Field of the invention

The present invention relates to a process for producing a monoterpene and to a cyanobacterial cell for the production of a monoterpene.

Background of invention

Isoprenoids (commonly known as terpenes) are comprised of diverse group of molecules found in all organisms, where they carry out important biological functions. For example, as quinones in electron transport, as components of membranes (prenyl-lipids in archaebacteria, sterols in eukaryotes), in subcellular targeting (prenylation of proteins), in hormone signaling in mammals (steroids), as photosynthetic pigments (carotenoids) and as semiochemical secondary metabolites in plants (monoterpenes, sesquiterpenes, diterpenes). They are the most abundant and structurally diverse natural products with more than 55,000 identified in bacteria, archaea and eukaryotes. Some are also commercially important as pharmaceutical ingredients, flavors, fragrances, cosmetic ingredients and also have been explored as precursors to alternative fuel.

However, many such compounds are present in nature in very small quantities or low yielding from their natural sources to be used widely for above applications. Moreover, most of the natural sources are not amenable to large-scale cultivation necessary to produce large quantities. Furthermore, the extractions from natural source involve the use of toxic organic chemicals necessitating the need for complicated handling and disposal procedures. Microbial fermentations involving genetically modified yeast or bacteria have recently gained lots of attention as a potential source for terpenes. This has been described in patent applications: 1) US 20110229958: Host Cells for Production of Isoprenoid Compounds; 2) US 20100112672: Production of isoprenoids and isoprenoid precursors and 3) EP 1392824: Improved isoprenoid production. However, standard fermentation processes require a carbon source, for which plants and algal species are employed to reduce carbon dioxide via photosynthesis (using the energy of the sun) to the level of sugars and cell material. After harvesting, these end products are converted to ethanol by yeast fermentation (in the case of crops) or converted chemically to biofuels (in the case of algae). The overall energy conservation of these methods is highly inefficient and therefore demands large surface areas. In addition, the crop processes are rather labor-intensive, are demanding with respect to water consumption and affect food stock prices with adverse consequences for food supplies. A more remotely similar process is based on the conversion of solar energy into hydrogen. Also this process suffers from a severely decreased efficiency.

U.S. Pat. No. 6,699,696 describes a process of producing ethanol by feeding carbon dioxide to a cyanobacterial cell, especially a Synechococcus comprising a nucleic acid molecule encoding an enzyme enabling the cell to convert pyruvate into ethanol, subjecting said cyanobacterial cell to sun energy and collecting ethanol. This system has several drawbacks among others the expression system used is temperature sensitive which demands to adapt the production system for such regulation.

WO 2009/078712 describes a process of producing ethanol, propanol, butanol, acetone, 1,3- propanediol, ethylene or D-lactate and where appropriate intermediary compounds in the pathway leading to any of these organic compounds. The process is carried out by feeding carbon dioxide to a culture of cyanobacterial cells and subjecting the culture to light, wherein the cells are capable of expressing a nucleic acid molecule under the control of a regulatory system which responds to a change in the concentration of a nutrient in the culture which confers on the cell the ability to convert a glycolytic intermediate into the above-mentioned organic compounds and/or into intermediary compounds.

Similar approaches for the production of some terpenes in the cyanobacteria Anabaena and Synechococcus have recently been suggested in "Genetically engineered cyanobacteria WO 2012116345 A2" and in "Methods for Isoprene and Pinene Production in Cyanobacteria US 20140030785 Al".

All isoprenoids are derived from five-carbon isoprene units and are synthesized from two universal C5 building blocks: isopentenyl diphosphate (IPP) and its isomer dimethylallyl diphosphate (DMAPP), which in turn can be produced by two distinct well studied routes: the mevalonate (MVA) pathway (see Figure 1) or the l-deoxy-D-xylulose-5-phosphate (DXB) pathway (the DXB pathway is also referred to as the MEP pathway based on another intermediate "2-C-methyl-D-erythritol-4-phosphate"); see Figure 2. Both pathways are distributed throughout nature. The MVA pathway is present in all eukaryotes (mammals, fungi etc.) and all archaea. Some Gram positive bacteria like Staphylococcus, Streptococcus, Enterococcus, Lactococcus, Lactobacillus, and Leuconostoc, and some Gram negative bacteria like Myxobacteria, also use the MVA pathway, whereas most other bacteria, including cyanobacteria, synthesize IPP and DMAPP using the MEP pathway. In plants, both pathways are present. The MEP pathway functions in the plastids whereas the MVA pathways functions in the cytosol. Isoprenoids are classified into groups according to the number of carbons in their skeletal structure: hemiterpenes (C5), monoterpenes (CIO), sesquiterpenes (CI 5), diterpenes (C20), triterpenes (C30) and tetraterpenes (C40); see Table 1.

The biosynthesis of isoprenoids can thus be divided into three major steps: 1) formation of the metabolic intermediates IPP and DMAPP 2) the linear condensation of the isoprene units to form polyprenyl diphosphates precursors of different lengths and 3) cyclization, modification and other reactions by which the polyprenyl diphosphates are converted to a variety of terpene end-products. Furthermore, modification (often oxidative) such as addition of functional groups such as carbonyl, ketone, hydroxyl, aldehyde and peroxide, leads to further diversity and such new compounds are often referred to as terpenoids. Terpenes and terpenoids are together referred to as isoprenoids.

Table 1. Classification of Terpenes

Monoterpenes have been known for several centuries as components of the fragrant oils obtained from leaves, flowers and fruits. Monoterpenes, with sesquiterpenes, are the main constituents of essential oils. While a few, such as camphor, occur in a near pure form, most occur as complex mixtures, often of isomers that are difficult to separate. These terpenes in essential oils have numerous actions, such as allelochemical functions between plants and between plants and predators. A role in wound healing has also been observed. Although the production of some terpenes in cyanobacteria from CO ₂ has recently been reported, there is still a need for an improved process for the biosynthesis of monoterpenes, preferably without the need of expensive or complicated starting materials, and/or the use of toxic organic chemicals necessitating the need for complicated handling and disposal procedure.

Description of the invention

In brief, the inventors of the present invention have arrived at a scalable process for the production of a monoterpene in cyanobacteria. The invention combines metabolic properties of photoautotrophic and chemotrophic microorganisms and is based on the employment of recombinant oxyphototrophs with high rates of conversion of Calvin cycle intermediates to a desired end product. One advantage resides in the fact that its core chemical reactions use carbon dioxide as the sole carbon-containing precursor and light (preferably sunlight), as the sole energy source, to drive carbon dioxide reduction. Moreover, the cyanobacterial cell factory is more suitable for production of a monoterpene than other microorganism used in fermentation processes such as E.coli and yeasts, since the abundantly available co-factor in the cyanobacterial cell is NADPH, rather than NADH in most chemotrophic organisms used for fermentation. NADPH is produced directly from photosynthesis and is also used in the fixing of CO ₂ via the Calvin-Benson-Bensham cycle. NADPH is abundant in phototrophic microorganisms like cyanobacteria. NADPH is mostly generated in - heterotrophic microorganisms via the pentose-phosphate cycle and its pool size is then relatively small compared to NADH. As most industrially relevant chemicals are produced by NADPH consuming pathways, the NADPH pools in photosynthetic organisms provide a strong driving force for production of chemicals. Production in a cyanobacterial cell according to the invention can be controlled by a nutrient- or light-sensitive promoter. Using a nutrient- sensitive promoter, production can be controlled by a medium component and can start at the most appropriate time, such as at the highest possible cell density. By using a light-mediated promoter, production can be controlled by light intensity. Whereas in current production processes for biochemicals, organisms are substrate (e.g., crops in ethanol production) or product (e.g., microalgae as biodiesel), herein microorganisms are used as highly specialized catalysts for the conversion of carbon dioxide as a substrate to a valuable end product. These catalysts can be subjected to further optimization strategies through physical- and chemical systems-biology approaches. The biochemical background of cyanobacterial cells for the production of valuable compounds is more extensively described in WO 2009/078712, especially in example 1. The various aspects of the present invention are more extensively described here below.

In a first aspect, the present invention relates to a cyanobacterial cell capable of expressing, preferably expressing, at least one functional enzyme selected from the group of enzymes consisting of a geranyl diphosphate synthase (GPPS) and a monoterpene synthase (MTS). Said cyanobacterial cell is herein further referred to as a cyanobacterial cell according to the present invention. The cyanobacterial cell according to the present invention is preferably capable of producing a monoterpene selected from the group consisting of: limonene, geranyl pyrophosphate, eucalyptol, pinene, menthol, camphor, linalool, citral, γ-terpinene, Ε-β- ocimene, terpineol, myrcene, citronellol, carvone and geraniol. More preferably, the monoterpene is limonene or linalool; most preferably, the monoterpene is limonene.

The term "functional enzyme" is herein preferably defined in the context of a geranyldiphosphate synthase (GPPS) as an enzyme that catalyzes the condensation of two C5 co-substrates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), to produce geranyl diphosphate (GPP) the precursor of all monoterpenes. Despite the multiple functions of monoterpenes, which are found most commonly in plants and insects where they act as essential oils and pheromones, respectively, our understanding of their biosynthesis remains limited. GPPS genes have been characterized from only a handful of plant species and, so far, only one study has reported on the existence of a GPPS in an insect. Unlike plants, microorganisms do not carry a specific GPPS. In yeast, both GPP and farnesyl diphosphate (FPP) synthase activities are shared by one single enzyme Farnesyl diphosphate synthase (FPPS) and these can consequently not be separated. However, dedicated GPPS enzymes have been reported in literature from plants. The term "functional enzyme" is herein preferably defined in the context of a monoterpene synthase as an enzyme able to convert the acyclic GPP produces by the GPPS enzyme into a variety of cyclic and acyclic forms. It may also refer to a mutant of a prenyl phoshphate synthase enzyme, more preferably a Farnesyl phosphate synthase enzyme, where in one or more amino acids have been mutated, such that the enzyme makes GPP as the preferred product.

A preferred cyanobacterial cell according to the invention is capable of expressing, preferably expressing, at least one functional enzyme selected from the group consisting of enzymes having ability to condense IPP and DMAPP to GPP. The enzyme may be native or may be heterologous to the cyanobacterial cell according to the present invention. The at least one functional enzyme is preferably selected from the group consisting of GPPS from Abies grandis, Picea abies, Arabidopsis thaliana and Saccharomyces cerevisiae. More preferably, the GPPS is from Abies grandis. The enzyme may be a mutant of a prenyphosphate synthase enzyme, with specificity for forming GPP. Further, the functional enzyme may be an N- terminal truncated version of the original protein, while substantially maintaining its monoterpene synthase activity.

In a cyanobacterial cell according to the present invention, at least one functional enzyme is preferably selected from the group consisting of monoterpene synthases, which are enzymes having the ability of converting GPP to various cyclic or acyclic monoterpenes. The at least one functional enzyme may be native or may be heterologous to the cyanobacterial cell and is preferably selected from the group consisting of monoterpene synthases from Mentha spicata, Mentha Canadensis, Abies grandis, Citrus sinensis, Mentha citrata, Citrus unshiu, Thymus caespititius, Origanum vulgare and Lotus japonicas. More preferably, the monoterpene synthase is from Mentha spicata or from Mentha citrata. Further, the functional enzyme may be an N-terminal truncated version of the original protein, while substantially maintaining its monoterpene synthase activity. Preferably, at least two functional enzymes are heterologous to the cyanobacterial cell.

Preferably, a cynabacterial cell according to the present invention is capable of expressing, preferably expressing, at least one functional enzyme selected from the group of enzymes consisting of a Geranyl diphosphate synthase (GPPS) and a monoterpenes synthase (MTS), wherein the at least one functional enzyme is selected from the group consisting of GPPS from Abies grandis, Picea abies, Arabidopsis thaliana, and Saccharomyces cerevisiae; and/or wherein the at least one functional enzyme is selected from the group consisting of monoterpene synthases from Mentha spicata, Mentha Canadensis, Abies grandis, and Citrus sinensis, Mentha citrata, Citrus unshiu, Thymus caespititius, Origanum vulgare and Lotus japonicas. More preferably, the GPPS is from Abies grandis and the monoterpene synthase is from Mentha spicata or the GPPS is from Abies grandis and the monoterpene synthase is from Mentha citrata. preferred cyanobacterial cell according to the present invention is capable of producing, preferably producing, a monoterpene, preferably a monoterpene selected from the group consisting of: limonene, geranyl pyrophosphate, eucalyptol, pinene, menthol, camphor, linalool, citral, γ-terpinene, Ε-β-ocimene, terpineol, myrcene, citronellol, carvone and geraniol. More preferably, the monoterpene is limonene or linalool; most preferably, the monoterpene is limonene. Preferably, a cyanobacterial cell according to the present invention is capable of producing, preferably producing, at least two terpenes, more preferably at least two monoterpenes. In a cyanobacterial cell according to the present invention, the at least one functional enzyme preferably comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2, SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 8, SEQ ID NO: 10, SEQ ID NO: 12, SEQ ID NO: 14, SEQ ID NO: 16, SEQ ID NO: 18, SEQ ID NO: 20, SEQ ID NO: 22, SEQ ID NO: 24, SEQ ID NO: 26 and SEQ ID NO: 28. More preferably, the at least one functional enzyme comprises or consists of a polypeptide that has an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 6 or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 18. Even more preferably, the at least one functional enzyme are at least two functional enzymes comprising or consisting of two polypeptides that have an amino acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 2 and SEQ ID NO: 6, or with SEQ ID NO: 2 and SEQ ID NO: 18, respectively.

In a cyanobacterial cell according to the present invention, the at least one functional enzyme is preferably encoded by a polynucleotide that has a nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91 %, 92%, 93%, 94%, 95%, 96%, 91%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 , SEQ ID NO: 3, SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 9, SEQ ID NO: 11 , SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17, SEQ ID NO: 19, SEQ ID NO: 21, SEQ ID NO: 23, SEQ ID NO: 25 and SEQ ID NO: 27. More preferably, the at least one functional enzyme is encoded by a polynucleotide that has a nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 5 or from the group consisting of SEQ ID NO: 1 and SEQ ID NO: 17. Even more preferably, the at least one functional enzyme are at least two functional enzymes that are encoded by a polynucleotide that has a nucleic acid sequence with at least 30%, 35%, 40%, 45%, 50%, 55%, 60%, 65%, 70%, 75%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or 100% sequence identity with SEQ ID NO: 1 and SEQ ID NO: 5, or with SEQ ID NO: 1 and SEQ ID NO: 17, respectively.

In the context of all embodiments of the present invention, the terms "a cyanobacterium", "a cyanobacterium cell" and "a cyanobacterial cell" are used interchangeably and refer to a blue- green algae, an oxygenic photosynthetic unicellular microorganism. Examples of cyanobacteria include the genera Aphanocapsa, Anabaena, Nostoc, Oscillatoria, Synechococcus, Synechocystis, Gloeocapsa, Agmenellum, Scytonema, Mastigocladus, Arthrosprira, and aplo siphon. A preferred order of cyanobacteria is Chroococcales. A more preferred cyanobacterium genus is Synechocystis. Synechocystis is well-studied, genetically well characterized and it does not require special media components for growth. Most importantly, it can grow mixotrophically, which means that it can grow on glucose in the absence of light. This makes Synechocystis robust for industrial applications. A more preferred strain of this genus is a Synechocystis PCC 6803 species. Even more preferably, the Synechocystis is a Pasteur Culture Collection (PCC) 6803 Synechocystis, which is a publicly available strain via ATCC for example. PCC 6803 has been stored at ATCC under ATCC27184. The phototrophic Synechocystis PCC 6803 is a fast growing cyanobacterium with no specific nutritional demands. Its physiological traits are well-documented: it is able to survive and grow in a wide range of conditions. For example, Synechocystis sp. PCC 6803 can grow in the absence of photosynthesis if a suitable fixed-carbon source such as glucose is provided. Perhaps most significantly, Synechocystis sp. PCC 6803 was the first photosynthetic organism for which the entire genome sequence was determined (available via the internet world wide web at kazusa.or.jp/cyano/cyano). In addition, an efficient gene deletion strategy (Shestakov SV et al., 2002; and Nakamura Y et al, 1999) is available for Synechocystis sp. PCC 6803, and this organism is furthermore easily transformable, also via natural transformation and homologous recombination (Grigirieva GA et al., 1982). In the context of all embodiments according to the invention, the cyanobacterium is preferably not from the genus Anabaena.

"Capable of producing monoterpene" preferably means herein that detectable amounts of monoterpene can be detected in a culture of a cyanobacterial cell according to the present invention cultured, under conditions conducive to the production of monoterpene, preferably in the presence of light and dissolved carbon dioxide and/or bicarbonate ions, during a preferred interval using a suitable assay for detecting monoterpenes. Detection may be in the culture broth (i.e. the medium including the cyanobacterial cell), in the medium or supernatant of the broth, in the cyanobacterial cell itself, and/or in the headspace of the culturing device. A preferred concentration of said dissolved carbon dioxide and/or bicarbonate ions is, the natural occurring concentration at neutral to alkaline conditions (pH 7 to 9) being approximately 1 mM. This is equivalent to 0.035% of carbon dioxide in ambient air. A more preferred concentration of carbon dioxide and/or bicarbonate ions is higher than this natural occurring concentration. Preferably, the concentration of bicarbonate ions is at least 0.5mM, 0.6mM, 0.7mM, 0.8mM, 0.9mM, lmM, 2mM, 5mM, lOmM, 15mM, 20mM, 25mM, 30mM, 35mM, 40mM, 45mM, 50mM, 60mM, 70mM, 80mM, 90mM or lOOmM. A preferred method to increase the carbon dioxide and/or bicarbonate ion concentrations in solution is by enrichment with carbon dioxide, preferably waste carbon dioxide from industrial plants, sparged into the culture broth. The concentration of carbon dioxide is preferably increased to at least 0.04%, 0.05%, 0.1%, 0.15%, 0.2%, 0.5%, 1%, 1.5%, 2%, 2.5%, 3%, 3.5%, 4%, 4.5%, or 5%.

Preferably, the monoterpene is thus detected in a cyanobacterial cell according to the present invention and/or in its culture broth or headspace, wherein said cyanobacterial cell is cultured under conditions conducive to the production of a monoterpene, preferably the conditions include culturing in the presence of sunlight and carbon dioxide during at least 1 day using a given assay for the intermediary compound.

The monoterpene produced within the cyanobacterial cell according to the invention may spontaneously diffuse into the culture broth or the headspace or both. Assays for the detection of a monoterpene are, but are not limited to, High Performance Liquid Chromatography (HPLC), Gas Chromatography (GC), Gas Chromatography-Mass Spectrometry (GC-MS), or Liquid Chromatography-Mass Spectrometry (LC-MS). A preferred assay for the detection of a monoterpene is Gas Chromatography-Mass Spectrometry (GC-MS). A detectable amount fof a monoterpene is preferably at least 1 ng/ml culture broth, 1 ng/gram dry weight of the culture broth or 1 ng/ml of culture supernatant which are preferably obtained under the culture conditions depicted here above and preferably using the above assay. Preferably, the amount is depicted as weight of product (ng, μg or mg)/gram dry weight of culture broth. Preferably, the amount is at least 2 ng, 3 ng, 4 ng, 5 ng, 6 ng, 7 ng, 8 ng, 9 ng, 10 ng, 15 ng, 20 ng, 25 ng, 30 ng, 35 ng, 40 ng, 45 ng, 50 ng, lOOng, 200ng, 300ng, 400ng, 500ng, 1 μg, 2 μg, 3 μg, 5 μg, 10 μg, 50 μg, 100 μg, 200 μg, 300 μg, 400 μg, 500 μg. lmg, 2 mg, 3 mg, 4 mg, 5 mg, 10 mg, or at least 100 mg/gram dry weight. Such amount is preferably obtained in at most 2 weeks, 1 week, 6 days, 5 days, 4 days, 3 days, 2 days, 1 day, 15 hours, 10 hours, 5 hours, 4 hours, 3 hours, 2 hours or 1 hour of culture. Preferably, a cyanobacterial cell according to the present invention comprises at least one nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one of the at least one functional enzyme as defined here above. Accordingly, a preferred cyanobacterial cell according to the invention comprises at least one nucleic acid molecule comprising or consisting of a polynucleotide encoding at least one of the at least one functional enzyme as defined here above.

The at least one functional enzyme as defined here above is encoded by a polynucleotide. In all embodiments according to the invention, each encoding polynucleotide may be present on a separate nucleic acid molecule. Alternatively, the encoding polynucleotides may be present on a single nucleic acid molecule.

A preferred cyanobacterial cell according to the invention is a cyanobacterial cell wherein the at least one functional enzyme is encoded by a nucleic acid molecule comprising or consisting of a polynucleotide wherein said nucleic acid molecule is preferably present in the cyanobacterial cell as an episomal entity, preferably said episomal entity is a plasmid, more preferably a self-replicating plasmid. The episomal entity and plasmid can be any episomal entity and plasmid known to the person skilled in the art or can be based on any episomal entity and plasmid known to the person skilled in the art and modified to comprise any nucleic acid and/or polynucleotide described herein.

Another preferred cyanobacterial cell according to the invention is a cyanobacterial cell wherein the at least one functional enzyme is encoded by a nucleic acid molecule comprising or consisting of a polynucleotide wherein said nucleic acid molecule is preferably integrated in the cyanobacterial genome, preferably via homologous recombination.

A cyanobacterial cell according to the present invention may comprise a single but preferably comprises multiple copies of each nucleic acid molecule.

A preferred cyanobacterial cell according to the present invention is a cyanobacterial cell, wherein a polynucleotide encoding the at least one functional enzyme is under control of a regulatory system which responds to a change in the concentration of a nutrient when culturing said cyanobacterial cell.

A promoter that may be used for the expression of a polynucleotide encoding the at least one functional enzyme may be foreign to the polynucleotide, i.e. a promoter that is heterologous to the polynucleotide encoding the at least one functional enzyme to which it is operably linked. Although a promoter preferably is heterologous to the polynucleotide to which it is operably linked, it is also possible that a promoter is native to the cyanobacterial cell according to the present invention. Preferably, a heterologous (to the nucleotide sequence) promoter is capable of producing a higher steady state level of a transcript comprising a coding sequence (or is capable of producing more transcript molecules, i.e. mRNA molecules, per unit of time) than is a promoter that is native to the coding sequence. A suitable promoter in this context includes both constitutive and an inducible natural promoters as well as engineered promoters. A promoter used in a cyanobacterial cell according to the present invention may be modified, if desired, to affect its control characteristics. A preferred promoter for constitutive expression is a Ptrc, as is further outlined below in the next paragraph.

The Ptrc promoter is an artificial promoter, which is constructed as a chimera of the E. coli trp operon and lacUV5 promoters (Brosius et al, J Biol Chem 1985). The promoter is thus regulated by the Lac repressor, Lacl. In Synechocystis, the Lacl regulated repression and induction does not function efficiently, but the Ptrc promoter does show high constitutive expression levels in the absence of Lacl (Huang H-H, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38:2577-2593).

The cyanobacterial cell according to the present invention can conveniently be used for the production of a monoterpene.

Accordingly, in a second aspect, the present invention relates to a process for producing a monoterpene comprising culturing a cyanobacterial cell according to the present invention, preferably a cyanobacterial cell as defined in the first aspect of the present invention, under conditions conducive to the production of a monoterpene and, optionally, isolating and/or purifying the monoterpene from the culture broth and/or its headspace. Said process is herein further referred to as a process according to the present invention.

Preferably, a process according to the present invention for producing a monoterpene comprises culturing a cyanobacterial cell according to the present invention, preferably a cyanobacterial cell as defined in the first aspect of the present invention, wherein the culture conditions comprise feeding carbon dioxide to the culture and/or subjecting the culture to light.

Usually, a process is started with a culture (also named culture or culture broth) of cyanobacterial cells having an optical density measured at 730 nm of approximately 0.2 to 2.0 (OD730 = 0.2 to 2) as measured in any conventional spectrophotometer with a measuring path length of 1 cm. Usually, the cell number in the culture doubles every 20 hours. A preferred process takes place in a tank with a depth of 30-50 cm exposed to sun light. Preferably, the light used is natural.

A preferred natural light is daylight, i.e. sunlight. Daylight (or sunlight) may have an intensity ranged between approximately 500 and approximately 1500 μΕίηβΐείη/ηι 2/s. In another embodiment, the light used is artificial. Such artificial light may have an intensity ranged between approximately 70 and approximately 800 μΕήΐδΐείη/ηι 2/s. Preferably, the cells are continuously under the light conditions as specified herein. However, the cells may also be exposed to high light intensities (such as e.g. daylight/sunlight) as defined elsewhere herein for a certain amount of time, after which the cells are exposed to a lower light intensity as defined elsewhere herein for a certain amount of time, and optionally this cycle is repeated. In a preferred embodiment, the cycle is the day/night cycle.

In a preferred process, the monoterpene is separated from the culture broth. This may be realized continuously with the production process or subsequently to it. Separation may be based on any separation method known to the person skilled in the art.

In a preferred process according to the present invention and in a preferred cyanobacterial cell according to the invention, the produced monoterpene is selected from the group consisting of: limonene, geranyl pyrophosphate, eucalyptol, pinene, menthol, camphor, linalool, citral, γ-terpinene, Ε-β-ocimene, terpineol, myrcene, citronellol, carvone and geraniol. More preferably, the monoterpene is limonene, linalool, γ-terpinene or Ε-β- ocimene; even more preferably limonene or linalool; most preferably the monoterpene is limonene. In a further preferred process according to the present invention and in a preferred cyanobacterial cell according to the present invention, at least two terpenes are produced, more preferably at least two monoterpenes as described herein are produced.

The monoterpene produced by a cyanobacterial cell according to the invention and by a process according to the invention have specific properties. Accordingly, there is provided for a monoterpene obtainable by a cyanobacterial cell according to the invention and by a process according to the invention. Prefereably, such monoterpene is a monoterpene selected from the group consisting of limonene, geranyl pyrophosphate, eucalyptol, pinene, menthol, camphor, linalool, γ-terpinene, Ε-β-oc imene, citral, terpineol, myrcene, citronellol, carvone and geraniol. More preferably, such monoterpene is a monoterpene selected from the group consisting of limonene, linalool, γ-terpinene and Ε-β-ocimene.

A monoterpene according to the invention can conveniently be used in a product. Accordingly, there is provided for a pharmaceutical composition, a fuel composition, a flavor composition, a flagrance composition or a cosmetic composition comprising a monoterpene obtainable by a cyanobacterial cell according to the invention and by a process according to the invention. Preferably, such composition comprises a monoterpene selected from the group consisting of limonene, geranyl pyrophosphate, eucalyptol, pinene, menthol, camphor, linalool, γ-terpinene, Ε-β-ocimene, citral, terpineol, myrcene, citronellol, carvone and geraniol. More preferably, such composition comprises a monoterpene selected from the group consisting of limonene, linalool, γ-terpinene and Ε-β-ocimene.

Definitions

"Sequence identity" or "identity" in the context of amino acid- or nucleic acid-sequence is herein defined as a relationship between two or more amino acid (peptide, polypeptide, or protein) sequences or two or more nucleic acid (nucleotide, polynucleotide) sequences, as determined by comparing the sequences. In the art, "identity" also means the degree of sequence relatedness between amino acid or nucleotide sequences, as the case may be, as determined by the match between strings of such sequences. Within the present invention, sequence identity with a particular sequence preferably means sequence identity over the entire length of said particular polypeptide or polynucleotide sequence. The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors.

"Similarity" between two amino acid sequences is determined by comparing the amino acid sequence and its conserved amino acid substitutes of one peptide or polypeptide to the sequence of a second peptide or polypeptide. In a preferred embodiment, identity or similarity is calculated over the whole SEQ ID NO as identified herein. "Identity" and "similarity" can be readily calculated by known methods, including but not limited to those described in Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988; Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993; Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994; Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987; and Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991; and Carillo, H., and Lipman, D., SIAM J. Applied Math., 48:1073 (1988).

Preferred methods to determine identity are designed to give the largest match between the sequences tested. Methods to determine identity and similarity are codified in publicly available computer programs. Preferred computer program methods to determine identity and similarity between two sequences include e.g. the GCG program package (Devereux, J., et al., Nucleic Acids Research 12 (1): 387 (1984)), BestFit, BLASTP, BLASTN, and FASTA (Altschul, S. F. et al, J. Mol. Biol. 215:403-410 (1990). The BLAST X program is publicly available from NCBI and other sources (BLAST Manual, Altschul, S., et al, NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al., J. Mol. Biol. 215:403-410 (1990). The well- known Smith Waterman algorithm may also be used to determine identity.

Preferred parameters for polypeptide sequence comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: BLOSSUM62 from Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919 (1992); Gap Penalty: 12; and Gap Length Penalty: 4. A program useful with these parameters is publicly available as the "Ogap" program from Genetics Computer Group, located in Madison, WI. The aforementioned parameters are the default parameters for amino acid comparisons (along with no penalty for end gaps).

Preferred parameters for nucleic acid comparison include the following: Algorithm: Needleman and Wunsch, J. Mol. Biol. 48:443-453 (1970); Comparison matrix: matches=+10, mismatch=0; Gap Penalty: 50; Gap Length Penalty: 3. Available as the Gap program from Genetics Computer Group, located in Madison, Wis. Given above are the default parameters for nucleic acid comparisons.

Optionally, in determining the degree of amino acid similarity, the skilled person may also take into account so-called "conservative" amino acid substitutions, as will be clear to the skilled person. Conservative amino acid substitutions refer to the interchangeability of residues having similar side chains. For example, a group of amino acids having aliphatic side chains is glycine, alanine, valine, leucine, and isoleucine; a group of amino acids having aliphatic-hydroxyl side chains is serine and threonine; a group of amino acids having amide- containing side chains is asparagine and glutamine; a group of amino acids having aromatic side chains is phenylalanine, tyrosine, and tryptophan; a group of amino acids having basic side chains is lysine, arginine, and histidine; and a group of amino acids having sulphur- containing side chains is cysteine and methionine. Preferred conservative amino acids substitution groups are: valine-leucine-isoleucine, phenylalanine-tyrosine, lysine-arginine, alanine-valine, and asparagine-glutamine. Substitutional variants of the amino acid sequence disclosed herein are those in which at least one residue in the disclosed sequences has been removed and a different residue inserted in its place. Preferably, the amino acid change is conservative. Preferred conservative substitutions for each of the naturally occurring amino acids are as follows: Ala to ser; Arg to lys; Asn to gin or his; Asp to glu; Cys to ser or ala; Gin to asn; Glu to asp; Gly to pro; His to asn or gin; He to leu or val; Leu to ile or val; Lys to arg; gin or glu; Met to leu or ile; Phe to met, leu or tyr; Ser to thr; Thr to ser; Trp to tyr; Tyr to trp or phe; and, Val to ile or leu.

A polynucleotide is represented by a nucleotide sequence. A polypeptide is represented by an amino acid sequence. A nnucleic acid construct is defined as a polynucleotide which is isolated from a naturally occurring gene or which has been modified to contain segments of polynucleotides which are combined or juxtaposed in a manner which would not otherwise exist in nature. Optionally, a polynucleotide present in a nucleic acid construct is operably linked to one or more control sequences, which direct the production or expression of said peptide or polypeptide in a cell or in a subject.

Polynucleotides described herein may be native or may be codon optimized. Codon optimization adapts the codon usage for an encoded polypeptide towards the codon bias of the organism where the polypeptide is to be produced in. Codon optimization generally helps to increase the production level of the encoded polypeptide in the host cell, such as in the preferred host herein: Cyanobacterium Synechocystis . Many algorithms are available to the person skilled in the art for codon optimization. A preferred method is the "guided random method based on a Monte Carlo algorithm available via the internet world wide web genomes.urv.es/OPTIMIZER/ (P. Puigbo, E. Guzman, A. Romeu, and S. Garcia- Vallve. Nucleic Acids Res. 2007 July; 35(Web Server issue): W126-W131).

A nucleotide sequence encoding an enzyme expressed or to be expressed in a cyanobacterial cell according to the invention or a promoter used in a cell according to the invention may be defined by its capability to hybridize with a nucleotide sequence such as SEQ ID NO: 1, 3, or 5 respectively, under moderate, or preferably under stringent hybridization conditions. Stringent hybridization conditions are herein defined as conditions that allow a nucleic acid sequence of at least about 25, preferably about 50 nucleotides, 75 or 100 and most preferably of about 200 or more nucleotides, to hybridize at a temperature of about 65° C. in a solution comprising about 1 M salt, preferably 6 ^X SSC or any other solution having a comparable ionic strength, and washing at 65° C. in a solution comprising about 0.1 M salt, or less, preferably 0.2xSSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having about 90% or more sequence identity. Moderate conditions are herein defined as conditions that allow a nucleic acid sequences of at least 50 nucleotides, preferably of about 200 or more nucleotides, to hybridize at a temperature of about 45° C. in a solution comprising about 1 M salt, preferably 6*SSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6 ^X SSC or any other solution having a comparable ionic strength. Preferably, the hybridization is performed overnight, i.e. at least for 10 hours, and preferably washing is performed for at least one hour with at least two changes of the washing solution. These conditions will usually allow the specific hybridization of sequences having up to 50% sequence identity. The person skilled in the art will be able to modify these hybridization conditions in order to specifically identify sequences varying in identity between 50% and 90%.

As used herein the term "heterologous sequence" or "heterologous nucleic acid" is one that is not naturally found operably linked as neighboring sequence of said first nucleotide sequence. As used herein, the term "heterologous" may mean "recombinant". "Recombinant" refers to a genetic entity distinct from that generally found in nature. As applied to a nucleotide sequence or nucleic acid molecule, this means that said nucleotide sequence or nucleic acid molecule is the product of various combinations of cloning, restriction and/or ligation steps, and other procedures that result in the production of a construct that is distinct from a sequence or molecule found in nature.

"Operably linked" is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to the nucleotide sequence coding for the polypeptide of the invention such that the control sequence directs the production/expression of the peptide or polypeptide of the invention in a cell and/or in a subject.

"Operably linked" may also be used for defining a configuration in which a sequence is appropriately placed at a position relative to another sequence coding for a functional domain such that a chimeric polypeptide is encoded in a cell and/or in a subject.

Expression will be understood to include any step involved in the production of the peptide or polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, post-translational modification and secretion.

As used herein, the term "promoter" refers to a nucleic acid fragment that functions to control the transcription of one or more nucleic acid molecules, located upstream with respect to the direction of transcription of the transcription initiation site of the nucleic acid molecule, and is structurally identified by the presence of a binding site for DNA-dependent R A polymerase, transcription initiation sites and any other DNA sequences, including, but not limited to transcription factor binding sites, repressor and activator protein binding sites, and any other sequences of nucleotides known to one of skill in the art to act directly or indirectly to regulate the amount of transcription from the promoter. A "constitutive" promoter is a promoter that is active under most environmental and developmental conditions. An "inducible" promoter is a promoter that is active under environmental or developmental regulation.

For expression of an enzyme in a cyanobacterial cell according to the inventions, as well as for additional genetic modification of a cyanobacterial cell according to the invention, the cell can be transformed with a nucleic acid or nucleic acid construct described herein by any method known to the person skilled in the art. Such methods are e.g. known from standard handbooks, such as Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3 ^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press, or F. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York (1987). Methods for transformation and genetic modification of cyanobacterial cells are known from e.g. U.S. Pat. No. 6,699,696 or U.S. Pat. No. 4,778,759. When a nucleic acid construct is used for expression of an enzyme in a cyanobacterial cell according to the invention, a selectable marker may be present in the nucleic acid construct comprising a polynucleotide encoding the enzyme. The term "marker" refers herein to a gene encoding a trait or a phenotype which permits the selection of, or the screening for, a cyanobacterial cell containing the marker. A marker gene may be an antibiotic resistance gene whereby the appropriate antibiotic can be used to select for transformed cells from among cells that are not transformed. Preferably however, a non-antibiotic resistance marker is used, such as an auxotrophic marker (URA3, TRP1, LEU2). A preferred cyanobacterial cell according to the invention, e.g. transformed with a nucleic acid construct, is marker gene free. Methods for constructing recombinant marker gene free microbial host cells are described in (Cheah et al., 2013) and are based on the use of bidirectional markers. Alternatively, a screenable marker such as Green Fluorescent Protein, lacZ, luciferase, chloramphenicol acetyltransferase, beta-glucuronidase may be incorporated into a nucleic acid construct according to the invention allowing to screen for transformed cells.

Optional further elements that may be present in a nucleic acid construct according to the invention include, but are not limited to, one or more leader sequences, enhancers, integration factors, and/or reporter genes, intron sequences, centromers, telomers and/or matrix attachment (MAR) sequences. A nucleic acid construct according to the invention can be provided in a manner known per se, which generally involves techniques such as restricting and linking nucleic acids/nucleic acid sequences, for which reference is made to the standard handbooks, such as Sambrook and ussel (2001) "Molecular Cloning: A Laboratory Manual (3 ^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

Methods for inactivation and gene disruption in a cyanobacterial cell are well known in the art (see e.g. Shestakov S V et al, (2002), Photosynthesis Research, 73 : 279-284 and Nakamura Y et al, (1999), Nucleic Acids Res. 27:66-68).

In this document and in its claims, the verb "to comprise" and its conjugations is used in its non- limiting sense to mean that items following the word are included, but items not specifically mentioned are not excluded. In addition, reference to an element by the indefinite article "a" or "an" does not exclude the possibility that more than one of the element is present, unless the context clearly requires that there be one and only one of the elements. The indefinite article "a" or "an" thus usually means "at least one".

The word "about" or "approximately" when used in association with a numerical value (e.g. about 10) preferably means that the value may be the given value (of 10) more or less 0.1% of the value.

The sequence information as provided herein should not be so narrowly construed as to require inclusion of erroneously identified bases. The skilled person is capable of identifying such erroneously identified bases and knows how to correct for such errors. In case of sequence errors, the sequence of the enzymes obtainable by expression of the genes as represented by SEQ ID NO's 1, 3, 5, 7, 9, 1 1, 13, 15, 17, 19, 21 , 23, 25 and 27 containing the enzyme encoding polynucleotide sequences should prevail.

All patent and literature references cited in the present specification are hereby incorporated by reference in their entirety. Table 2. Sequences

SEQ ID Gene/Polypeptide Sequence

NO

1 geranyl ATGGCTTACAGTGCTATGGCAACCATGGGTTACAAT diphosphate GGTATGGCAGCTAGCTGCCATACCCTGCATCCTACC synthase from AGCCCATTAAAACCTTTTCATGGAGCTTCAACCTCA Abies grandis CTGGAAGCTTTTAATGGCGAGCATATGGGCCTCCTC

CGAGGGTATTCGAAGAGGAAGCTATCTTCATATAAA

AATCCGGCATCTAGATCCTCAAACGCTACAGTTGCC

CAGTTGCTTAATCCTCCACAAAAAGGGAAGAAGGC

CGTTGAATTTGATTTCAACAAGTACATGGATTCCAA

GGCAATGACAGTGAATGAGGCGTTGAATAAGGCTA TCCCACTTCGTTATCCCCAGAAAATATATGAATCCA

TGAGGTATTCTCTTCTGGCAGGAGGGAAACGAGTTC

GTCCTGTTCTGTGCATTGCAGCATGTGAGCTTGTTG

GAGGAACCGAGGAGCTTGCGATTCCAACTGCCTGTG

CAATCGAAATGATCCACACAATGTCTTTGATGCATG

ATGACTTGCCTTGCATAGACAATGATGATTTACGGC

GAGGGAAACCTACAAACCATAAGATCTTCGGGGAA

GATACTGCTGTTACTGCAGGGAATGCGCTTCATTCT

TACGCCTTTGAGCATATTGCAGTTTCCACAAGCAAA

ACTGTGGGGGCTGATAGGATTTTGAGGATGGTATCT

GAACTGGGTAGAGCAACAGGCTCTGAAGGGGTTAT

GGGTGGCCAGATGGTCGATATTGCCAGCGAAGGGG

ATCCTTCTATTGACCTTCAGACTCTGGAATGGATTC

ATATTCACAAGACTGCAATGCTCTTGGAGTGCTCGG

TTGTGTGTGGGGCGATCATCGGTGGTGCTTCGGAGA

TTGTGATCGAGAGAGCTCGAAGGTATGCCCGTTGCG

TGGGGCTTCTTTTTCAGGTTGTGGATGACATACTCG

ATGTCACGAAATCATCAGACGAACTGGGCAAGACT

GCAGGAAAGGATTTGATTAGTGATAAGGCAACTTAT

CCAAAGCTCATGGGTTTGGAGAAAGCAAAGGAGTT

TTCTGATGAATTGTTGAACAGAGCTAAGGGAGAGTT

ATCTTGCTTCGATCCAGTGAAGGCAGCACCTCTGTT

GGGTCTTGCAGATTACGTGGCATTCAGACAAAATTG

A

geranyl MA YS AM ATMGY GM AAS CHTLHPT SPLKPFHG ASTS diphosphate LEAFNGEHMGLLRGYSKRKLSSYKNPASRSSNATVAQ synthase from LLNPPQKGKKAVEFDF KYMDSKAMTV EALNKAIP Abies grandis LRYPQKIYESMRYSLLAGGKRVRPVLCIAACELVGGT

EELAIPTACAIEMIHTMSLMHDDLPCIDNDDLRRGKPT

NHKIFGEDTAVTAGNALHSYAFEHIAVSTSKTVGADPJ

LRMVSELGRATGSEGVMGGQMVDIASEGDPSIDLQTL

EWIHIHKTAMLLECSVVCGAIIGGASEIVIERARRYARC

VGLLFQVVDDILDVTKSSDELGKTAGKDLISDKATYP

KLMGLEKAKEFSDELLNRAKGELSCFDPVKAAPLLGL

ADYVAFRQN

farnesyl ATGGCTTCAGAAAAAGAAATTAGGAGAGAGAGATT diphosphate CTTGAACGTTTTCCCTAAATTAGTAGAGGAATTGAA synthase from CGCATCGCTTTTGGCTTACGGTATGCCTAAGGAAGC

Saccharomyces ATGTGACTGGTATGCCCACTCATTGAACTACAACAC cerevisiae

TCCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGT GGACACGTATGCTATTCTCTCCAACAAGACCGTTGA ACAATTGGGGCAAGAAGAATACGAAAAGGTTGCCA TTCTAGGTTGGTGCATTGAGTTGTTGCAGGCTTACTT CTTGGTCGCCGATGATATGATGGACAAGTCCATTAC

CAGAAGAGGCCAACCATGTTGGTACAA

GGTTCCTGAAGTTGGGGAAATTGCCATCAATGACGC

ATTCATGTTAGAGGCTGCTATCTACAAGCTTTTGAA

ATCTCACTTCAGAAACGAAAAATACTACATAGATAT

CACCGAATTGTTCCATGAGGTCACCTTCCAAACCGA

ATTGGGCCAATTGATGGACTTAATCACTGCACCTGA

AGACAAAGTCGACTTGAGTAAGTTCTCCCTAAAGAA

GCACTCCTTCATAGTTACTTTCAAGACTGCTTACTAT

TCTTTCTACTTGCCTGTCGCATTGGCCATGTACGTTG

CCGGTATCACGGATGAAAAGGATTTGAAACAAGCC

AGAGATGTCTTGATTCCATTGGGTG

AATACTTCCAAATTCAAGATGACTACTTAGACTGCT

TCGGTACCCCAGAACAGATCGGTAAGATCGGTACA

GATATCCAAGATAACAAATGTTCTTGGGTAATCAAC

AAGGCATTGGAACTTGCTTCCGCAGAACAAAGAAA

GACTTTAGACGAAAATTACGGTAAGAAGGACTCAG

TCGCAGAAGCCAAATGCAAAAAGATTTTCAATGACT

TGAAAATTGAACAGCTATACCACGAATATGAAGAG

TCTATTGCCAAGGATTTGAAGGCCAAAATTTCTCAG

GTCGATGAGTCTCGTGGCTTCAAAGCTGATGTCTTA

ACTGCGTTCTTGAACAAAGTTTACAAGAGA

AGCAAATAG

farnesyl MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEAC diphosphate DWYAHSLNY TPGGKLNRGLSVVDTYAILSNKTVEQ synthase from LGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRR

Saccharomyces GQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFRNE cerevisiae

KYYIDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFS

LKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDL

QARDVLIPLGEYFQIQDDYLDCFGTPEQIG IGTDIQDN

KCSWVIN ALELASAEQRKTLDENYGKKDSVAEAKC

KKIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFK

ADVLTAFLNKVYKRSK

4S-limonene ATGGCTCTCAAAGTGTTAAGTGTTGCAACTCAAATG synthase from GCGATTCCTAGCAACCTAACGACATGTCTTCAACCC Mentha spicata TCACACTTCAAATCTTCTCCAAAACTGTTATCTAGC

ACTAACAGTAGTAGTCGGTCTCGCCTCCGTGTGTAT

TGCTCCTCCTCGCAACTCACTACTGAAAGACGATCC

GGAAACTACAACCCTTCTCGTTGGGATGTCAACTTC

ATCCAATCGCTTCTCAGTGACTATAAGGAGGACAAA

CACGTGATTAGGGCTTCTGAGCTGGTCACTTTGGTG

AAGATGGAACTGGAGAAAGAAACGGATCAAATTCG

ACAACTTGAGTTGATCGATGACTTGCA GAGGATGGGGCTGTCCGATCATTTCCAAAATGAGTT

CAAAGAAATCTTGTCCTCTATATATCTCGACCATCA

CTATTACAAGAACCCTTTTCCAAAAGAAGAAAGGG

ATCTCTACTCCACATCTCTTGCATTTAGGCTCCTCAG

AGAACATGGTTTTCAAGTCGCACAAGAGGTATTCGA

TAGTTTCAAGAACGAGGAGGGTGAGTTCAAAGAAA

GCCTTAGCGACGACACCAGAGGATTGTTGCAACTGT

ATGAAGCTTCCTTTCTGTTGACGGAAGGCGAAACCA

CGCTCGAGTCAGCGAGGGAATTCGCCACCAAATTTT

TGGAGGAAAAAGTGAACGAGGGTGGTG

TTGATGGCGACCTTTTAACAAGAATCGCATATTCTT

TGGACATCCCTCTTCATTGGAGGATTAAAAGGCCAA

ATGCACCTGTGTGGATCGAATGGTATAGGAAGAGG

CCCGACATGAATCCAGTAGTGTTGGAGCTTGCCATA

CTCGACTTAAATATTGTTCAAGCACAATTTCAAGAA

GAGCTCAAAGAATCCTTCAGGTGGTGGAGAAATAC

TGGGTTTGTTGAGAAGCTGCCCTTCGCAAGGGATAG

ACTGGTGGAATGCTACTTTTGGAATACTGGGATCAT

CGAGCCACGTCAGCATGCAAGTGCAAGGATAATGA

TGGGCAAAGTCAACGCTCTGATTACGGTG

ATCGATGATATTTATGATGTCTATGGCACCTTAGAA

GAACTCGAACAATTCACTGACCTCATTCGAAGATGG

GATATAAACTCAATCGACCAACTTCCCGATTACATG

CAACTGTGCTTTCTTGCACTCAACAACTTCGTCGAT

GATACATCGTACGATGTTATGAAGGAGAAAGGCGT

CAACGTTATACCCTACCTGCGGCAATCGTGGGTTGA

TTTGGCGGATAAGTATATGGTAGAGGCACGGTGGTT

CTACGGCGGGCACAAACCAAGTTTGGAAGAGTATTT

GGAGAACTCATGGCAGTCGATAAGTGGGCCCTGTAT

GTTAAC GC AC AT ATTCTTC CGAGT AAC

AGATTCGTTCACAAAGGAGACCGTCGACAGTTTGTA

CAAATACCACGATTTAGTTCGTTGGTCATCCTTCGTT

CTGCGGCTTGCTGATGATTTGGGAACCTCGGTGGAA

GAGGTGAGCAGAGGGGATGTGCCGAAATCACTTCA

GTGCTACATGAGTGACTACAATGCATCGGAGGCGG

AGGCGCGGAAGCACGTGAAATGGCTGATAGCGGAG

GTGTGGAAGAAGATGAATGCGGAGAGGGTGTCGAA

GGATTCTCCATTCGGCAAAGATTTTATAGGATGTGC

AGTTGATTTAGGAAGGATGGCGCAGTTGATGTACCA

TAATGGAGATGGGCACGGCACACAACACC

CTATTATACATCAACAAATGACCAGAACCTTATTCG

AGCCCTTTGCATGA

4S-limonene MALKVLS VATQMAIPSNLTTCLQPSHFKS SPKLLS STN synthase from Mentha spicata SSSRSRLRVYCSSSQLTTERRSGNYNPSRWDVNFIQSL

LSDYKEDKHVIRASELVTLVKMELEKETDQIRQLELID

DLQRMGLSDHFQNEFKEILSSIYLDHHYYK PFP EER

DLYSTSLAFRLLREHGFQVAQEVFDSFKNEEGEFKESL

SDDTRGLLQLYEASFLLTEGETTLESAREFATKFLEE

VNEGGVDGDLLTRIAYSLDIPLHWRIKRPNAPVWIEW

YRKRPDMNPVVLELAILDLNIVQAQFQEELKESFRWW

RNTGFVEKLPFARDRLVECYFWNTGIIEPRQHASARIM

MGKVNALITVIDDIYDVYGTLEELEQFTDLIRRDINSID

QLPDYMQLCFLALNNFVDDTSYDVMKEKGVNVIPYL

RQSWVDLADKYMVEARWFYGGHKPSLEEYLENSWQ

SISGPCMLTHIFFRVTDSFTKETVDSLYKYHDLVRWSS

FVLRLADDLGTSVEEVSRGDVPKSLQCYMSDY ASEA

EARKHVKWLIAEVWKKMNAERVSKDSPFGKDFIGCA

VDLGRMAQLMYHNGDGHGTQHPIIHQQMTRTLFEPF

A

geranyl ATGGGTTACAATGGCATGGTAGTTAGCTCCAACCTT diphosphate GGCCTGTATTATTTGAACATTGCCTCTCGAGAAT synthase from GTAACCTGAAAAGAATTTCAATCCCATCACCTTTTC Picea abies ATGGCGTTTCAACCTCATTGGGCTCTTCTACTAG

TAAACATCTGGGCCTCCGTGGCCATTTGAAGAAAGA

GTTGTTGTCACATAGACTTCTGCTATCATCAACT

AGATCGTCGAAAGCACTTGTCCAGCTAGCTGATCTG

TCTGAACAGGTGAAGAATGTTGTTGAGTTTGATT

TCGATAAGTACATGCATTCCAAGGCAATTGCAGTGA

ATGAGGCATTGGATAAGGTTATCCCACCACGTTA

TCCCCAGAAAATATATGAATCCATGAGGTATTCTCT

TCTAGCAGGAGGGAAACGAGTTCGTCCTATTTTG

TGCATTGCTGCGTGCGAGCTTATGGGAGGAACCGAG

GAGCTTGCGATGCCAACTGCCTGTGCAATTGAAA

TGATCCACACAATGTCTTTGATTCATGATGACTTGC

CTTACATAGACAATGATGATTTACGCCGAGGGAA

GCCCACAAACCATAAGGTCTTCGGGGAGGATACTG

CTATTATTGCAGGGGATGCACTTCTTTCACTCGCC

TTTGAACACGTTGCAGTCTCCACGAGCAGAACTCTG

GGGACTGATATAATTTTGAGGCTGCTATCTGAAA

TCGGTAGAGCAACAGGCTCTGAAGGGGTTATGGGT

GGCCAGGTTGTCGATATCGAGAGCGAAGGCGATCC

TTCTATTGATCTCGAGACTCTCGAATGGGTTCATATT

CACAAGACTGCAGTGCTCTTGGAGTGCTCGGTT

GTGTGTGGGGCGATCATGGGTGGTGCTTCGGAGGAT

GATATTGAGAGAGCTCGAAGGTATGCCCGTTGCG

TGGGGCTTCTGTTTCAGGTTGTGGATGACATACTCG ATGTCTCTCAATCATCAGAAGAATTGGGCAAGAC

GGCAGGGAAGGATTTGATTAGTGATAAAGCCACTT

ATCCCAAGCTGATGGGTTTGGAGAAAGCAAAGGAA

TTTGCTGATGAATTGTTGAACAGAGGTAAGCAGGAG

TTATCTTGCTTCGACCCAACTAAGGCTGCACCTT

TGTTTGCTCTGGCAGATTACATTGCTTCAAGACAAA

ACTGA

geranyl MGY GMVVSSNLGLYYLNIASRECNLKRISIPSPFHGV diphosphate STSLGSSTSKHLGLRGHLKKELLSHRLLLSSTRSSKAL synthase from VQLADLSEQVKNVVEFDFDKYMHSKAIAVNEALDKV Picea abies IPPRYPQKIYESMRYSLLAGGKRVRPILCIAACELMGG

TEELAMPTACAIEMIHTMSLIHDDLPYIDNDDLRRGKP

TNHKVFGEDTAIIAGDALLSLAFEHVAVSTSRTLGTDII

LRLLSEIGRATGSEGVMGGQVVDIESEGDPSIDLETLE

WVHIH T AVLLEC S VVC GAIMGGASEDDIERARRY AR

CVGLLFQVVDDILDVSQSSEELGKTAGKDLISDKATYP

KLMGLEKAKEFADELLNRGKQELSCFDPTKAAPLFAL

ADYIASRQN

geranyl ATGTTATTCACGAGGAGTGTTGCTCGGATTTCTTCTA diphosphate AGTTTCTGAGAAACCGTAGCTTCTATGGCTCCT synthase from CTCAATCTCTCGCCTCTCATCGGTTCGCAATCATTCC Arabidopsis CGATCAGGGTCACTCTTGTTCTGACTCTCCACA thaliana

CAAGGGTTACGTTTGCAGAACAACTTATTCATTGAA

ATCTCCGGTTTTTGGTGGATTTAGTCATCAACTC

TATCACCAGAGTAGCTCCTTGGTTGAGGAGGAGCTT

GACCCATTTTCGCTTGTTGCCGATGAGCTGTCAC

TTCTTAGTAATAAGTTGAGAGAGATGGTACTTGCCG

AGGTTCCAAAGCTTGCCTCTGCTGCTGAGTACTT

CTTCAAAAGGGGTGTGCAAGGAAAACAGTTTCGTTC

AACTATTTTGCTGCTGATGGCGACAGCTCTGGAT

GTACGAGTTCCAGAAGCATTGATTGGGGAATCAAC

AGATATAGTCACATCAGAATTACGCGTAAGGCAAC

GGGGTATTGCTGAAATCACTGAAATGATACACGTCG

CAAGTCTACTGCACGATGATGTCTTGGATGATGC

CGATACAAGGCGTGGTGTTGGTTCCTTAAATGTTGT

AATGGGTAACAAGATGTCGGTATTAGCAGGAGAC

TTCTTGCTCTCCCGGGCTTGTGGGGCTCTCGCTGCTT

TAAAGAACACAGAGGTTGTAGCATTACTTGCAA

CTGCTGTAGAACATCTTGTTACCGGTGAAACCATGG

AGATAACTAGTTCAACCGAGCAGCGTTATAGTAT

GGACTACTACATGCAGAAGACATATTATAAGACAG

CATCGCTAATCTCTAACAGCTGCAAAGCTGTTGCC

GTTCTCACTGGACAAACAGCAGAAGTTGCCGTGTTA GCTTTTGAGTATGGGAGGAATCTGGGTTTAGCAT

TCCAATTAATAGACGACATTCTTGATTTCACGGGCA

CATCTGCCTCTCTCGGAAAGGGATCGTTGTCAGA

TATTCGCCATGGAGTCATAACAGCCCCAATCCTCTT

TGCCATGGAAGAGTTTCCTCAACTACGCGAAGTT

GTTGATCAAGTTGAAAAAGATCCTAGGAATGTTGAC

ATTGCTTTAGAGTATCTTGGGAAGAGCAAGGGAA

TACAGAGGGCAAGAGAATTAGCCATGGAACATGCG

AATCTAGCAGCAGCTGCAATCGGGTCTCTACCTGA

AACAGACAATGAAGATGTCAAAAGATCGAGGCGGG

CACTTATTGACTTGACCCATAGAGTCATCACCAGA

AACAAGTGA

geranyl MLFTRSVARISSKFLRNRSFYGSSQSLASHRFAIIPDQG diphosphate HSCSDSPH GYVCRTTYSLKSPVFGGFSHQL synthase from YHQSSSLVEEELDPFSLVADELSLLSNKLREMVLAEVP Arabidopsis KLASAAEYFFKRGVQGKQFRSTILLLMATALD thaliana

VRVPEALIGESTDIVTSELRVRQRGIAEITEMIHVASLL

HDDVLDDADTRRGVGSLNVVMGNKMSVLAGD

FLLSRACGALAALK TEVVALLATAVEHLVTGETMEI

TSSTEQRYSMDYYMQKTYYKTASLISNSCKAVA

VLTGQTAEVAVLAFEYGR LGLAFQLIDDILDFTGTSA

SLG GSLSDIRHGVITAPILFAMEEFPQLREV

VDQVEKX)PRNVDIALEYLG SKGIQRARELAMEHANL

AAAAIGSLPETDNEDVKRSRRALIDLTHRVITR

NK

limonene synthase ATGGCTCTCAAAGTGTTAAGTGTTGCAACTCAAATG from Mentha GCGATTCCTAGCAAGCTAACGAGATGTCTTCAAC canadensis CCTCACACTTGAAATCCTCTCCAAAATTGTTATCTA

GCACTAACAGTAGTAGTCGGTCTCGCCTCCGTGT

GTATTGCTCCTCCTCGCAACTCACTACTGAGAGACG

ATCCGGAAACTACAACCCTTCTCGTTGGGATGTC

GAATTCATCCAATCCCTCCACAGTGATTATGAGGAG

GACAAACATGCGATTAGGGCTTCTGAGCTGGTCA

CTTTGGTGAAGATGGAATTGGAGAAAGAAACGGAT

CATATTCGACAACTTGAGTTGATCGATGACTTGCA

GAGGATGGGGCTGTCCGATCATTTCCAGAATGAGTT

CAAAGAAATCTTGTCCTCTATATATCTCGACCAT

CACTATTACAAGAACCCTTTTCCAGAAGAAGAAAA

GGGATCTCTACTCACATCTCTTGCATTTAGGCTCC

TCAGAGAACATGGTTTTCAAGTCGCACAAGAGGTAT

TCGACAGTTTCAAGAACGAGGAGGGTGAGTTCAA

AGAAAGCCTTAGCGACGACACTAGAGGATTGTTGC

GACTGTATGAAGCTTCCTTTCTGTTGACGGAAGGC GAAACCACGCTCGAGTCAGCGAGGGAATTCGCCAC

CAAATTTTTGGAGGAAAGAGTGAACGAGGGTGGTG

TTGATGGCGACCTTTTAACAAGAATCGCATATTCTT

TGGACATCCCACTTCATTGGAGGATTAAAAGGCC

AAATGCACCTACGTGGATCGAATGGTATAGGAAGA

GGCCCGACATGAATCCAGTAGTGTTGGAGCTTGCC

ATACTCGACTTAGATATTGTTCAAGCACAATTTCAA

GAAGAGCTCAAAGAATCCTTCAGGTGGTGGAGAA

ATACTGGTTTTGTTGAGAAGCTGCCCTTCGCAAGGG

ATAGATTGGTGGAATGCTACTTTTGGAATACTGG

GATCATCGAGCCACGTCAGCATGCAAGTGCAAGGA

TAATGATGGGCAAAGTCAACGCTCTGATTACGGTG

ATCGATGATATTTATGATGTCTACGGCACCTTAGAA

GAACTCGAACAATTCACAGAACTCATTCGGAGAT

GGGATATAAACTCAATCGACCAACTTCCCGATTACA

TGCAACTGTGCTTTCTTGCACTCAACAACTTCGT

CGATGATACATCGTACGATGTTATGAAGGAGAAAG

GCGTCAACGTTATACCCTACCTGCGGCAATCGTGG

GTGGATTTGGCGGATAAGTATATGGTAGAGGCACG

GTGGTTCTACGGCGGGCACAAACCAAGTTTGGAAG

AGTATTTGGAGAACTCATGGCAGTCGATAAGTGGGC

CCTGTATGTTAACGCACATATTCTTCCGAGTAAC

AGATTCGTTCACAAAGGAGACCGTCGACAGCTTGTA

CAAATACCACGATTTAGTTCGTTGGTCGTCCTTC

GTTCTGCGGCTTGCTGATGATTTGGGAACCTCGGTG

GAAGAGGTGAGCAGAGGCGATGTGCCGAAATCAC

TTCAGTGCTACATGAGTGACTACAATGCATCGGAGG

CGGAGGCGCGGAAGCACGTGAAATGGCTGATAGC

GGAGGTGTGGAAGAAGATGAATGCGGAGAGGGTGC

CGAAGGATTCTCCATTCGGCAAAGATTTTATAGGA

TGTGCAGCTGATTTAGGAAGGATGGCGCAGTTGATG

TACCATAATGGAGATGGGCACGGCACACAACATC

CT AT AATAC ATC AAC AAATGAC C AG AAC CTTATTC G

AGCCCTTTGCATGA

limonenc synthase MALKVLSVATQMAIPSKLTRCLQPSHLKSSPKLLSSTN from Mentha S S SPvSPvLRVYC S S S QLTTERRSGNYNPSRWD V canadensis EFIQSLHSDYEEDKHAIRASELVTLVKMELEKETDHIR

QLELIDDLQRMGLSDHFQNEFKEILS SIYLDH

HYYK PFPEEE GSLLTSLAFRLLREHGFQVAQEVFDS

FK EEGEF ESLSDDTRGLLRLYEASFLLTEG

ETTLESAREFATKFLEERV EGGVDGDLLTRIAYSLDIP

LHWRIKRPNAPTWIEWYRKRPDMNPVVLELA

ILDLDIVQAQFQEELKESFRWWRNTGFVEKLPFARDR LVECYFW TGIIEPRQHASARIMMGKVNALITV

IDDIYDVYGTLEELEQFTELIRRWDINSIDQLPDYMQLC

FLAL F VDDT S YD VMKEKGV VIP YLRQ S W

VDLAD YMVEARWFYGGHKPSLEEYLENSWQSISGP

CMLTHIFFRVTDSFTKETVDSLY YHDLVRWSSF

VLRLADDLGTSVEEVSRGDVP SLQCYMSDYNASEAE

ARKHVKWLIAEVWKKMNAERVPKDSPFGKDFIG

CAADLGRMAQLMYHNGDGHGTQHPIIHQQMTRTLFE

PFA

4S-limonene ATGGCTCTCCTTTCTATCGTATCTTTGCAGGTTCCCA synthase from AATCCTGCGGGCTGAAATCGTTGATCAGTTCCA Abies grandis GCAATGTGCAGAAGGCTCTCTGTATCTCTACAGCAG

TCCCAACACTCAGAATGCGTAGGCGACAGAAAGC

TCTGGTCATCAACATGAAATTGACCACTGTATCCCA

TCGTGATGATAATGGTGGTGGTGTACTGCAAAGA

CGCATAGCCGATCATCATCCCAACCTGTGGGAAGAT

GATTTCATACAATCATTGTCCTCACCTTATGGGG

GATCTTCGTACAGTGAACGTGCTGAGACAGTCGTTG

AGGAAGTAAAAGAGATGTTCAATTCAATACCAAA

TAATAGAGAATTATTTGGTTCCCAAAATGATCTCCT

TACACGCCTTTGGATGGTGGATAGCATTGAACGT

CTGGGGATAGATAGACATTTCCAAAATGAGATAAG

AGTAGCCCTCGATTATGTTTACAGTTATTGGAAGG

AAAAGGAAGGCATTGGGTGTGGCAGAGATTCTACT

TTTCCTGATCTCAACTCGACTGCCTTGGCGCTTCG

AACTCTTCGACTGCACGGATACAATGTGTCTTCAGA

TGTGCTGGAATACTTCAAAGATGAAAAGGGGCAT

TTTGCCTGCCCTGCAATCCTAACCGAGGGACAGATC

ACTAGAAGTGTTCTAAATTTATATCGGGCTTCCC

TGGTCGCCTTTCCCGGGGAGAAAGTTATGGAAGAG

GCTGAAATCTTCTCGGCATCTTATTTGAAAAAAGT

CTTACAAAAGATTCCGGTCTCCAATCTTTCAGGAGA

GATAGAATATGTTTTGGAATATGGTTGGCACACG

AATTTGCCGAGATTGGAAGCAAGAAATTATATCGA

GGTCTACGAGCAGAGCGGCTATGAAAGCTTAAACG

AGATGCCATATATGAACATGAAGAAGCTTTTACAAC

TTGCAAAATTGGAGTTCAATATCTTTCACTCTTT

GCAACTAAGAGAGTTACAATCTATCTCCAGATGGTG

GAAAGAATCAGGTTCGTCTCAACTGACTTTTACA

CGGCATCGTCACGTGGAATACTACACTATGGCATCT

TGCATTTCTATGTTGCCAAAACATTCAGCTTTCA

GAATGGAGTTTGTCAAAGTGTGTCATCTTGTAACAG

TTCTCGATGATATATATGACACTTTTGGAACAAT GAACGAACTCCAACTTTTTACGGATGCAATTAAGAG

ATGGGATTTGTCAACGACAAGGTGGCTTCCAGAA

TATATGAAAGGAGTGTACATGGACTTGTATCAATGC

ATTAATGAAATGGTGGAAGAGGCTGAGAAGACTC

AAGGCCGAGATATGCTCAACTATATTCAAAATGCTT

GGGAAGC CCT ATTTGATAC CTTTATGC AAGAAGC

AAAGTGGATCTCCAGCAGTTATCTCCCAACGTTTGA

GGAGTACTTGAAGAATGCAAAAGTTAGTTCTGGT

TCTCGCATAGCCACATTACAACCCATTCTCACTTTG

GATGTACCACTTCCTGATTACATACTGCAAGAAA

TTGATTATCCATCCAGATTCAATGAGTTAGCTTCGTC

CATCCTTCGACTACGAGGTGACACGCGCTGCTA

CAAGGCGGATAGGGCCCGTGGAGAAGAAGCTTCAG

CTATATCGTGTTATATGAAAGACCATCCTGGATCA

ATAGAGGAAGATGCTCTCAATCATATCAACGCCATG

ATCAGTGATGCAATCAGAGAATTAAATTGGGAGC

TTCTCAGACCGGATAGCAAAAGTCCCATCTCTTCCA

AGAAACATGCTTTTGACATCACCAGAGCTTTCCA

TCATGTCTACAAATATCGAGATGGTTACACTGTTTC

CAACAACGAAACAAAGAATTTGGTGATGAAAACC

GTTCTTGAACCTCTCGCTTTGTAA

4S-iimonenc MALLSIVSLQVPKSCGLKSLISSSNVQKALCISTAVPTL synthase from RMRRRQKA.LVINMKLTTVSHRDDNGGGVLQR Abies grandis PJADHHPNL WEDDFIQ SLS SPYGGSS YSERAETVVEEV

KEMFNSIP NRELFGSQNDLLTRLW VDSIER

LGIDRHFQNEIRVALDYVYSYWKEKEGIGCGRDSTFP

DLNSTALALRTLRLHGY VSSDVLEYFKDEKGH

FACPAILTEGQITRSVLNLYRASLVAFPGEKVMEEAEIF

S AS YLKKVLQKIPV SNL S GEIEYVLEYG WHT

NLPRLE AR YIEVYEQS GYE SLNEMP YMNMKKLLQL

AKLEFNIFHSLQLRELQSISRWWKESGSSQLTFT

RHRHVEYYTMASCISMLPKHSAFRMEFV VCHLVTV

LDDIYDTFGTMNELQLFTDAIKRWDLSTTRWLPE

YMKGVYMDLYQCINEMVEEAE TQGRDMLNYIQNA

WE ALFDTFMQEA WIS S SYLPTFEEYLKNAKVS SG

SRIATLQPILTLDVPLPDYILQEIDYPSRFNELASSILRLR

GDTRCYKADRARGEEASAISCYMKDHPGS

IEED ALNHrNAMI SD AIRELN WELLRPD S SPI S SKKHA

FDITRAFHHVY YRDGYTVS ETKNLVMKT

VLEPLAL

R-limonene ATTTGAGAATCTTTGCCAAGTATAACTGTAAGCTAG synthase from CTTACACTACATCTGTATATCCAATGTCTTCTTG Citrus sinensis CATTAATCCCTCAACCTTGGTTACCTCTATAAATGGT TTCAAATGTCTTCCTCTTGCAACAAATAAAGCA

GCCATCAGAATCATGGCCAAAAATAAGCCAGTCCA

ATGCCTTGTCAGCGCCAAATATGATAATTTGACAG

TTGATAGGAGATCAGCAAACTACCAACCTTCAATTT

GGGACCATGATTTTTTGCAGTCATTGAATAGCAA

CTATACGGATCAAACATACAGAAGACGAGCAGAAG

AGCTGAAGGGAAAAGTGAAGACAGCGATTAAGGAT

GTAACCGAGCCTCTGGATCAGTTGGAGCTGATTGAC

AACTTGCAAAGACTTGGATTGGCTTATCATTTTG

AGACTGAGATTCGAAACATATTGCATAATATCTACA

ACAATAATAAAGATTATATTTGGAGAAAAGCAAA

TCTGTATGCAACCTCCCTTGAATTCAGACTACTTAG

ACAACATGGCTATCCTGTTTCTCAAGAGGTTTTC

AGTGGTTTTAAAGACGACAAGGGAGGCTTCATTTGT

GATGATTTCAAGGGAATACTGAGCTTGCATGAAG

CCTCGTATTACAGCTTAGAAGGAGAAAGCATCATGG

AGGAGGCCTGGCAATTCACCAGTAAGCATCTTAA

AGAAAC GATG ATC ATC AGC AAC AGC AAGGAAGAGT

ATGTATTTGTAGCAGAACAAGCGAAGCGTGCGCTG

GAGCTCCCTCTGCATTGGAAAGTGCCAACGTTGGAG

GCAAGGTGGTTCATACACGTTTATGAGAAAAGAG

AGGACAAGAACCACCTTTTACTTGAGCTCGCTAAGT

TGGAGTTTAACACTTTGCAGGCAATTTACCAGGA

AGAACTTAAAGACATTTCAGGATGGTGGAAGGATA

CAGGTCTTGGAGAGAAATTGAGCTTTGCGAGGAAC

AGGTTGGTAGCGTCCTTCTTATGGAGCATGGGGATC

GCGTTTGAGCCTCAATTCGCCTACTGCAGGAGAG

TGCTCACAATCTCGATAGCCCTAATTACAGTGATTG

ATGACATTTATGATGTCTATGGAACATTGGATGA

ACTTGAGATATTCACTGATGCTGTTGCGAGGTGGGA

CATCAATTATGCTTTGAAGCACCTTCCGGGATAT

ATGAAAATGTGTTTTCTTGCGCTTTACAACTTTGTTA

ATGAATTTGCTTATTACGTTCTCAAACAACAGG

ATTTTGATATGCTTCTGAGCATAAAAAATGCATGGC

TTGGCTTAATACAAGCCTACTTGGTGGAGGCGAA

ATGGTACCATAGCAAGTACACACCGAAACTGGAAG

AATACTTGGAAAATGGATTGGTGTCAATAACGGGC

CCTTTAATTATAGCGATTTCATATCTTTCTGGTACAA

ATCCAATCATTAAGAAGGAACTGGAATTTCTAG

AAAGTAATCCAGATATAGTTCACTGGTCATCCAAGA

TTTTCCGTCTGCAAGATGATTTGGGAACTTCATC

GGACGAGATACAGAGAGGGGATGTACCGAAATCAA

TCCAGTGTTACATGCATGAAACTGGTGCCTCGGAG GAAGTTGCTCGTGAGCACATCAAGGATATGATGAG

ACAGATGTGGAAGAAGGTGAATGCATACACAGCGG

ATAAAGACTCTCCCTTGACTCGAACAACTACTGAGT

TCCTCTTGAATCTTGTGAGAATGTCCCATTTTAT

GTATCTACATGGAGATGGGCATGGTGTTCAAAACCA

AGAGACTATCGATGTCGGTTTTACATTGCTTTTT

CAGCCCATTCCCTTGGAGGACAAAGACATGGCTTTC

ACAGCATCTCCTGGCACCAAAGGCTGATGATGAA

TTATAATGCACGATGCGTTGCGAATTCCCAGAGAGT

GCAGTTTCAGTTGATGTTGGCCTCCGCTTTTCTT

TCTTCTGAGGGATCTCTTTTCGATAATAAAATAAAT

TCCCTCATTCATCAAGGTTTATAAATGAAAAAGA

AATGATATATACATATATGTTACTTTTATTGAGAAT

AAAAGTCTTCAGGATATGCAAATA

R-!imonenc MSSCINPSTLVTSINGF CLPLATN AAIRIMAKNKPV synthase from QCLVSAKYDNLTVDRRSANYQPSIWDHDFLQS Citrus sinensis LNSNYTDQTYRRPvAEELKG VKTAIKDVTEPLDQLELI

DNLQRLGLAYHFETEIRNILHNIYNN KDYIW

RKANLYATSLEFRLLRQHGYPVSQEVFSGFKDDKGGF

ICDDFKGILSLHEASYYSLEGESIMEEAWQFTS

KHLKETMIISNSKEEYVFVAEQAKRALELPLHWKVPT

LEARWFIHVYEKREDKNHLLLELAKLEFNTLQA

IYQEELKDISGWWKDTGLGEKLSFARNRLVASFLWSM

GIAFEPQFAYCRRVLTISIALITVIDDIYDVYG

TLDELEIFTDAVARWDI YALKHLPGYMKMCFLALY

NFV EFAYYVLKQQDFDMLLSIKNAWLGLIQAYL

VEAKWYHSKYTPKLEEYLENGLVSITGPLIIAISYLSGT

NPIIKKELEFLESNPDIVHWSSKIFRLQDDL

GTSSDEIQRGDVPKSIQCYMHETGASEEVAREHIKDM

MRQMWKKVNAYTADKDSPLTRTTTEFLLNLVRM

SHFMYLHGDGHGVQNQETIDVGFTLLFQPIPLEDKDM

AFTASPGT G

linalool synthase ATGTGTACTATTATTAGCGTAAATCATCATCATGTG from Mentha GCGATCCTTAGCAAGCCTAAAGTAAAACTTTTCC citrata ACACCAAAAACAAGAGATCAGCTTCAATTAATCTCC

CATGGAGTCTCTCTCCTTCTTCATCCGCCGCCTC

TCGCCCCATCAGTTGTTCTATCTCCTCAAAACTATAT

ACCATCAGTTCGGCTCAGGAGGAAACCCGACGT

TCCGGAAACTACCACCCTTCAGTTTGGGATTTTGAT

TTCATTCAATCTCTCGACACTGATCACTATAAGG

AGGAGAAGCAGTTAGAGAGGGAGGAAGAGCTGATC

ATGGAGGTGAAGAAGTTGTTGGGGGCAAAAATGGA

GGCAACTAAGCAGTTGGAGTTGATTGATGACTTGCA GAATTTGGGATTGTCTTATTTTTTCCGAGACGAG

ATTAAG AATATCTTG AATTCT ATATAT AAAATTTTC C

AAAATAATAATAGTACTAAAGTAGGGGATTTGC

ATTTCACGTCTCTTGGATTCAGGCTCCTCCGGCAGC

ATGGTTTCAACGTTTCACAAGGAGTATTTGATTG

CTTCAAGAACGAGCATGGTAGCGATTTCGAGAAAA

CCCTAATTGGGGAAGATACGAAAGGAGTGCTGCAA

CTTTACGAAGCATCATTCCTTTTGAGAGAAGGTGAA

GATACATTGGAGGTAGCTAGAAAATTCTCCACCG

AATTTCTCGAGGAAAAACTCAAAGCCGGAATCGAT

GGTGATAATCTATCATCATCGATTGGCCATTCTTT

GGAGATCCCTCTTCACTGGAGGATTCAAAGACTAGA

GGAAAGATGGTTCTTAGATGCTTACTCAAGGAGG

AAAGACATGAACCCTATCATTTTCGAGCTCGCCAAA

CTCGACTTCAATATTATTCAAGCAACGCAGCAAG

AAGAACTCAAAGATCTCTCAAGGTGGTGGAATGATT

CAAGCCTACCTCAAAAACTCCCATTTGTGAGGGA

TAGGCTGGTGGAAAGCTACTATTGGGCCCTTGGGTT

GTTTGAGGCTCACAAATTTGGATATGAAAGAAAA

ACTGCTGCAAAGATTATTACCCTAATTACAGCTCTT

GATGATGTTTATGATATTTATGGCACACTCGACG

AGCTCCAACTATTTACACACGTCATTCGAAGATGGG

ATACTGAATCAGCCACCCAACTTCCTTATTACTT

GCAATTATTCTATTTCGTACTATACAACTTTGTTTCC

GAGGTGGCGTACCACATTCTAAAGGAAGAGGGT

TTCATCAGCATCCCATTTCTACACAGAGCGTGGGTG

GATTTGGTTGAAGGATATTTACAAGAGGCAAAGT

GGTACTACACTAAATATACACCAACCATGGAAGAA

TATTTGAACTATGCCAGCATCACAATAGGGGCTCC

TGCAGTAATATCCCAAATTTATTTTATGCTAGCCAA

ATCGAAAGAGAAACCGGTGATCGAGAGTTTTTAC

GAATACGACGAAATAATTCGCCTTTCGGGAATGCTC

GTGAGGCTTCCCGATGACCTAGGAACACTACCGT

TTGAGATGAAGAGAGGCGACGTGGCGAAATCAATC

CAGATTTACATGAAGGAACAGAATGCAACACGGGA

AGAAGCAGAAGAACACGTGAGGTTTATGATTAGGG

AGGCGTGGAAGGAGATGAACACAACTATGGCGGCG

AATTCTGATTTGAGAGGTGATGTGGTTATGGCTGCA

GCTAATCTTGGAAGGGATGCACAGTTTATGTATC

TCGACGGAGACGGTAACCACTCTCAGTTACAACACC

GGATTGCGAACTTGCTGTTCAAGCCATATGTCTGA

linalool synthase MCTII S V HHHVAIL SKPKVi LFHTi NKRS ASI LP WS from Mentha LSPSSSAASRPISCSISSKLYTISSAQEETRRSGNYHPSV citrata WDFDFIQSLDTDHYKEEKQLEREEELIMEV KLLGAK

MEATKQLELIDDLQNLGLSYFFRDEIKNILNSIYKIFQN

N STKVGDLHFTSLGFRLLRQHGFNVSQGVFDCFK E

HGSDFE TLIGEDTKGVLQLYEASFLLREGEDTLEVAR

KFSTEFLEEKLKAGIDGDNLSSSIGHSLEIPLHWRIQRL

EERWFLDAYSRRKDMNPIIFELAKLDFNIIQATQQEEL

KDLSRWWNDSSLPQKLPFVRDRLVESYYWALGLFEA

HKFGYERKTAAKIITLITALDDVYDIYGTLDELQLFTH

VIRRWDTESATQLPYYLQLFYFVLY FVSEVAYHILKE

EGFISIPFLHRAWVDLVEGYLQEAKWYYTKYTPTMEE

YLNYASITIGAPAVISQIYFMLAKSKEKPVIESFYEYDEI

IRLSGMLVRLPDDLGTLPFEMKRGDVAKSIQIYMKEQ

NATREEAEEHVRFMIREAWKEMNTTMAANSDLRGDV

VMAAANLGRDAQFMYLDGDGNHSQLQHRIANLLFKP

YV

γ-terpinene ATGGCTCTTAATCTGCTATCTTCACTACCTGCGGCA synthase from GGCAATTTCACCATATTATCATTACCATTATCAA Citrus unshiu GCAAAGTTAATGGCTTTGTTCCTCCTATTACTCGAGT

CCAATATCCCATGGCTGCATCCACTACTTCTAT

TAAGCCTGTCGATCAAACCATTATTAGGCGATCTGC

CGATTACGGGCCAACCATTTGGAGTTTTGATTAT

ATTCAATCACTTGACAGTAAATATAAAGGAGAATCG

TATGCCAGACAATTGGAAAAGCTGAAGGAACAAG

TAAGCGCGATGCTACAGCAGGATAATAAAGTGGTG

GATTTGGATCCTTTACATCAACTTGAGCTCATCGA

TAATCTGCACAGACTTGGAGTATCTTATCACTTTGA

GGATGAAATAAAAAGAACTTTGGATAGGATACAC

AACAAGAATACTAATGAAAATTTATATGCCACAGC

ACTCAAATTTAGAATCCTAAGGCAATATGGTTACA

ATACACCTGTAAAAGAAACTTTTTCACATTTCATGG

ATGAGAAGGGGAGCTTTAAGTCATCAAGCCACAG

TGACGACTGCAAAGGAATGTTAGCTCTGTATGAAGC

TGCATACCTCCTGGTAGAAGAAGAAAGCAGTATC

TTTCGTGACGCTATAAGGTTCACCACCGCATATCTC

AAAGAATGGGTGGTCAAGCATGATATTGACAAAA

ATGATGATGAATATCTTTGTACATTAGTTAAACATG

CTTTGGAACTTCCATTACATTGGAGGATGCGAAG

ATTGGAGGCAAGGTGGTTCATCGATGTATACGAAA

GTGGACCAGACATGAACCCTATCTTGCTCGAGCTC

GCTAAACTTGACTATAATATTGTGCAAGCAATACAC

CAAGAGGATCTCAAATATGTGTCAAGGTGGTGGA

TGAAAACAGGACTTGGGGAGAAGTTGAATTTTGCA

AGAGACAGAGTAGTGGAGAATTTCTTCTGGACCGT GGGAGATATATTCGAACCTCAGTTTGGATATTGTAG

AAGGATGTCTGCAATGGTTAATTGTCTTTTAACA

TCAATCGATGATGTTTATGATGTCTATGGGACCTTG

GACGAACTTGAGCTATTCACTGATGCAGTTGAGA

GATGGGACGCTACTGCAACAGAGCAACTTCCGTACT

ATATGAAGCTGTGCTTTCATGCTCTCTATAATTC

CGTAAATGAAATGGGTTTTATTGCTCTCAGAGATCA

AGAAGTTGGCATGATCATTCCTTATCTTAAGAAA

GCGTGGGCAGATCAATGCAAATCATATTTAGTGGAG

GCAAAGTGGTACAACAGCGGCTACATACCAACTC

TTCAAGAATATATGGAAAACGCTTGGATTTCAGTAA

CAGCACCTGTAATGCTACTCCATGCGTATGCTTT

TACAGCAAATCCAATAACAAAGGAGGCCTTGGAAT

TCTTGCAGGATTCTCCCGATATAATTCGTATTTCA

TCAATGATTGTACGACTTGAAGACGATTTGGGAACA

TCATCGGATGAGCTGAAGAGGGGAGATGTTCCCA

AATCAATTCAATGTTACATGCATGAAACTGGAGTTT

C AGAGGATGAGGCTC GTGAAC AT ATAC GAGATTT

GATTGCTGAGACATGGATGAAGATGAACAGTGCTC

GATTCGGAAACCCACCTTACTTGCCCGATGTTTTC

ATTGGGATTGCAATGAATTTGGTGAGGATGTCTCAA

TGCATGTACCTATATGGAGATGGACACGGTGTAC

AAGAA AATAC C AAGG ATC GTGT ATTGTCTTT ATTT A

TTGATCCCATTCCTTAA

γ-terpinene MALNLLSSLPAAGNFTILSLPLSSKVNGFVPPITRVQYP synthase from MAASTTSIKPVDQTIIRRSADYGPTIWSFDY

Citrus unshiu IQSLDS YKGESYARQLEKLKEQVSAMLQQDNKVVD

LDPLHQLELIDNLHRLGVSYHFEDEIKRTLDRIH

NKNTNENLYATALKFMLRQYGYNTPVKETFSHFMDE

KGSFKSSSHSDDCKGMLALYEAAYLLVEEESSI

FRDAIRFTTAYLKEWVVKHDIDK DDEYLCTLVKHAL

ELPLHWRMRRLEARWFIDVYESGPDMNPILLEL

AKLDYNIVQAIHQEDLKYVSRWWMKTGLGEKLNFAR

DRVVENFFWTVGDIFEPQFGYCRRMSAMVNCLLT

SIDDVYDVYGTLDELELFTDAVERWDATATEQLPYY

MKLCFHALYNSVNEMGFIALRDQEVGMIIPYLKK

AWADQCKSYLVEAKWYNSGYIPTLQEYMENAWISVT

AP VMLLHAYAFTANPIT EALEFLQD SPDIIRI S

SMIVRLEDDLGTSSDELKRGDVPKSIQCYMHETGVSE

DEAREHIRDLIAETWMKMNSARFGNPPYLPDVF

IGIAMNLVRMSQCMYLYGDGHGVQENTKDRVLSLFID

PIP

γ-terpinene ATGGCCTCACTGCAAGTCGAGGAAGAAACCCGGCG synthase from Thymus caespititius TTCTGGGAACTACCAGGCTTCCATTTGGGACAATG CTTTCATTCAATCTTTCAATACAAATAAATATAGGG ACGAGAAGCACTTGAACAGGAAAGAAGAGCTGAT TGCACAAGTGAAGGTACTGTTGAACACAAAAATGG AGGCTGTTAAGCAATTGGAGTTGATTGATGACTTG AGAAATCTAGGGTTGACTTATTATTTTCAAGATGAG TTTAAGAAGATTCTTACTTGTATATATAATGATC ACAAATGTTTCAAAAACGAACAAGTTGGGGATTTGT ACTTCACATCTCTTGGATTCAGACTCCTCAGACT ACACGGTTTCGATGTCTCAGAAGAAGTGTTTAGCTT TTTTAAGAACGAGGATGGTAGTGATTTCAAGGCG AGCCTTGGTGAAAATACGAAGGACGTATTGCAACTT TACGAGGCATCGTTCCTTGTAAGGGTAGGTGAAG TTACACTGGAGCAAGCAAGGGTATTTTCCACTAAAA TTCTGGAAAAGAAAGTCGATGAGGGAATTAATGA TGAAAAATTATTAGCATGGATTCAACATTCTTTGGC TCTCCCTCTTCACTGGAGGATTCAAAGGCTAGAG GCGAGATGGTTCTTAGATGCTTACGCGGCGAGGAA GGACATGAATCCTCTTATCTTCGAGCTCGGGAAAA TAGACTTCCATATTATTCAAGAAACACAACTAGAAG AAGTCCAAGAGGTTTCGAGGTGGTGGACTAATTC TAACCTCGCCGAGAAACTGCCATTTGTGAGAGATAG AATTGTGGAGTGCTACTTTTGGGCGCTTGGGCTC TTTGAGCCACATGAATATGGATACCAGAGAAAAAT GGCCGCAATTATCATCACTTTCGTTACAATCATAG ACGATGTTTACGACGTCTATGGAACACTCGACGAAC TGCAGCTATTCACGGACGCGATTCGAAAATGGGA CTTTGAATCAATAAGCACACTTCCATATTACATGCA AGTTTGCTATTTGGCACTCTACACCTATGCTTCT GAGCTGGCTTATGATATTCTCAAAGATCAGGGTTTC AACAGCATCTCATACCTACAGAGATCGTGGCTGA GTTTGGTCGAAGGGTTTTTCCAAGAGGCAAAATGGT ACTACGCTGGATACACGCCGACCCTAGCAGAATA CCTAGAGAACGCCAAAGTTTCAATATCGTCTCCAAC TATTATATCTCAAGTTTACTTCACTCTCCCGAAT TCGACTGAGAGAACGGTTGTCGAGAACGTCTACGG ATACCACAACATACTCTATCTTTCCGGCATGATTT TAAGGCTTGCTGATGATCTTGGTACAACTCAGTTTG AGCTGAAGAGAGGGGACGTGCAAAAGGCGATCCA GTGCTACATGAAGGACAACAATGCCACAGAGAAAG AAGGGCAAGAGCACGTGAAGTATCTGTTGCTAGAG GCGTGGAAGGAGATGAACACGGCGATGGCGGACCC CGACTGCCCGTTGTCTGAGGATCTGGTGGATGCAG CAGCTAATCTGGGAAGAGCATCTCAGTTCATATATC TCGAAGGAGATGGCCATGGCGTGCAGCACTCGGA GATTCATAACCAGATGGGAGGCCTTATTTTCGAGCC ATATGTGTGA

γ-terpinene MASLQVEEETRRSGNYQASIWDNAFIQSFNTNKYRDE synthase from KHLNRKEELIAQVKVLLNTKMEAVKQLELIDDL Thymus caespititius R LGLTYYFQDEFKKILTCIYNDH CFK EQVGDLYF

TSLGFRLLRLHGFDVSEEVFSFFKNEDGSDFKA

SLGENTKDVLQLYEASFLVRVGEVTLEQARVFSTKILE

KKVDEGINDEKLLAWIQHSLALPLHWRIQRLE

ARWFLDAYAARKDMNPLIFELGKIDFHIIQETQLEEVQ

EVSRWWTNSNLAEKLPFVRDRIVECYFWALGL

FEPHEYGYQRKMAAIIITFVTIIDDVYDVYGTLDELQLF

TDAIR WDFESISTLPYYMQVCYLALYTYAS

ELAYDILKDQGFNSISYLQRSWLSLVEGFFQEAKWYY

AGYTPTLAEYLENAKVSISSPTIISQVYFTLPN

STERTVVENVYGYHNILYLSGMILRLADDLGTTQFEL

KRGDVQKAIQCYMKO NATEKEGQEHVKYLLLE

AWKEMNTAMADPDCPLSEDLVDAAANLGRASQFIYL

EGDGHGVQHSEIHNQMGGLIFEPYV

γ-terpinene ATGGCTACCCTTAGCATGCAAGTGTCCATACTTAGC synthase from AAGGAAGTGAAAAATGTCAACAACATTGGCATGA Origanum vulgare GAGCATCTAAACCAATGGTGGCGAGGCGCGTCTCTA

CCACTCGTCTCCGGCCTATTTGCTCCGCCTCACT

GCAAGTCGAAGAAGAAACCCGACGTTCCGGAAACT

ACCAGGCTTCAATTTGGAACAATGATTACGTTCAA

TCTTTCAACACAAATCAATATAAGGACGAGAAGCA

CTTGAAAAAGAAAGAAGAGCTGATTGCACAAGTAA

AGATATTGTTGAACACAAAAATGGAGGCTGTTAAA

CAATTGGAGTTGATTGAAGACTTGAGAAATCTAGG

GTTGACTTATTATTTTCAAGATGAGGTTAAGAAGAT

TCTTACTTCTATATATAATGATCACAAATGTTTC

AAAAACGAACAAGTTGGGGATTTGTATTTTACTTCT

CTTGGATTCAGACTCCTCAGACTGCACGGTTTCG

ATGTCTCAGAAGAGGTGTTTGACTTTTTTAAGAACG

AGGATGGTAGTGATTTCAAGGCGAGCCTTGGTGA

AAATATAAAAGACGTATTGCAGCTTTACGAAGCATC

TTTCCTTATAAGGGAAGGTGAAGTTATACTGGAG

CAAGCAAGAGTATTTTCCACCAAACATCTTGAAAAG

AAAGTTGATGAGGGAATTAATGATGAAAAATTAT

TAGCATGGATTCGCCATTCTTTGGCTCTCCCTCTTCA

TTGGAGGATTCAAAGGCTAGAGGCGAGGTGGTT

CTTAGATGCTTACAGGGCGAGGAAAGACATGATTCC TCTTATTTTCGAGCTCGGGAAAATCGACTTCCAT

ATCATTCAAGAAACACAACTAGAAGAACTCCAAGA

AGTCTCAAAGTGGTGGACTAATTCAAACCTCGCCG

AGAAACTCCCATTTGTGAGAGATAGAATTGTGGAGT

GCTACTTTTGGGCGCTTGGGCTCTTTGAACCACA

TGAGTATGGTTATCAGAGGAAAATGGCTGCCATTAT

TATCACTTTCGTTACGATCATAGACGATGTTTAC

GACGTCTACGGTACACTCGACGAACTGCAGCTATTC

ACCGACGCGATTCGAAAATGGGACTTTCAATCAA

TAAGCACACTTCCATACTACATGCAAGTTTGCTATT

TGGCACTCTACACCTATGCTTCTGAACTGGCTTA

TGATATTCTCAAAGATCAAGGTTTCAACAGTATTGC

TTATCTACAAAGATCGTGGCTGAGTTTGGTGGAA

GGATTTTTCCAAGAGGCAAAATGGTACTACGCCGGG

TACACGCCAACCCTAGCAGAATACCTAGAGAACG

CCAAAGTTTCAATATCATCTCCTACTATTATCTCTCA

AGTTTACTTCACTCTTCCGAATTCGACTGAGAG

AACGGTTGTCGAAAACGTCTTCGGATACCACAACAT

ACTCTACCTTTCCGGAATGATTTTAAGGCTTGCA

GATGATCTTGGCACTACTCAGTTTGAGCTGAAGAGA

GGGGACGTGCAAAAGGCAATCCAGTGTTACATGA

AGGACAACAATGCTACAGAGAAAGAAGGGGCTGAG

CATGTGAAGTATCTGTTACGAGAAGCGTGGAAGGA

GATGAACACGGCGATGGCGGACCCCGAGTGCCCGT

TGTCGGAAGATCTGGTGGATGCTGCTGCTAATCTG

GGGAGAGCATCTCAGTTCATATATCTGGAAGGAGAT

GGCCACGGCGTTCAGCACTCAGAGATTCATAACC

AAATGGGAGGCCTTATTTTCGAGCCATATGTGTGA

γ-terpinene MATLSMQVSILSKEVKNV NIGMRASKPMVARRVST synthase from TRLRPIC SASLQVEEETRRSGNYQASIW DYVQ Origanum vulgare SFNTNQYKDEKHLKKKEELIAQVKILLNTKMEAVKQL

ELIEDLR LGLTYYFQDEVKKILTSIY DHKCF

K EQVGDLYFTSLGFRLLRLHGFDVSEEVFDFFK ED

GSDFKASLGENIKDVLQLYEASFLIREGEVILE

QARVFSTKHLEKKVDEGINDEKLLAWIRHSLALPLHW

RIQRLEARWFLDAYRARKDMIPLIFELGKIDFH

IIQETQLEELQEVSKWWTNSNLAEKLPFVRDRIVECYF

WALGLFEPHEYGYQRKMAAIIITFVTIIDDVY

DVYGTLDELQLFTDAIRKWDFQSISTLPYYMQVCYLA

LYTYASELAYDILKDQGFNSIAYLQRSWLSLVE

GFFQEA WYYAGYTPTLAE YLEN A V SI S SPTIIS Q V Y

FTLPNSTERTVVENVFGYHNILYLSGMILRLA

DDLGTTQFELKRGDVQKAIQCYMKD ATEKEGAEH VKYLLREAWKEMNTAMADPECPLSEDLVDAAANL GRASQFIYLEGDGHGVQHSEIHNQMGGLIFEPYV

Ε-β-ocimene ATGGCCCAGAGCTTTTCCATGGTGCTCAATTCGTCC synthase from TTCACTTCACATCCTATTTTTTGCAAACCTCAAA Lotus japonicas AGCTAATTATAAGAGGGCATAATCTACTTCAAGGGC

ACAGAATTAATTCCCCAATTCCATGCTATGCAAG

CACTAGCAGCACAAGTGTGTCACAAAGAAAATCAG

CCAATTACCAACCTAACATTTGGAATTACGATTAT

TTGCAGTCCTTAAAGCTTGGTTATGCGGATGCACAT

TATGAGGATATGGCTAAAAAGTTGCAAGAGGAAG

TGAGAAGAATAATTAAGGATGACAAAGCAGAGATT

TGGACTACACTAGAGCTTATTGATGATGTGAAACG

CTTGGGTCTTGGCTATCACTTTGAAAAGGAGATAAG

AGAGGTTCTTAACAAGTTTCTATCTTTGAACACA

TGTGTTCATAGAAGCTTGGATAAGACTGCTCTATGC

TTTAGGCTCTTGAGAGAATACGGCTCCGATGTAT

CAGCAGATATTTTTGAGAGATTCTTGGACCAAAATG

GTAATTTCAAGACAAGTCTTGTCAATAATGTAAA

AGGAATGTTGAGTCTCTATGAGGCATCATTTCTTTCT

TATGAAGGAGAACAGATTTTGGATAAGGCCAAT

GCTTTCACTAGCTTTCATCTCAAGAGCATCCATGAA

GAAGATATAAATAACATTCTCTTAGAACAAGTGA

ATCATGCATTGGAGCTTCCACTACATCGTCGTATCC

ACAGGCTTGAGGCCCGGTGGTACACTGAGTCATA

TTCAAGAAGAAAGGATGCAAATTGGGTGTTGCTTGA

AGCAGCTAAACTGGATTTCAACATGGTTCAATCA

ACACTGCAAAAAGATCTCCAAGAAATGTCAAGGTG

GTGGAAGGGGATGGGGCTTGCCCCAAAGTTAAGCT

TCAGTCGTGATAGATTAATGGAGTGCTTCTTTTGGA

CGGTTGGGATGGCTTTTGAGCCAAAATACAGTGA

TCTTCGCAAAGGTTTAACCAAAGTCACCTCTTTAAT

AACTACAATTGATGACATTTATGATGTGCATGGA

ACCTTGGAAGAATTAGAGCTTTTCACAGCAATTGTG

GAAAGTTGGGACATTAAAGCAATGCAAGTTCTCC

CAGAATACATGAAGATAAGCTTCTTAGCCCTCTACA

ACACAGTCAATGAATTGGCTTATGATGCACTTAG

AGAACAAGGGCATGATATCCTACCCTACCTCACTAA

AGCATGGTCTGATATGTTGAAAGCTTTCCTACAA

GAAGCAAAGTGGTGCCGAGAAAAACACTTGCCAAA

ATTTGAGCATTATCTCAATAATGCTTGGGTCTCAG

TGTCTGGTGTAGTTATACTAACTCATGCCTATTTCTT

GCTGAATCACAACACAACAAAGGAGGTACTTGA

GGCCTTGGAAAATTACCATGCTCTGTTAAAAAGACC ATCCATAATTTTTCGACTTTGCAATGATTTGGGT

ACATCAACGGCGGAGTTACAGAGAGGTGAAGTAGC

AAATTCAATTTTATCCTGCATGCATGAAAATGATA

TTGGTGAAGAGAGTGCTCACCAACACATTCATAGTT

TGCTTAATGAAACTTGGAAGAAGATGAATAGAGA

TAGGTTCATCCACTCACCTTTCCCAGAACCTTTTGTG

GAAATAGCAACCAACCTAGCCAGAATTGCTCAG

TGTACGTACCAAACTGGAGATGGGCATGGAGCCCC

GGATAGTATAGCAAAGAATCGAGTCAAATCATTGA

TAATTGAACCCATTGTTCTCAATGGAGACATATATT

AA

Ε-β-ocimene MAQSFSMVLNSSFTSHPIFCKPQKLII GHNLLQGHRIN synthase from SPIPCYASTSSTSVSQRKSANYQPNIW YDY

Lotus japonicas LQSLKLGYADAHYEDMAKKLQEEVRRIIKDDKAEIWT

TLELIDDVKRLGLGYHFEKEIREVLNKFLSLNT

CVHRSLD TALCFRLLREYGSDVSADIFERFLDQNGNF

KTSLV VKGMLSLYEASFLSYEGEQILDKAN

AFTSFHLKSIHEEDI NILLEQVNHALELPLHRRIHRLE

ARWYTESYSRRi DANWVLLEAAKLDFNMVQS

TLQKDLQEMSRWWKGMGLAPKLSFSRDRLMECFFW

TVGMAFEPKYSDLRKGLTKVTSLITTIDDIYDVHG

TLEELELFTAIVESWDIKAMQVLPEYMKISFLALYNTV

NELAYDALREQGHDILPYLTi AWSDMLKAFLQ

EAKWCREKHLPKFEHYLN AWVSVSGVVILTHAYFL

LNHNTTKEVLEALE YHALLKRPSIIFRLCNDLG

TSTAELQRGEVANSILSCMHENDIGEESAHQHIHSLLN

ETWKKMNRDRFIHSPFPEPFVEIATNLARIAQ

CTYQTGDGHGAPDSIAK RVKSLIIEPIVLNGDIY

Ε-β-ocimene ATGCCTAAACGACAGGCTCAACGGCGTTTCACTCGC synthase from AAGACTGACTCGAAAACACCATCCCAGCCTCTGG Arabidopsis TATCCCGTCGCTCTGCAAACTATCAACCGTCTCTTTG thaliana GCAGCACGAATATCTCCTCTCGCTCGGTAATAC

ATATGTGAAAGAGGACAACGTCGAGAGAGTTACGT

TATTGAAGCAGGAAGTGAGTAAAATGCTCAATGAA

ACGG AAGGTTTACTC GAAC AGCTAG AGCTC ATC GAC

ACTTTACAAAGGCTTGGAGTTTCTTACCATTTTG

AACAAGAAATCAAGAAGACACTAACGAATGTGCAT

GTTAAAAATGTGCGAGCACACAAAAACCGGATAGA

TCGAAACCGATGGGGAGATTTATACGCGACCGCCCT

TGAGTTCCGACTCCTAAGGCAACATGGTTTCAGT

ATCGCACAAGATGTTTTTGACGGAAATATTGGAGTT

GATTTGGATGATAAAGACATCAAGGGTATTCTTT

CACTATACGAAGCTTCATATCTCTCGACCAGAATCG ATACTAAATTGAAAGAGAGCATATACTATACAAC

AAAACGACTTAGAAAATTTGTGGAGGTAAATAAGA

ATGAGACCAAATCTTACACTCTTCGAAGGATGGTT

ATACATGCGTTAGAGATGCCGTACCACCGGAGAGT

GGGAAGACTAGAAGCAAGATGGTACATAGAAGTGT

ACGGAGAGAGACACGACATGAACCCTATCTTGCTTG

AACTCGCGAAACTTGATTTTAATTTCGTACAAGC

TATCCATCAAGACGAGCTCAAATCCCTCTCTAGTTG

GTGGAGCAAGACGGGATTAACAAAACACCTCGAT

TTCGTTAGAGATCGAATAACGGAGGGTTATTTCTCG

AGTGTTGGAGTAATGTATGAGCCCGAGTTTGCAT

ATCACCGACAAATGCTTACAAAGGTTTTCATGCTCA

TTACAACTATCGACGATATATACGATATTTATGG

GACACTTGAGGAGCTCCAACTATTCACGACCATAGT

TGAAAAATGGGATGTGAATCGTCTTGAAGAACTT

CCCAACTACATGAAGTTATGTTTTCTCTGCCTCGTCA

ACGAAATCAATCAGATTGGATATTTTGTACTCA

GAGATAAAGGGTTTAATGTGATTCCTTACCTCAAAG

AATCTTGGGCAGATATGTGTACAACGTTTTTGAA

AGAGGCAAAGTGGTATAAAAGTGGTTACAAACCTA

ACTTCGAAGAATACATGCAAAATGGTTGGATCTCA

AGCTCAGTCCCTACAATACTTCTACACTTGTTCTGTC

TCTTATCCGACCAAACCTTAGACATTCTTGGCT

CCTACAATCACTCTGTAGTTCGAAGCTCCGCCACCA

TCCTCCGTCTCGCTAACGATCTCGCCACTTCTTC

GGAGGAATTAGCGAGAGGCGACACTATGAAATCCG

TACAATGTCACATGCATGAAACTGGAGCTTCGGAG

GCAGAGTCACGCGCGTACATTCAAGGAATTATCGGT

GTGGCTTGGGATGACTTAAACATGGAGAAAAAGA

GTTGTAGGCTACATCAAGGTTTCCTAGAAGCTGCGG

CTAATCTTGGACGTGTGGCTCAGTGCGTTTATCA

GTACGGTGATGGCCATGGCTGTCCTGACAAGGCTAA

GACCGTCAATCATGTCCGGTCCTTGCTCGTCCAC

CCTCTTCCACTCAATTAA

Ε-β-ocimene MPKRQAQRRFTRKTDSKTPSQPLVSRRSANYQPSLWQ synthase from HEYLLSLGNTYVKEDNVE VTLLKQEVSKMLNE

Arabidopsis TEGLLEQLELIDTLQRLGVSYHFEQEIKKTLTNVHVKN thaliana VRAHKNRIDRNRWGDLYATALEFRLLRQHGFS

IAQDVFDGNIGVDLDDKDI GILSLYEASYLSTRIDTKL

KESIYYTTKRLRKFVEVNKNETKSYTLRRMV

IHALEMPYHRRVGRLEARWYIEVYGERHDMNPILLEL

AKLDFNFVQ AIHQDELKSLS S WWSKTGLTKHLD

F VRDRITEGYF S S VGVMYEPEF AYHRQMLTKVFMLIT TIDDIYDIYGTLEELQLFTTIVEKWDV RLEEL

PNYMKLCFLCLV EINQIGYFVLRDKGFNVIPYLKESW

ADMCTTFLKEA WYKSGYKPNFEEYMQNGWIS

SSVPTILLHLFCLLSDQTLDILGSYNHSWRSSATILRLA

NDLATSSEELARGDTM SVQCHMHETGASE

AESRAYIQGIIGVAWDDLNMEK SCRLHQGFLEAAAN

LGRVAQCVYQYGDGHGCPDKA TV HVRSLLVH

PLPLN

Figure legends

Figure 1 The Mevalonate pathway

Figure 2 The MEP pathway

Figure 3 Plasmid map of integration vector

Figure 4 Plasmid map of broad host self-replicating plasmid

Figure 5 GC-results: Counts vs acquisition time. Part where limonene elutes (around 6.75 minutes) is zoomed

Figure 6 GC-results: FID units pA vs acquisition time. Part where linalool elutes (around 7.2 minutes) is zoomed

Figure 7 Linalool production from a batch culture

Examples

The present invention is further described by the following examples which should not be construed as limiting the scope of the invention.

Unless stated otherwise, the practice of the invention will employ standard conventional methods of molecular biology, virology, microbiology or biochemistry.

Such techniques are described in Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press; in Sambrook and Russell (2001) Molecular Cloning: A Laboratory Manual, Third Edition, Cold Spring Harbor Laboratory Press, NY; in Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA; and in Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK); Oligonucleotide Synthesis (N. Gait editor); NucleicAcid Hybridization (Hames and Higgins, eds.). Example 1. Enzymes for production of the monoterpene limonene.

The inventors have introduced a specific two-enzyme pathway into a cyanobacterial cell to produce limonene.

Limonene is a simple cyclic CIO terpene with no rare groups. Limonene is chiral and exists in two enantiomeric forms R-limonene and S-limonene. In nature R-limonene is the most abundant and is commercially harvested from citrus rinds. The other enantiomer S-limonene enantiomer is also found in nature and is the precursor for menthol.

Limonene like other monoterpenes is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by Geranyl diphosphate synthase (GPPS) to form geranyl diphosphate (GPP). In the second step, the enzyme Limonene Synthase (LS) catalyzes the cyclization of GPP to limonene. To make limonene in Synechocystis, we chose the following two GPP synthases (1) The gene gpps from Abies grandis (SEQ ID NO: 1, 2) and (2) 197A mutant of erg20 gene, the FPP synthase from Saccharomyces cerevisiae (SEQ ID NO: 3, 4). We chose the limonene synthase (LS) from Mentha spicata which is specific for S-limonene (SEQ ID NO: 5, 6). The LS from Mentha spicata and the GPPS from Abies grandis were also truncated to remove the N-terminal plastidic targeting signals. Example 2. Biochemical Background of a cyanobacterial cell according to the present invention

The genes encoding the LS from Mentha spicata and the GPPS from Abies grandis were co don-optimized for expression in Synechocystis and obtained through chemical synthesis. While the erg20 gene was amplified from Saccharomyces cerevisiae and the mutation Kl 97A was introduced by overlap-extention PCR. These genes were each cloned with a trc promoter into an integration vector, containing sequences to facilitate (double) homologous recombination with the neutral site slrO 168 in the cyanobacterial genome, and a kanamycin marker, which confers resistance to kanamycin. The genes were introduced either as operons, with both genes sharing the same trc promoter or as independent transcription cassettes, with a trc promoter for each gene. This led to making of 4 plasmids,

1. LS from Mentha spicata and the GPPS from Abies grandis as operon (LG-op)

2. LS from Mentha spicata and the GPPS from Abies grandis as cassette (GL-cas)

3. LS from Mentha spicata and the ERG20 mutant from Saccharomyces cerevisiae as operon (EL-op) 4. LS from Mentha spicata and the ERG20 mutant from Saccharomyces cerevisiae as cassette (EL-cas)

Each plasmid was transformed into Synechocystis PCC 6803 as described in patent application EP2563927.

Example 3. Production of limonene by a cyanobacterial cell

Mutant cultures were grown to an OD730 = 1 to 3 and were used for limonene measurements. 2mL or 4mL of a select culture was transferred to a 20 mL glass vial and sealed. 10 to 20 mM of bicarbonate was also added to each vial and the vial incubated in low light intensity (~40 μΕ), 30° C, and shaking at 120 rpm light overnight. Next day, the vial was loaded onto an automated GCMS (Agilent Technologies 7200 Accurate-Mass Q-TOF GCMS). In the first step, the vial was heated for 10 min at 55 deg C, to release all volatiles into the headspace. Then a needle carrying a SPE (solid phase extraction) cartridge was inserted into the vial and incubated for 10 minutes, to allow all volatiles to bind. The needle was them injected into the GC and volatiles loaded onto the column and separated and determined by MS. Similar experiments were done with a known amount of Limonene to obtain a standard curve as well as with a wild- type culture as negative control. Limonene elutes at a retention time of around 6.75 minutes. All four strains obtained in example 2: LG- op, GL-cas, EL-op, and EL-cas, were tested. Figure 5 shows the counts vs acquisition time plots obtained from GC analysis. From the figure, it is evident that LG-op, GL-cas, EL-op, and EL-cas all produce limonene while the wild-type strain did not produce any limonene. In addition, the results show that best combination of enzymes is the LS from Mentha spicata and the GPPS from Abies grandis. Moreover, yields are higher when the genes are inserted into Synechocystis as individual transcription cassettes wherein each gene is operably linked to its native promoter.

Example 4. Enzymes for production of the monoterpene linalool

The inventors have introduced a specific two-enzyme pathway into a cyanobacterial cell to produce linalool.

Linalool is a non cyclic CIO terpene with a hydroxyl-group. It is chiral and exists in two enantiomeric forms (R)-(-)-linalool also known as licareol and (S)-(+)-linalool also known as coriandrol. (S)-(+)-Linalool is perceived as sweet, floral, petit grain- like and the (R)-form as more woody and lavender-like. In nature R-linalool found in lavender oil while the other enantiomer S-linalool is found in coriander oil.

Linalool like limonene and other monoterpenes is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by Geranyl diphosphate synthase (GPPS) to form geranyl diphosphate (GPP). In the second step, the enzyme Linalool Synthase (LS) catalyzes the isomerization/hydrolysis of GPP to linalool. To make linalool in Synechocystis, we chose the following GPP synthase : The gene gpps from Abies grandis (SEQ ID NO: 1, 2). We chose the linalool synthase (LinS) from Mentha citrata which is specific for R-linalool (SEQ ID NO: 17, 18). The LinS from Mentha citrata and the GPPS from Abies grandis were also truncated to remove the N-terminal plastidic targeting signals.

Example 5. Enzymes for production of γ-terpinene

Together with the GPPS (SEQ ID NO: 1, 2) mentioned above, γ-terpinene can be made using the the γ-terpinene synthase from Citrus unshiu (SEQ ID NO: 19, 20); Thymus caespititius (SEQ ID NO: 21, 22), Origanum vulgare (SEQ ID NO: 23, 24).

Example 6. Enzymes for production of Ε-β-ocimene synthase

Together with the GPPS (SEQ ID NO: 1, 2) mentioned above, Ε-β-ocimene can be made using the the Ε-β-ocimene synthase from Lotus japonicus (SEQ ID NO: 25, 26) and Arabidopsis thaliana (SEQ ID NO: 27, 28).

Example 6. Biochemical Background of a cyanobacterial cell producing linalool according to the present invention

The genes encoding the Linalool synthase (LinS) from Mentha citrata and the GPPS from Abies grandis as described in Example 4 were co don-optimized for expression in Synechocystis and obtained through chemical synthesis. These genes were each cloned with a trc promoter into an integration vector (Figure 3), containing sequences to facilitate (double) homologous recombination with the neutral site slr0168 in the cyanobacterial genome, and a kanamycin marker, which confers resistance to kanamycin. The genes were introduced as independent transcription cassettes, with a trc promoter for each gene. The genes were also cloned into a RSFlOlO-based conjugative plasmid pVZ (Figure 4) as independent transcription cassettes. This led to the provision of two plasmids,

1. Integration plasmid with Linalool synthase (LinS) from Mentha citrata and the GPPS from Abies grandis (integrated) 2. Conjugative plasmid pVZ with Linalool synthase (LinS) from Mentha citrata and the GPPS from Abies grandis (plasmid)

Each plasmid was transformed into Synechocystis PCC 6803 as described in patent application EP2563927.

Example 7. Production of linalool by a cyanobacterial cell

Mutant cultures were grown from an OD730 = 0.5 to about OD730 = 10, a 1L photobioreactors. The photobioreactors were bubbled with air/carbon-dioxide mixture and linalool formed was trapped on Supelpak SV reisn. The bound terpene was eluted with hexane and the eluate was analyzed by GC FID. Standard solution of linalool in hexane were used to obtain a calibration curve for quantitative determination. A wild- type culture was used a negative control. Linalool elutes at a retention time of around 7.2 minutes. Both strains obtained in example 2: integrated and plasmid were tested. Figure 6 shows the FID units vs acquisition time plots obtained from GC analysis. From the figure, it is evident that both strains tested produce linalool while the wild-type strain did not produce any linalool. Figure 7 shows that linalool can be produced in continuously growing cultures and maximum production rates of about 120 μg/gDW/L/day were achieved.

Reference list

1. Computational Molecular Biology, Lesk, A. M., ed., Oxford University Press, New York, 1988.

2. Biocomputing: Informatics and Genome Projects, Smith, D. W., ed., Academic Press, New York, 1993.

3. Computer Analysis of Sequence Data, Part I, Griffin, A. M., and Griffin, H. G., eds., Humana Press, New Jersey, 1994.

4. Sequence Analysis in Molecular Biology, von Heine, G., Academic Press, 1987.

5. Sequence Analysis Primer, Gribskov, M. and Devereux, J., eds., M Stockton Press, New York, 1991.

6. Carillo, H., and Lipman, D., SIAM J. Applied Math., 48: 1073, 1988.

7. Devereux, J., et al., Nucleic Acids Research 12 (1): 387, 1984.

8. Altschul, S. F. et al., J. Mol. Biol. 215:403-410, 1990. 9. BLAST Manual, Altschul, S., et al., NCBI NLM NIH Bethesda, MD 20894; Altschul, S., et al, J. Mol. Biol. 215:403-410, 1990.

10. Needleman and Wunsch, J. Mol. Biol. 48:443-453, 1970.

11. Hentikoff and Hentikoff, Proc. Natl. Acad. Sci. USA. 89:10915-10919, 1992.

12. Puigbo, E. Guzman, A. Romeu, and S. Garcia- Vallve. Nucleic Acids Res. 2007 July; 35 (Web Server issue): W126-W131.

13. Sambrook and Russel (2001) "Molecular Cloning: A Laboratory Manual (3 ^rd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press.

14. Ausubel et al, eds., "Current protocols in molecular biology", Green Publishing and Wiley Interscience, New York, 1987.

15. Cheah et al, (2013) Biotechnol Prog 2013, 29:23-30.

16. Shestakov S V et al, (2002), Photosynthesis Research, 73: 279-284

17. Nakamura Y et al, (1999), Nucleic Acids Res. 27:66-68

18. Sambrook et al. (1989) Molecular Cloning, A Laboratory Manual (2nd edition), Cold Spring Harbor Laboratory, Cold Spring Harbor Laboratory Press

19. Volumes 1 and 2 of Ausubel et al. (1994) Current Protocols in Molecular Biology, Current Protocols, USA.

20. Volumes I and II of Brown (1998) Molecular Biology LabFax, Second Edition, Academic Press (UK).

21. Brosius et al, J Biol Chem 1985

22. Huang H-H, Camsund D, Lindblad P, Heidorn T: Design and characterization of molecular tools for a Synthetic Biology approach towards developing cyanobacterial biotechnology. Nucleic Acids Res 2010, 38:2577-2593.