Cyanobacterial production of plant essential oils.

MAIN CONCLUSION: Synechocystis (a cyanobacterium) was employed as an alternative host for the production of plant essential oil constituents. ß-Phellandrene synthase (PHLS) genes from different plants, when expressed in Synechocystis, enabled synthesis of variable monoterpene hydrocarbon blends, converting Synechocystis into a cell factory that photosynthesized and released useful products. Monoterpene synthases are secondary metabolism enzymes that catalyze the generation of essential oil constituents in terrestrial plants. Essential oils, including monoterpene hydrocarbons, are of interest for their commercial application and value. Therefore, heterologous expression of monoterpene synthases for high-capacity essential oil production in photosynthetic microorganism transformants is of current interest. In the present work, the cyanobacterium Synechocystsis PCC 6803 was employed as an alternative host for the production of plant essential oil constituents. As a case study, ß-phellandrene synthase (PHLS) genes from different plants were heterologously expressed in Synechocystis. Genomic integration of individual PHLS-encoding sequences endowed Synechocystis with constitutive monoterpene hydrocarbons generation, occurring concomitant with photosynthesis and cell growth. Specifically, the ß-phellandrene synthase from Lavandula angustifolia (lavender), Solanum lycopersicum (tomato), Pinus banksiana (pine), Picea sitchensis (Sitka spruce) and Abies grandis (grand fir) were active in Synechocystis transformants but, instead of a single product, they generated a blend of terpene hydrocarbons comprising ß-phellandrene, α-phellandrene, ß-myrcene, ß-pinene, and δ-carene with variable percentage ratios ranging from < 10 to > 90% in different product combinations and proportions. Our results suggested that PHLS enzyme conformation and function depends on the cytosolic environment in which they reside, with the biochemical properties of the latter causing catalytic deviations from the products naturally observed in the corresponding gene-encoding plants, giving rise to the terpene hydrocarbon blends described in this work. These findings may have commercial application in the generation of designer essential oil blends and will further assist the development of heterologous cyanobacterial platforms for the generation of desired monoterpene hydrocarbon products.

Bornyl-diphosphate synthase from Lavandula angustifolia: A major monoterpene synthase involved in essential oil quality.

Lavender essential oils (EOs) of higher quality are produced by a few Lavandula angustifolia cultivars and mainly used in the perfume industry. Undesirable compounds such as camphor and borneol are also synthesized by lavender leading to a depreciated EO. Here, we report the cloning of bornyl diphosphate synthase of lavender (LaBPPS), an enzyme that catalyzes the production of bornyl diphosphate (BPP) and then by-products such as borneol or camphor, from an EST library. Compared to the BPPS of Salvia officinalis, the functional characterization of LaBPPS showed several differences in amino acid sequence, and the distribution of catalyzed products. Molecular modeling of the enzyme's active site suggests that the carbocation intermediates are more stable in LaBPPS than in SoBPPS leading probably to a lower efficiency of LaBPPS to convert GPP into BPP. Quantitative RT-PCR performed from leaves and flowers at different development stages of L. angustifolia samples show a clear correlation between transcript level of LaBPPS and accumulation of borneol/camphor, suggesting that LaBPPS is mainly responsible of in vivo biosynthesis of borneol/camphor in fine lavender. A phylogenetic analysis of terpene synthases (TPS) pointed out the basal position of LaBPPS in the TPSb clade, suggesting that LaBPPS could be an ancestor of others lavender TPSb. Finally, borneol could be one of the first monoterpenes to be synthesized in the Lavandula subgenus. Knowledge gained from these experiments will facilitate future studies to improve the lavender oils through metabolic engineering or plant breeding. Accession numbers: LaBPPS: KM015221.

Functional characterization of terpene synthases and chemotypic variation in three lavender species of section Stoechas.

Lavandula pedunculata (Mill.) Cav. subsp. lusitanica, Lavandula stoechas L. subsp. stoechas and Lavandula viridis l'Hér. are three lavender taxa that belong to the botanical section Stoechas and are widely used as aromatherapy, culinary herb or folk medicine in many Mediterranean regions. The analysis of their bioactive volatile constituents revealed the presence of 124 substances, the most abundant being the bicyclic monoterpenes fenchone, camphor and 1,8-cineole that give these three species their respective chemotypes. Most noteworthy was fenchone which, with its reduced form fenchol, made 48% of the total volatile constituents of L. pedunculata while present at 2.9% in L. stoechas and undetectable in L. viridis. In order to provide a molecular explanation to the differences in volatile compounds of these three species, two monoterpene synthases (monoTPS) and one sesquiterpene synthase (sesquiTPS) were cloned in L. pedunculata and functionally characterized as fenchol synthase (LpFENS), α-pinene synthase (LpPINS) and germacrene A synthase (LpGEAS). The two other lavender species contained a single orthologous gene for each of these three classes of TPS with similar enzyme product specificities. Expression profiles of FENS and PINS genes matched the accumulation profile of the enzyme products unlike GEAS. This study provides one of the rare documented cases of chemotype modification during plant speciation via changes in the level of plant TPS gene expression, and not functionality.

Deoxyxylulose 5-phosphate reductoisomerase is not a rate-determining enzyme for essential oil production in spike lavender.

Spike lavender (Lavandula latifolia) is an economically important aromatic plant producing essential oils, whose components (mostly monoterpenes) are mainly synthesized through the plastidial methylerythritol 4-phosphate (MEP) pathway. 1-Deoxy-D-xylulose-5-phosphate (DXP) synthase (DXS), that catalyzes the first step of the MEP pathway, plays a crucial role in monoterpene precursors biosynthesis in spike lavender. To date, however, it is not known whether the DXP reductoisomerase (DXR), that catalyzes the conversion of DXP into MEP, is also a rate-limiting enzyme for the biosynthesis of monoterpenes in spike lavender. To investigate it, we generated transgenic spike lavender plants constitutively expressing the Arabidopsis thaliana DXR gene. Although two out of the seven transgenic T0 plants analyzed accumulated more essential oils than the controls, this is hardly imputable to the DXR transgene effect since a clear correlation between transcript accumulation and monoterpene production could not be established. Furthermore, these increased essential oil phenotypes were not maintained in their respective T1 progenies. Similar results were obtained when total chlorophyll and carotenoid content in both T0 transgenic plants and their progenies were analyzed. Our results then demonstrate that DXR enzyme does not play a crucial role in the synthesis of plastidial monoterpene precursors, suggesting that the control flux of the MEP pathway in spike lavender is primarily exerted by the DXS enzyme.

Molecular cloning and functional characterization of borneol dehydrogenase from the glandular trichomes of Lavandula x intermedia.

Several varieties of Lavandula x intermedia (lavandins) are cultivated for their essential oils (EOs) for use in cosmetic, hygiene and personal care products. These EOs are mainly constituted of monoterpenes including camphor, which contributes an off odor reducing the olfactory appeal of the oil. We have recently constructed a cDNA library from the glandular trichomes (the sites of EO synthesis) of L. x intermedia plants. Here, we describe the cloning of a borneol dehydrogenase cDNA (LiBDH) from this library. The 780 bp open reading frame of the cDNA encoded a 259 amino acid short chain alcohol dehydrogenase with a predicted molecular mass of ca. 27.5 kDa. The recombinant LiBDH was expressed in Escherichia coli, purified by Ni-NTA agarose affinity chromatography, and functionally characterized in vitro. The bacterially produced enzyme specifically converted borneol to camphor as the only product with K(m) and k(cat) values of 53 µM and 4.0 × 10(-4) s(-1), respectively. The LiBDH transcripts were specifically expressed in glandular trichomes of mature flowers indicating that like other Lavandula monoterpene synthases the expression of this gene is regulated in a tissue-specific manner. The cloning of LiBDH has far reaching implications in improving the quality of Lavandula EOs through metabolic engineering.

Differential accumulation of volatile terpene and terpene synthase mRNAs during lavender (Lavandula angustifolia and L. x intermedia) inflorescence development.

Despite the commercial importance of Lavandula angustifolia Mill. and L. x intermedia Emeric ex Loisel floral essential oils (EOs), no information is currently available on potential changes in individual volatile organic compound (VOC) content during inflorescence development. Calyces were found to be the main sites of VOC accumulation. The 20 most abundant VOCs could be separated into three sub-groups according to their patterns of change in concentration The three groups of VOCs sequentially dominated the global scent bouquet of inflorescences, the transition between the first and second groups occurring around the opening of the first flower of the inflorescence and the one between the second and third groups at the start of seed set. Changes in calyx VOC accumulation were linked to the developmental stage of individual flowers. Leaves accumulated a smaller number of VOCs which were a subset of those seen in preflowering inflorescences. Their nature and content remained constant during the growing season. Quantitative real time polymerase chain reaction assessments of the expression of two terpene synthase (TPS) genes, LaLIMS and LaLINS, revealed similar trends between their patterns of expression and those of their VOC products. Molecular and chemical analyses suggest that changes in TPS expression occur during lavender inflorescence development and lead to changes in EO composition. Both molecular data and terpene analysis support the findings that changes in biosynthesis of terpene occurred during inflorescence development.