mTC/Ss. Rational engineering of mTC/Ss is hampered by the lack of correlation between the active site sequence and cyclisation type. We used available mutagenesis data to show that amino acids involved in product outcome are clustered and spatially conserved within the mTC/S family. Consensus sequences for three such plasticity regions were introduced in different mTC/S with increasingly complex cyclisation cascades, including the model enzyme limonene synthase (LimS). In all three mTC/S studied, mutations in the first two regions mostly give rise to products that result from premature quenching of the linalyl or α-terpinyl cations, suggesting that both plasticity regions are involved in the formation and stabilisation of cations early in the reaction cascade. A LimS variant with mutations in the second region (S454G, C457V, M458I), produced mainly more complex bicyclic products. QM/MM MD simulations reveal that the second cyclisation is not due to compression of the C2-C7 distance in the α-terpinyl cation, but is the result of an increased distance between C8 of the α-terpinyl cation and two putative bases (W324, H579) located on the other side of the active site, preventing early termination by deprotonation. Such insights into the impact of mutations can only be obtained using integrated experimental and computational approaches, and will aid the design of altered mTC/S activities towards clean monoterpenoid products.

The expression of monoterpene synthase genes and their respective end products are affected by gibberellic acid in Thymus vulgaris.

Thymus vulgaris L. (Lamiaceae), a well-known aromatic medicinal herb, has many important essential constituents in its oil, including γ-terpinene, carvacrol, thymol, and p-cymene. Gibberellins comprise hundreds of components, which regulate several various growths and underlying developmental processes, such as cell division and elongation, shoot elongation, seed germination, and gene expression. In this study, we investigated the influence of sprayed gibberellic acid (GA3) treatments on the internode length, leaf morphology, length of new shoot, expression of monoterpene synthase genes and monoterpenes content during two plant growth stages. Our results showed that increasing of internode length was a clear effect of GA3 that was varied with internode position. The results also showed that all internodes displayed a dramatic increase in the highest concentration of GA3. Also, the foliar application of GA3 resulted in not only an increased expression level of monoterpene synthase genes, but also the improved production of a monoterpene, especially in the moderate concentration of GA3 that they were up-regulated. In the lowest GA3 concentrations, relative expression levels were similar or lower than the control plants and a notable downregulation in those genes was observed in the application of the highest concentration of GA3 rather than the moderate concentrations. Overall, the expression of two out of five monoterpene synthase genes, TPS and CYP71D181, showed a correlation with the level of γ-terpinene and carvacrol, respectively, indicating that they are regulated at the transcriptional levels.