WO2016008885A1 - Biosynthesis of sesquiterpenes in cyanobacteria - Google Patents

Abstract

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

Description

Biosynthesis of sesquiterpenes in cyanobacteria

Field of the invention

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

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, Enter ococcus, 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

Sesquiterpenes have been known for several centuries as components of the fragrant oils obtained from leaves, flowers and fruits. Sesquiterpenes, with monoterpenes, are the main constituents of essential oils. 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 C0₂ has recently been reported, there is still a need for an improved process for the biosynthesis of sesquiterpenes, 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 sesquiterpene 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 sesquiterpene 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 C0₂ 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 farnesyl diphosphate synthase (FPPS) and a sesquiterpene synthase (STS). 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 sesquiterpene selected from the group consisting of: artemisinin, bisabolol, farnesene, valencene, santalene and bergamotene. More preferably, the sesquiterpene is santalene, bisabolol, valencene or farnesene; most preferably, the sesquiterpene is valencene or santalene.

The term "functional enzyme" is herein preferably defined in the context of a farnesyldiphosphate synthase (FPPS) as an enzyme that first catalyzes the condensation of two C5 co-substrates, isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), to produce geranyl diphosphate (GPP) and then catalyzes a second condensation step of GPP and IPP to produce farnesyl diphosphate (FPP). The FPP product serves as precursor to sesquiterpenes, sterols, dolichols, and is used for protein farnesylation. In addition, farnesyl diphosphate serves as the primer for the biosynthesis of dolichols and the isoprenoid moiety for polyprenyl quinones. The latter reaction is catalyzed by solanesyl diphosphate synthase (SPPS), yielding the C45-moiety of ubiquinones and plastoquinones. FPPSs were purified to homogeneity from a number of organisms, including Saccharomyces cerevisiae, chicken, pig, human, and green pepper, Capsicum annuum. The term "functional enzyme" is herein preferably defined in the context of a sesquiterpene synthase as an enzyme able to convert the acyclic FPP produces by the FPPS enzyme into a variety of cyclic and acyclic forms.

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 and further to FPP. The at least one functional 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 FPPS from E. coli, Methanothermobacter thermautotrophicus, Mentha x piperita and Saccharomyces cerevisiae. More preferably, the FPPS is from Saccharomyces cerevisiae. In a cyanobacterial cell according to the present invention, at least one functional enzyme is preferably selected from the group consisting of sesquiterpene synthases, which are enzymes having the ability of converting FPP to various cyclic or acyclic sesquiterpenes. The enzyme may be native or may be heterologous to the cyanobacterial cell and is preferably selected from the group consisting of sesquiterpene synthases from Malus domestica, Ricinus communis, Solanum habrochaites, Santalum spicatum, Artemisia annua, Callitropis nootkatensis, Citrus sinensis and Santalum album. More preferably, the sesquiterpene synthase is selected from the group consisting of the Farnesene synthase from Malus domestica, the bisabolol synthase from Artemisia annua, the valencene synthase from Callitropis nootkatensis, the valencene synthase from Citrus sinensis and the santalene synthase from Santalum album. More preferably, the sesquiterpene synthase is the santalene synthase from Santalum album or the valencene synthase from Callitropis nootkatensis. Further, the functional enzyme may be an N-terminal truncated version of the original protein, while substantially maintaining its sesquiterpene synthase activity. Preferably, at least two functional enzymes are heterologous to the cyanobacterial cell.

Preferably, a cynanobacterial 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 Farnesyl diphosphate synthase (FPPS) and a sesquiterpene synthase (STS), wherein the at least one functional enzyme is selected from the group consisting of FPPS from E. coli, Methanothermobacter thermautotrophicus, Mentha x piperita and Saccharomyces cerevisiae and/or wherein the at least one functional enzyme is selected from the group consisting of sesquiterpene synthases of Malus domestica, Ricinus communis, Solanum habrochaites, Santalum spicatum, Artemisia annua, Callitropis nootkatensis, Citrus sinensis and Santalum album. More preferably, the FPPS is from Saccharomyces cerevisiae and the sesquiterpene synthase is from Malus domestica, Artemisia annua, Callitropis nootkatensis, Citrus sinensis or Santalum album. More preferably, the FPPS is from E.coli and the sesquiterpene synthase is from Malus domestica, Artemisia annua, Callitropis nootkatensis, Citrus sinensis or Santalum album. Most preferably, the FPPS is from Saccharomyces cerevisiae and the sesquiterpene synthase is from Santalum album, the FPPS is from Saccharomyces cerevisiae and the sesquiterpene synthase is from Artemisia annua, the FPPS is from Saccharomyces cerevisiae and the sesquiterpene synthase is from Callitropis nootkatensis, the FPPS is from Saccharomyces cerevisiae and the sesquiterpene synthase is from Citrus sinensis, the FPPS is from E.coli and the sesquiterpene synthase is from Santalum album, the FPPS is from E.coli and the sesquiterpene synthase is from Artemisia annua, the FPPS is from E.coli and the sesquiterpene synthase is from Callitropis nootkatensis, or the FPPS is from E.coli and the sesquiterpene synthase is from Citrus sinensis.

A preferred cyanobacterial cell according to the present invention is capable of producing, preferably producing, a sesquiterpene, preferably a sesquiterpene selected from the group consisting of: artemisinin, bisabolol, farnesene, valencene, santalene, bergamotene. More preferably, the sesquiterpene is farnesene, valencene, bisabolol and santalene; most preferably, the sesquiterpene is valencene or santalene. Preferably, a cyanobacterial cell according to the present invention is capable of producing, preferably producing, at least two terpenes, more preferably at least two sesquiterpenes, even more preferably at least santalene and bergamotene, even more preferably santalene and bergamotene. In an embodiment, a preferred cyanobacterial cell according to the present invention is capable of producing, preferably producing, bisabolol and at least one other terpene, preferably a sesquiterpene selected from the group consisting of a-bisabolene , cis-γ -bisabolene , cis-a-bisabolene , β- bisabolene and β-sesquiphellandrene.

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 and SEQ ID NO: 24. 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%o, 97%), 98%o, 99% or 100% sequence identity with a sequence selected from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 10, or from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 14, or from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 20, or from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 22, or from the group consisting of SEQ ID NO: 4 and SEQ ID NO: 24, or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 10, or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 14, or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 20, or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 22, or from the group consisting of SEQ ID NO: 2 and SEQ ID NO: 24. 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: 4 and SEQ ID NO: 14, or with SEQ ID NO: 2 and SEQ ID NO: 10, or with SEQ ID NO: 2 and SEQ ID NO: 14, or with SEQ ID NO: 4 and SEQ ID NO: 10, or with SEQ ID NO: 4 and SEQ ID NO: 20, or with SEQ ID NO: 4 and SEQ ID NO: 22, or with SEQ ID NO: 4 and SEQ ID NO: 24, 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%o, 97%), 98%o, 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: 19, SEQ ID NO: 21 and SEQ ID NO: 23. 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: 3, SEQ ID NO: 9, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23. 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: 9, or with SEQ ID NO: 1 and 13, or with SEQ ID NO: 3 and SEQ ID NO: 9, or with SEQ ID NO: 3 and SEQ ID NO: 13, or with SEQ ID NO: 3 and SEQ ID NO: 17, or with SEQ ID NO: 3 and SEQ ID NO: 19, or with SEQ ID NO: 3 and SEQ ID NO: 21, or with SEQ ID NO: 3 and SEQ ID NO: 23 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 Haplosiphon. 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 sesquiterpene" preferably means herein that detectable amounts of sesquiterpene can be detected in a culture of a cyanobacterial cell according to the present invention cultured, under conditions conducive to the production of sesquiterpene, preferably in the presence of light and dissolved carbon dioxide and/or bicarbonate ions, during a preferred interval using a suitable assay for detecting sesquiterpenes. 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 sesquiterpene 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 the sesquiterpene, 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 sesquiterpene 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 terpenes 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 sesquiterpene is Gas Chromatography-Mass Spectrometry (GC-MS). A detectable amount of a sesquiterpene 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 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 μ_§, 2 μ_§, 3 μ_§, 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 lac\JV5 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 sesquiterpenes.

Accordingly, in a second aspect, the present invention relates to a process for producing a sesquiterpene 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 sesquiterpene and, optionally, isolating and/or purifying the sesquiterpene 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 sesquiterpene 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 μΕίηβίείη/ιη 2/s. In another embodiment, the light used is artificial. Such artificial light may have an intensity ranged between approximately 70 and approximately 800 μΕίηβίείη/ιη 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 sesquiterpene 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 present invention, the produced sesquiterpene is selected from the group consisting of: artemisinin, bisabolol, farnesene, valencene, santalene and bergamotene. More preferably, the sesquiterpene is farnesene, bisabolol, valencene or santalene; most preferably, the sesquiterpene is valencene or santalene. 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 sesquiterpenes are produced, even more preferably at least santalene and bergamotene are produced, even more preferably santalene and bergamotene are produced. In an embodiment, a preferred process according to the invention produces the sesquiterpene bisabolol and at least one other terpene, preferably a sesquiterpene selected from the group consisting of a-bisabolene , cis-γ -bisabolene , cis-a- bisabolene , β-bisabolene and β-sesquiphellandrene.

The sesquiterpene 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 sesquiterpene obtainable by a cyanobacterial cell according to the invention and by a process according to the invention. Preferably, such sesquiterpene is a sesquiterpene selected from the group consisting of artemisinin, bisabolol, farnesene, valencene, santalene and bergamotene.

A sesquiterpene 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 sesquiterpene obtainable by a cyanobacterial cell according to the invention and by a process according to the invention. Preferably, such composition comprises a sesquiterpene according to the invention selected from the group consisting of artemisinin, bisabolol, farnesene, valencene, santalene and bergamotene.

Definitions

"Sequence identity" or "identity" in the context of amino acid- or nucleic acid-sequence herein defined as a relationship between two or more amino acid (peptide, polypeptide, 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 nucleic 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 alogorithm 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^XSSC 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^XSSC or any other solution having a comparable ionic strength, and washing at room temperature in a solution comprising about 1 M salt, preferably 6^XSSC 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 RNA 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^r 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 Russel (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, 11, 13, 15, 17, 19, 21 and 23 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 farnesyl ATGGACTTTCCGCAGCAACTCGAAGCCTGCGTTAAG diphosphate CAGGCCAACCAGGCGCTGAGCCGTTTTATCGCCCCA synthase from CTGCCCTTTCAGAACACTCCCGTGGTCGAAACCATG

CAGTATGGCGCATTATTAGGTGGTAAGCGCCTGCGA

Escherichia coli

CCTTTCCTGGTTTATGCCACCGGTCATATGTTCGGCG

TTAGCACAAACACGCTGGACGCACCCGCTGCCGCCG

TTGAGTGTATCCACGCTTACTCATTAATTCATGATG

ATTTACCGGCAATGGATGATGACGATCTGCGTCGCG

GTTTGCCAACCTGCCATGTGAAGTTTGGCGAAGCAA

ACGCGATTCTCGCTGGCGACGCTTTACAAACGCTGG

CGTTCTCGATTTTAAGCGATGCCGATATGCCGGAAG

TGTCGGACCGCGACAGAATTTCGATGATTTCTGAAC

TGGCGAGCGCCAGTGGTATTGCCGGAATGTGCGGTG

GTCAGGCATTAGATTTAGACGCGGAAGGCAAACAC

GTACCTCTGGACGCGCTTGAGCGTATTCATCGTCAT

AAAACCGGCGCATTGATTCGCGCCGCCGTTCGCCTT

GGTGCATTAAGCGCCGGAGATAAAGGACGTCGTGC

TCTGCCGGTACTCGACAAGTATGCAGAGAGCATCGG

CCTTGCCTTCCAGGTTCAGGATGACATCCTGGATGT

GGTGGGAGATACTGCAACGTTGGGAAAACGCCAGG

GTGCCGACCAGCAACTTGGTAAAAGTACCTACCCTG

CACTTCTGGGTCTTGAGCAAGCCCGGAAGAAAGCCC GGGATCTGATCGACGATGCCCGTCAGTCGCTGAAAC AACTGGCTGAACAGTCACTCGATACCTCGGCACTGG AAGCGCTAGCGGACTACATCATCCAGCGTAATAAAT AA

famesyl MDFPQQLEACVKQANQALSRFIAPLPFQNTPVVETMQ diphosphate YGALLGGKRLRPFLVYATGHMFGVSTNTLDAPAAAV

ECIHAYSLIHDDLPAMDDDDLRRGLPTCHVKFGEANA

synthase from

ILAGDALQTLAFSILSDADMPEVSDPvDRISMISELASAS Escherichia coli

GIAGMCGGQALDLDAEGKHVPLDALEPvIHPvHKTGALI

RAAVRLGALSAGDKGPvRALPVLDKYAESIGLAFQVQ

DDILDVVGDTATLGKRQGADQQLGKSTYPALLGLEQ

AR KARDLIDDARQSLKQLAEQSLDTSALEALADYIIQ

RNK

famesyl ATGGCTTCAGAAAAAGAAATTAGGAGAGAGAGATT diphosphate CTTGAACGTTTTCCCTAAATTAGTAGAGGAATTGAA synthase from CGCATCGCTTTTGGCTTACGGTATGCCTAAGGAAGC

ATGTGACTGGTATGCCCACTCATTGAACTACAACAC

Saccharomyces

TCCAGGCGGTAAGCTAAATAGAGGTTTGTCCGTTGT

cerevisiae GGACACGTATGCTATTCTCTCCAACAAGACCGTTGA

ACAATTGGGGCAAGAAGAATACGAAAAGGTTGCCA

TTCTAGGTTGGTGCATTGAGTTGTTGCAGGCTTACTT

CTTGGTCGCCGATGATATGATGGACAAGTCCATTAC

CAGAAGAGGCCAACCATGTTGGTACAAGGTTCCTG

AAGTTGGGGAAATTGCCATCAATGACGCATTCATGT

TAGAGGCTGCTATCTACAAGCTTTTGAAATCTCACT

TCAGAAACGAAAAATACTACATAGATATCACCGAA

TTGTTCCATGAGGTCACCTTCCAAACCGAATTGGGC

CAATTGATGGACTTAATCACTGCACCTGAAGACAAA

GTCGACTTGAGTAAGTTCTCCCTAAAGAAGCACTCC

TTCATAGTTACTTTCAAGACTGCTTACTATTCTTTCT

ACTTGCCTGTCGCATTGGCCATGTACGTTGCCGGTA

TCACGGATGAAAAGGATTTGAAACAAGCCAGAGAT

GTCTTGATTCCATTGGGTGAATACTTCCAAATTCAA

GATGACTACTTAGACTGCTTCGGTACCCCAGAACAG

ATCGGTAAGATCGGTACAGATATCCAAGATAACAA

ATGTTCTTGGGTAATCAACAAGGCATTGGAACTTGC

TTCCGCAGAACAAAGAAAGACTTTAGACGAAAATT

ACGGTAAGAAGGACTCAGTCGCAGAAGCCAAATGC

AAAAAGATTTTCAATGACTTGAAAATTGAACAGCTA

TACCACGAATATGAAGAGTCTATTGCCAAGGATTTG

AAGGCCAAAATTTCTCAGGTCGATGAGTCTCGTGGC

TTCAAAGCTGATGTCTTAACTGCGTTCTTGAACAAA

GTTTACAAGAGAAGCAAATAG

famesyl MASEKEIRRERFLNVFPKLVEELNASLLAYGMPKEAC diphosphate DWYAHSLNYNTPGGKLNRGLSVVDTYAILSNKTVEQ synthase from LGQEEYEKVAILGWCIELLQAYFLVADDMMDKSITRR

GQPCWYKVPEVGEIAINDAFMLEAAIYKLLKSHFR E

Saccharomyces

KYYIDITELFHEVTFQTELGQLMDLITAPEDKVDLSKFS

cerevisiae

LKKHSFIVTFKTAYYSFYLPVALAMYVAGITDEKDLK

QARDVLIPLGEYFQIQDDYLDCFGTPEQIGKIGTDIQDN

KC S WVINKALEL AS AEQRKTLDEN YGKKD S V AE A C

KKIFNDLKIEQLYHEYEESIAKDLKAKISQVDESRGFK

ADVLTAFLNKVYKRSK

farnesyl ATGGCGAATCTCAACGGAGCGGCGTCGGATCTGAG diphosphate GAAGACGTTTCTGGGGGTTTATTCTACGCTGAAATC synthase from TGAGCTCTTGAATGACCCTGCTTTCGAGTGGACTGA

TGGCTCTCGCCAATGGGTCGAGCGTATGTTGGACTA

Mentha x piperita

CAATGTACCTGGAGGGAAGTTAAACCGAGGCTTGTC

AGTTATTGATAGCTACCAGTTACTGAAAGAAGGAA

AAGATCTAACTGATGATGAAGTGTTTCTAGCTAGCG

CTCTTGGCTGGTGTATTGAATGGCTTCAGGCATATTT

TCTTGTCCTTGACGATATAATGGATAATTCTCACAC

ACGACGTGGTCAGCCATGCTGGTTTAAAGTTCCCAA

GGTTGGTATGATTGCCATAAATGATGGAATCATTCT

CAGAAACCATATCCCCAGAATTCTGAAGAAGCACTT

CAGATCAAAGCCTTACTATGTAGATCTGCTGGATCT

GTTCAATGAGGTGGAGTTTCAAACTGCCTCTGGACA

GATGATAGATTTAATTACTACTATTGAAGGAGAAAA

AGATTTGTCAAAATACTCCTTGCCTCTTCATCGCCGC

ATTGTTCAGTACAAGACTGCCTACTACTCATTTTACC

TTCCAGTTGCTTGTGCATTGCTCATGGCGGGTGAGA

ACCTGGAGAACCATCCAACTGTAAAAGATGTGCTTA

TCGATATGGGAATATACTTTCAAGTACAGGATGACT

ACTTAGATTGCTTCGGTGAGCCCGAAAAGATTGGGA

AGATTGGAACAGATATTGAAGATTTCAAATGTTCTT

GGCTGGTTGTAAAGGCCCTAGAGCTTTGCAATGAAG

AACAAAAGAAAACTCTTTTCGAACATTATGGAAAA

GAAAATCCAGCTGATGTTGCTAAAATCAAAGCCCTC

TACAATGACATCAATCTCCAAGGCATGTTTGCTGAT

TTCGAGAGCAAGAGCTACGAGAAAATAACAAGCTC

CATCGAAGCTCATCCCAGCAAATCTGTGCAAGCAGT

GCTCAAGTCTTTCTTGGGAAAGATATACAAGAGGCA

GAAATAA

farnesyl MANLNGAASDLRKTFLGVYSTLKSELLNDPAFEWTD diphosphate GSRQWVERMLDYNVPGGKLNRGLSVIDSYQLLKEGK synthase from DLTDDEVFLASALGWCIEWLQAYFLVLDDIMDNSHTR

RGQPCWFKVPKVGMIAINDGIILRNHIPRILKKHFRSKP

Mentha x piperita

YYVDLLDLFNEVEFQTASGQMIDLITTIEGEKDLSKYS LPLHR IVQYKTAYYSFYLPVACALLMAGENLENHPT VKDVLIDMGIYFQVQDDYLDCFGEPEKIGKIGTDIEDF KCSWLVVKALELCNEEQK TLFEHYGKENPADVAKI KALYNDINLQGMFADFESKSYEKITSSIEAHPSKSVQA VL SFLGKIYK Q

farnesyl ATGACGGAAGTATTAGATATTCTCAGAAAGTACTCT diphosphate GAAGTGGCCGATAAGAGGATAATGGAGTGCATCA synthase from GCGACATAACACCTGATACGCTCCTTAAGGCATCAG

AACACCTCATAACAGCAGGTGGTAAGAAGATAAG

Methanothermobac

ACCTTCACTTGCACTCCTTAGCTGTGAGGCTGTAGG

ter CGGAAACCCTGAGGACGCTGCAGGGGTGGCTGCG thermautotrophicus GCCATAGAACTGATACACACATTCTCACTCATTCAC

GATGATATAATGGATGATGATGAGATGAGGCGAG

GGGAACCCTCAGTCCATGTCATATGGGGTGAACCCA

TGGCCATACTTGCAGGGGACGTCCTCTTCTCCAA

GGCCTTCGAGGCCGTCATCAGGAACGGGGACTCAG

AAAGGGTGAAGGATGCCCTTGCAGTGGTGGTTGAT

TCATGCGTTAAGATATGTGAGGGCCAGGCCCTTGAC

ATGGGCTTTGAGGAGAGGCTGGATGTCACAGAGG

ATGAGTACATGGAGATGATCTACAAGAAGACCGCA

GCCCTCATTGCAGCAGCAACAAAGGCAGGTGCCAT

AATGGGGGGCGCATCAGAGAGAGAGGTTGAGGCCC

TGGAGGACTACGGCAAATTCATCGGCCTCGCATTC

CAGATACACGACGACTACCTGGATGTGGTGAGTGAT

GAGGAATCTCTGGGCAAACCTGTGGGCAGTGACA

TAGCAGAGGGCAAGATGACCCTCATGGTTGTAAAG

GCCCTTGAGGAGGCATCAGAGGAGGACAGGGAGAG

GCTCATATCGATCCTCGGATCAGGTGATGAGGGCAG

TGTCGCTGAGGCCATAGAGATCTTTGAACGCTAC

GGTGCAACCCAGTATGCCCATGAGGTGGCCCTTGAC

TATGTGAGGATGGCCAAGGAGCGCCTTGAGATAC

TTGAAGACTCAGATGCAAGGGACGCACTCATGAGG

ATAGCTGACTTTGTACTTGAAAGGGAACACTAG

farnesyl MTEVLDILR YSEVADKRIMECISDITPDTLLKASEHLI diphosphate TAGGKKIRPSLALLSCEAVGGNPEDAAGVAAAIELIHT synthase from F SLIHDDIMDDDEMRRGEP S VH VI WGEPM AIL AGD VL

FSKAFEAVIR GDSERVKDALAVVVDSCVKICEGQAL

Methanothermobac

DMGFEEPvLDVTEDEYMEMIYKKTAALIAAATKAGAI ter MGGASEREVEALEDYGKFIGLAFQIHDDYLDVVSDEE thermautotrophicus SLGKPVGSDIAEGKMTLMVVKALEEASEEDRERLISIL

GSGDEGSVAEAIEIFERYGATQYAHEVALDYVRMAKE

RLEILEDSDARDALMPvIADFVLEREH farnesene synthase ATGGAATTCAGAGTTCACTTGCAAGCTGATAATGAG from Malus x CAGAAAATTTTTCAAAACCAGATGAAACCCGAAC domestica CTGAAGCCTCTTACTTGATTAATCAAAGACGGTCTG

CAAATTACAAGCCAAATATTTGGAAGAACGATTT

CCTAGATCAATCTCTTATCAGCAAATACGATGGAGA

TGAGTATCGGAAGCTGTCTGAGAAGTTAATAGAA

GAAGTTAAGATTTATATATCTGCTGAAACAATGGAT

TTAGTAGCTAAGTTGGAGCTCATTGACAGCGTCC

GAAAACTAGGCCTCGCGAACCTCTTCGAAAAGAAA

ATCAAGGAAGCCCTAGACAGCATTGCAGCTATCGA

AAGCGACAATCTCGGCACAAGAGACGATCTCTATG

GTGCTGCATTACACTTCAAGATCCTCAGGCAGCAT

GGCTATAAAGTTTCACAAGATATATTTGGTAGATTC

ATGGATGAAAAGGGCACATTAGAGAACCACCATT

TCGCGCATTTAAAAGGAATGCTGGAACTTTTCGAGG

CCTCAAACCTGGGTTTCGAAGGTGAAGATATTTT

AGATGAGGCGAAAGCTTCCTTGACGCTAGCTCTCAG

AGATAGTGGTCATATTTGTTATCCAGACAGTAAC

CTTTCCAGGGACGTAGTTCATTCCCTGGAGCTTCCA

TCACACCGCAGAGTGCAGTGGTTTGATGTCAAAT

GGCAAATCAACGCCTATGAAAAAGACATTTGTCGC

GTCAACGCCACGTTACTCGAATTAGCAAAGCTTAA

TTTCAACGTAGTTCAGGCCCAACTCCAAAAAAACTT

AAGGGAAGCATCCAGGTGGTGGGCAAATCTGGGC

TTCGCAGACAACTTGAAATTTGCAAGAGATGGACTG

GTTGAATGTTTCTCATGTGCTGTGGGAGTAGCAT

TCGAGCCTGAGCACTCATCTTTTAGAATATGTCTTA

CCAAAGTCATCAACTTAGTACTGATCATAGACGA

CGTCTATGATATTTATGGCTCAGAGGAAGAGCTAAA

GCACTTCACCAATGCTGTTGATAGGTGGGATTCT

AGGGAAACTGAGCAGCTTCCAGAGTGTATGAAGAT

GTGTTTCCAAGTACTCTACAACACTACTTGTGAAA

TTGCTCGTGAAATTGAGGAGGAGAATGGTTGGAAC

CAAGTATTACCTCAATTGACCAAAGTGTGGGCAGA

TTTTTGTAAAGCATTATTGGTGGAGGCAGAGTGGTA

T AAT AAG AGC CAT AT AC C AAC C CTT G AAG AGT AC

CTAAGAAACGGATGCATTTCATCATCAGTTTCAGTG

CTTTTGGTTCACTCGTTTTTCTCTATAACTCATG

AGGGAACCAAAGAGATGGCTGATTTTCTTCACAAG

AATGAAGATCTTTTGTATAATATCTCTCTCATCGT

TCGTCTCAACAATGATTTGGGAACTTCCGCGGCTGA

ACAAGAGAGAGGGGATTCTCCTTCATCAATCGTA

TGTTACATGAGAGAAGTGAATGCCTCTGAAGAAAC

AGCTAGGAAGAACATTAAGGGCATGATAGACAATG

CATGGAAGAAAGTAAATGGAAAATGCTTCACAACA

AACCAAGTGCCTTTTCTGTCATCATTCATGAACAA TGCCACAAACATGGCACGTGTGGCGCACAGCCTTTA CAAAGATGGAGATGGGTTTGGTGACCAAGAGAAA GGGCCTCGGACCCACATCCTGTCTTTACTATTCCAA CCTCTTGTAAACTAG

famesene synthase MEFRVHLQADNEQKIFQNQMKPEPEASYLINQRRSAN from Malus x YKPNIWK DFLDQSLISKYDGDEYRKLSEKLIEEVKIYI domestica SAETMDLVAKLELIDSVRKLGLANLFEKKIKEALDSIA

AIESDNLGTRDDLYGAALHFKILRQHGYKVSQDIFGRF

MDEKGTLENHHFAHLKGMLELFEASNLGFEGEDILDE

AKASLTLALRDSGHICYPDSNLSRDVVHSLELPSHRRV

QWFDVKWQINAYEKDICRVNATLLELAKLNFNVVQA

QLQK LREASRWWANLGFADNLKFARDGLVECFSCA

VGVAFEPEHSSFRICLTKVINLVLIIDDVYDIYGSEEELK

HFTNAVDRWDSRETEQLPECMKMCFQVLYNTTCEIA

REIEEENGWNQVLPQLTKVWADFCKALLVEAEWYNK

SHIPTLEEYLRNGCISSSVSVLLVHSFFSITHEGTKEMA

DFLHK EDLLYNISLIVRL NDLGTSAAEQERGDSPSSI

VCYMREVNASEETARK IKGMIDNAWK VNGKCFTT

NQVPFLSSFMNNATNMARVAHSLYKDGDGFGDQEKG

PRTHILSLLFQPLVN

famesene synthase ATGAAAGTTGGGCAACCAGTTCTACAATGCCAGACC from Ricinus AATTCTGAAGCCTTTGGCATGATGCAAGAAAGGC communis GGTCCGGAAATTATAAGCCTAACATTTGGAAATATG

ATTTTCTACAATCTCTTTCTAGCAAATATGATGA

AGAGAAATACAAAACACAAGCTGAGAGGTTAAAAG

AGGATGCTAAGCATCTTTTCATTGAAGCAGTAGAC

TTGCAGGGTAAACTAGAGCTTGTTGATTGCATCATA

AAAGTAGGTTTGGCAAGCCACTTCAAGGATGAAA

TCAAGAAAGCTTTAGATACTATAGCCTCCTCTATCA

AGAACGACAAATCTGATGCAATAAAGAATCGGTA

TGTTACTGCATTATGCTTTAGACTTCTGAGGCAGCA

TGGTTATGAAGTCTCACAAGATGTTTTCAGTGAT

TTTTTGGACGAAAATGGTACTTTCTTGAAAGCCAAA

AGTATGGACGTTAAAGGAGTTCTGGAGCTTTTTG

AGGCTTCATATCTGGCTCTAGAAAGTGAAAATATCT

TAGATGATGCTAAGGCTTTTTCGACTACAATTCT

GAAAGATATCAACTCTGCTACAACAGAAAGCAACC

TTTACAAACAAGTCGTTCATGCTTTGGAACTTCCA

TTTCATTGGAGAGTGAGATGGTTTGATGTGAAATGG

CATATTAAAACGTTCCAGAAGGACAAAAGCATAA

ATAAAACCTTACTTGATTTGGCTAAAGTTAACTTTA

ACGTTGTCCAAGCCACACTCCAAAACGACCTAAA

GGAGATTTCCAGGTGGTGGAGGAATTTGGGTCTAAT

AGAGAATTTGAAATTTTCAAGGGATCGATTGGTG GAGAGCTTCTTGTGCACTGTAGGACTTGTATTTGAG

CCTCAGTACAGCTCTTTTAGAAGATGGCTTACCA

AAGTGGTTATCATGATATTGGTCATTGATGATGTTT

ATGACATTTATGGTTCACTGGAAGAACTACAGCA

TTTCACTAATGCTATTAATAGATGGGATACTGCAGA

ATTAGAACAACTTCCAGAATATATGAAAATATGC

TTTAAAACACTCCACACTATCACCGGTGAAACTGCT

CATGAGATGCAAAGGGAAAAGAGATGGGACCAAG

AACAAACTGAAACTCACCTGAAGAAAGTGTGGGCA

GATTTCTGTCAGGCACTGTTCGTGGAAGCCAAATG

GTTTAATAAAGGATATACACCATCCGTGCAAGAATA

TCTGAAAACTGCTTGTATTTCTTCATCAGGCAGC

CTCCTTTCTGTTCATTCGTTCTTCTTAATTATGAATG

AAGGAACTCGGGAAATGCTACATTTTCTTGAAA

AAAATCAAGAGATGTTTTATAATATATCTCTCATCA

TCCGCCTCTGCAACGATTTAGGCACTTCAGTGGC

AGAACAAGAGAGAGGAGATGCTGCTTCATCAATAG

TTTGTCATATGAGAGAAATGGAAGTTTTAGAGGAA

GAAGCTAGGAGCTATTTAAAGGGAATAATAGGCAA

TTACTGGAAGAAAGTAAATGAAAAATGCTTCACCC

AATCACCTGAGATGCAATTATTTATCAATATTAATG

TCAATATGGCCCGTGTGGTGCATAATCTATATCA

AAACAGAGATGGATTTGGTGTTCAAGACCATCAAA

ATAAGAAGCAGATCCTATCCCTCCTTGTTCATCCT

TTCAAACTAGACTGA

famesene synthase MKVGQPVLQCQTNSEAFGMMQERRSGNYKPNIWKY from Ricinus DFLQSLSSKYDEEKYKTQAERLKEDAKHLFIEAVDLQ communis GKLELVDCIIKVGLASHFKDEIKKALDTIASSIK DKSD

AIK RYVTALCFRLLRQHGYEVSQDVFSDFLDENGTF

LKAKSMDVKGVLELFEASYLALESENILDDAKAFSTTI

LKDINSATTESNLYKQVVHALELPFHWRVRWFDVKW

HIKTFQKDKSINKTLLDLAKVNFNVVQATLQNDLKEIS

RWWRNLGLIENLKFSRDRLVESFLCTVGLVFEPQYSSF

RRWLTKVVIMILVIDDVYDIYGSLEELQHFTNAINRWD

TAELEQLPEYMKICFKTLHTITGETAHEMQREKRWDQ

EQTETHLK VWADFCQALFVEAKWFNKGYTPSVQEY

LKTACIS S SGSLLS VHSFFLIMNEGTREMLHFLEK QE

MFYNISLIIRLCNDLGTSVAEQERGDAASSIVCHMREM

EVLEEEARSYLKGIIGNYWK VNEKCFTQSPEMQLFIN

INVNMARVVHNLYQNRDGFGVQDHQNK QILSLLVH

PFKLD

santalene synthase ATGGATTCTTCCACCGCCACCGCCATGACAGCTCCA from Santalum TTCATTGATCCTACTGATCATGTGAATCTCAAAA

CTGATACGGATGCCTCAGAGAATCGAAGGATGGGA album AATTATAAACCCAGCATTTGGAATTATGATTTTTT

ACAATCACTTGCAACTCATCACAATATTGTGGAAGA

GAGGCATCTAAAGCTAGCTGAGAAGCTGAAGGGC

CAAGTGAAGTTTATGTTTGGGGCACCAATGGAGCCG

TTAGCAAAGCTGGAGCTTGTGGATGTGGTTCAAA

GGCTTGGGCTAAACCACCTATTTGAGACAGAGATCA

AGGAAGCGCTGTTTAGTATTTACAAGGATGGGAG

CAATGGATGGTGGTTTGGCCACCTTCATGCGACATC

TCTCCGATTTAGGCTGCTACGACAGTGTGGGCTT

TTTATTCCCCAAGATGTGTTTAAAACGTTCCAAAAC

AAGACTGGGGAATTTGATATGAAACTTTGTGACA

ACGTAAAAGGGCTGCTGAGCTTATATGAAGCTTCAT

ACTTGGGATGGAAGGGTGAAAACATCCTAGATGA

AGCCAAGGCCTTCACCACCAAGTGCTTGAAAAGTGC

ATGGGAAAATATATCCGAAAAGTGGTTAGCCAAA

AGAGTGAAGCATGCATTGGCTTTGCCTTTGCATTGG

AGAGTCCCTCGAATCGAAGCTAGATGGTTCATTG

AGGCATATGAGCAAGAAGCGAATATGAACCCAACA

CTACTCAAACTCGCAAAATTAGACTTTAATATGGT

GCAATCAATTCATCAGAAAGAGATTGGGGAATTAG

CAAGGTGGTGGGTGACTACTGGCTTGGATAAGTTA

GCCTTTGCCAGGAATAATTTACTGCAGAGCTATATG

TGGAGCTGCGCGATTGCTTCCGACCCGAAGTTCA

AACTTGCTAGAGAAACTATTGTCGAAATCGGAAGTG

TACTCACAGTTGTTGACGATGGATATGACGTCTA

TGGTTCAATCGACGAACTTGATCTCTACACAAGCTC

CGTTGAAAGGTGGAGCTGTGTGGAAATTGACAAG

TTGCCAAACACGTTAAAATTAATTTTTATGTCTATGT

TCAACAAGACCAATGAGGTTGGCCTTCGAGTCC

AGCATGAGCGAGGCTACAATAGCATCCCTACTTTTA

TCAAAGCGTGGGTTGAACAGTGTAAATCATACCA

GAAAGAAGCAAGATGGTTCCACGGGGGACACACGC

CTCCATTGGAAGAATATAGCTTGAATGGACTTGTT

TCCATAGGATTCCCTCTCTTGTTAATCACGGGCTAC

GTGGCAATCGCTGAGAACGAGGCTGCACTGGATA

AAGTGCACCCCCTTCCTGATCTTCTGCACTACTCCTC

CCTCCTTAGTCGCCTCATCAATGATATAGGAAC

GTCTCCGGATGAGATGGCAAGAGGCGATAATCTGA

AGTCAATCCATTGTTACATGAACGAAACTGGGGCT

TCCGAGGAAGTTGCTCGTGAGCACATAAAGGGAGT

AATCGAGGAGAATTGGAAAATACTGAATCAGTGCT

GCTTTGATCAATCTCAGTTTCAGGAGCCTTTTATAAC

CTTCAATTTGAACTCTGTTCGAGGGTCTCATTT

CTTCTATGAATTTGGGGATGGCTTTGGGGTGACGGA TAGCTGGACAAAGGTTGATATGAAGTCCGTTTTG ATCGACCCTATTCCTCTCGGCGAGGAGTAG

santalene synthase MDSSTATAMTAPFIDPTDHVNLKTDTDASENRRMGN from Santalum YKPSIWNYDFLQSLATHHNIVEERHLKLAEKLKGQVK album FMFGAPMEPLAKLELVDVVQRLGLNHLFETEIKEALFS

IYKDGSNGWWFGHLHATSLRFRLLRQCGLFIPQDVFK

TFQNKTGEFDMKLCDNVKGLLSLYEASYLGWKGENI

LDEAKAFTTKCLKSAWENISEKWLAKRVKHALALPL

HWRVPRIEARWFIEAYEQEANMNPTLLKLAKLDFNM

VQSIHQKEIGELARWWVTTGLDKLAFAR NLLQSYM

WSCAIASDPKFKLARETIVEIGSVLTVVDDGYDVYGSI

DELDLYTSSVERWSCVEIDKLPNTLKLIFMSMFNKTNE

VGLRVQHERGYNSIPTFIKAWVEQCKSYQKEARWFH

GGHTPPLEEYSLNGLVSIGFPLLLITGYVAIAENEAALD

KVHPLPDLLHYSSLLSRLINDIGTSPDEMARGDNLKSIH

CYMNETGASEEVAREHIKGVIEENWKILNQCCFDQSQ

FQEPFITFNLNSVRGSHFFYEFGDGFGVTDSWTKVDM

KSVLIDPIPLGEE

santalene synthase ATGATAGTTGGCTATAGAAGCACAATCATAACCCTT from Solanum TCTCATCCTAAGCTAGGCAATGGGAAAACAATTT habrochaites CATCCAATGCAATTTTCCAGAGATCATGTAGAGTAA

GATGCAGCCACAGTACCCCTTCATCAATGAATGG

TTTCGAAGATGCAAGGGATAGAATAAGGGAAAGTT

TTGGGAAAGTAGAGTTATCTCCTTCTTCCTATGAC

ACAGCATGGGTAGCTATGGTCCCTTCAAAACATTCA

CTAAATGAGCCATGTTTTCCACAATGTTTGGATT

GGATTATTGAAAATCAAAGAGAAGATGGATCTTGG

GGACTAAACCCTAGCCATCCATTGCTTCTCAAGGA

CTCACTTTCTTCCACTCTTGCATGTTTGCTTGCACTA

ACCAAATGGAGAGTTGGAGATGAGCAAATCAAA

AGAGGCCTTGGCTTTATTGAAACCCAGAGTTGGGCA

ATTGATAACAAGGATCAAATTTCACCTCTAGGAT

TTGAAATTATATTTCCCAGTATGATCAAGTCTGCAG

AAAAACTAAACTTAAATCTAGCAATTAACAAAAG

AGATTCAACAATTAAAAGAGCATTACAGAATGAGT

TCACGAGGAATATTGAATATATGAGTGAAGGAGTT

GGTGAATTATGTGATTGGAAGGAAATAATAAAGTT

ACATCAAAGGCAAAATGGTTCATTATTTGATTCAC

CAGCCACTACTGCAGCTGCCTTGATTTACCATCAGC

ATGATAAAAAATGCTATGAATATCTTAATTCAAT

CTTGCAACAACACAAAAATTGGGTTCCCACTATGTA

TCCAACAAAGATACATTCATTGCTTTGCTTGGTT

GATACACTTCAAAATCTTGGAGTACATCGGCATTTT

AAATCAGAAATAAAGAAAGCTCTAGATGAAATAT ACAGGCTATGGCAACAAAAGAATGAACAAATTTTC

TCAAATGTCACCCATTGTGCTATGGCTTTTCGACT

TCTAAGGATGAGCTACTATGATGTCTCCTCAGATGA

ACTAGCAGAATTTGTGGATGAAGAACATTTTTTT

GCAATAAGTGGGAAATATACAAGTCATGTTGAAATT

CTTGAACTCCACAAAGCATCACAATTGGCTATTG

ATCATGAGAAAGATGACATTTTGGATAAGATTAACA

ATTGGACAAGAACATTTATGGAGCAAAAACTCTT

AAACAATGGCTTCATAGATAGGATGTCAAAAAAGG

AGGTGGAACTTGCTTTGAGGAAGTTTTATACCATA

TCTGATCTAGCAGAAAATAGAAGATGTATAAAGTC

ATACGAAGAGAACAATTTTAAAATCTTAAAAGCAG

CTTATAGGTCACCTAACATTTACAATAAGGACTTGT

TTATATTTTCAATACGCAACTTTGAATTATGCCA

AGCTCAACACCAAGAAGAACTTCAACAATTCAAGA

GGTGGTTTGAAGATTATAGATTGGACCAACTCGGA

ATTGCAGAACGATATATACATGATACTTACTTATGT

GCTGTTATTGTTGTCCCCGAGCCTGAATTATCCG

ATGCTCGTCTCTTGTACGCGAAATACGTCTTGCTCCT

GACTATTGTCGATGATCAGTTCGACAGTTTTGC

ATCTACAGATGAATGTCTCAACATCATTGAATTAGT

AGAAAGGTGGGATGACTATGCAAGTGTAGGTTAT

AAATCTGAGAAGGTTAAAGTTTTCTTTTCAACTTTGT

ACAAATCAATAGAGGAGCTTGTAACAATTGCTG

AAATTAAACAAGGACGATCTGTCAAAAATCACCTTC

TTAATTTGTGGCTTGAATTGGTGAAGTTGATGTT

GATGGAACGAGTAGAGTGGTTTTCTGGCAAGACAA

TCCCAAGCATAGAAGAGTATTTGTATGTTACATCT

ATAACATTTGGTGCAAGATTGATTCCTCTCACAACA

CAATATTTTCTTGGAATAAAAATATCCGAAGATA

TTTTAGAAAGTGATGAAATATATGGTTTATGCAACT

GTACCGGTAGAGTCCTTCGAATCCTTAATGATTT

ACAAGATTCCAAGAAAGAACAAAAGGAGGACTCAG

TAACTATAGTCACATTACTAATGAAAAGTATGTCT

GAGGAAGAAGCTATAATGAAGATAAAGGAAATCTT

GGAAATGAATAGAAGAGAGTTATTGAAAATGGTTT

TAGTTCAAAAAAAGGGAAGCCAATTGCCTCAAATA

TGCAAAGATATATTTTGGAGGACAAGCAACTGGGC

TGATTTCATTTATTTACAAACTGATGGATATAGAAT

TGCAGAGGAAATGAAGAATCACATTGATGAAGTC

TTTTACAAACCACTCAATCATTAA

santalene synthase MIVGYRSTIITLSHPKLGNGKTISSNAIFQRSCRVRCSHS from Solanum TPSSMNGFEDARDRIRESFGKVELSPSSYDTAWVAMV

PSKHSLNEPCFPQCLDWIIENQREDGSWGLNPSHPLLL habrochaites KDSLSSTLACLLALTKWRVGDEQIKRGLGFIETQSWAI

DNKDQISPLGFEIIFPSMIKSAEKLNLNLAINKRDSTIKR

ALQNEFTRNIEYMSEGVGELCDWKEIIKLHQRQNGSLF

DSPATTAAALIYHQHDK CYEYLNSILQQHK WVPT

MYPTKIHSLLCLVDTLQNLGVHRHFKSEIKKALDEIYR

LWQQK EQIFSNVTHCAMAFRLLRMSYYDVSSDELA

EFVDEEHFFAISGKYTSHVEILELHKASQLAIDHEKDDI

LDKINNWTRTFMEQKLL NGFIDRMSKKEVELALRKF

YTISDLAENRRCIKSYEE NFKILKAAYRSPNIYNKDLF

IFSIRNFELCQAQHQEELQQFKRWFEDYRLDQLGIAER

YIHDTYLCAVIVVPEPELSDARLLYAKYVLLLTIVDDQ

FDSFASTDECLNIIELVERWDDYASVGYKSEKVKVFFS

TLYKSIEELVTIAEIKQGRSVK HLLNLWLELVKLML

MERVEWFSGKTIPSIEEYLYVTSITFGARLIPLTTQYFL

GIKISEDILESDEIYGLCNCTGRVLRILNDLQDSKKEQK

EDSVTIVTLLMKSMSEEEAIMKIKEILEMNRRELLKMV

LVQK GSQLPQICKDIFWRTSNWADFIYLQTDGYRIAE

EMK HIDEVFYKPLNH

Bisabolol synthase ATGGATGCCTTTGCCACTTCCCCGACCACGGCTCTC from Santalum TTCGAAACAGTTAACTGCAATGCACATGTCGCTC spicatum CTATGGCTGGGGAAGATTCGTCCGAGAATCGACCG

GCCAGCAATTACAAGCCAAGCACTTGGGACTATGA

GTTCCTCCAGTCACTCGCCACCACTAACAACACAGT

GGGGGAGAAGCATACGAGGATGGCTGACAAGCTG

AAGGAGGAAGTGAAGTCTATGATGAAGGGGACAAT

GGAACCCGTGGCGAAGCTTGAGTTGATCAATATAG

TTCAGCGGCTCGGCCTGAAATATAGGTTTGAGTCTG

AGATAAAGGAAGAATTGTTTAGTCTTTACAAAGA

TGGTACCGATGCATGGTGGGTGGGTAATTTGCATGC

TACGGCCCTCCGGTTTCGGCTTCTACGTGAAAAT

GGGATATTTGTTCCCCAAGATGTGTTTGAGACTTTT

AAGGACAAAAGTGGTGAATTTAAGAGTCAATTAT

GCAAGGATGTGAGGGGACTCCTAAGCTTGTATGAA

GCCTCTTACTTGGGTTGGGAAGGTGAGGAGTTGCT

TGATGAGGCAAAGAAGTTTAGTACTACAAATTTGAA

CAATGTGAAGGAAAGCATATCTTCCAATACCTTG

GGAAGATTGGTGAAGCATGCTTTGAATCTGCCATTG

CATTGGTCAGCAGCAAGATATGAGGCTCGGTGGT

TTATTGATGAGTATGAGAGAGAGGAAAATGTGATC

CCTAATTTACTAAAATATGCCAAGTTGGATTTCAA

TGTTGTGCAATCAATTCATCAGAAGGAGCTTGGCAA

CCTGGCAAGGTGGTGGGTGGAGACGGGCTTGGAT

AAGCTAGGCTTTGTGAGGAACACTTTAATGCAAAAC

TTCATGTGGGGCTGTGCCATGGCGTTTGAGCCAC AGTACGGCAAAGTCAGAGATGCCGCAGTCAAACTG

GGCAGTCTCATTACTATGGTTGATGATGTTTATGA

TGTCTATGGCACCTTGGAGGAATTGGAGATCTTCAC

AGATATTGTTGACAGGTGGGACATCAATGGAATT

GACAAACTACCAAGAAATATAAGTATGATTGTACTT

ACGATGTTCAACACTGCAAATCAGATAAGTTATG

ACCTCTTGAGAGACCGAGGCTTCAACAGCATTCCTC

ATATAGCCGAAGCTTGGGCAACTCTATGCAAGAC

GTACCTCAAGGAAGCAAAATGGTACCACAGCGGAT

ACAAGCCCACGCTTGAGGAGTATTTGGAGAACGGA

TTAGTTTCCATTTCCTTTGTTCTTAGTCTCGTTACAG

CATACTTGCAGACCGAAAGGCTTGAGAATTTAA

CCTATGAGAGCGCTGCGTACGTTAATTCTGTACCGC

CTCTTGTCCGATACTCTGGTCTTCTCAACCGCCT

CTATAATGATCTTGGAACCTCTTCGGCCGAGATAGC

GAGAGGGGACACGCTCAAGTCGATCCAGTGTTAC

ATGACCCAAACTGGCGCAACCGAGGAGGTTGCACG

CGAGCACATCAAAGGGTTGGTGCATGAGGCTTGGA

AAGGCATGAACAGGTGCTTGTTTGAGCAAACCCCAC

TTGCTGAGCCATTTGTGGGCTTCAATGTGAACAC

TGTTCGTGGCTCTCAATTCTTCTACCAGCATGGAGA

TGGGTATGCTGTCACAGAGAGTTGGACTAAGGAC

CTTTCCCTCTCAGTTCTCATCCACCCTATTCCACTGA

ACGAGGAGGATTGA

Bisabolol synthase MD AF AT SPTT ALFET VNCN AH V APM AGED S SENRP AS from Santalum NYKPSTWDYEFLOSLATT NTVGEKHTRMADKL spicatum KEEVKSMMKGTMEPVAKLELINIVORLGLKYRFESEI

KEELFSLYKDGTDAWWVGNLHATALRFRLLREN

GIFVPODVFETFKDKSGEFKSOLCKDVPvGLLSLYEASY

LGWEGEELLDEAKKFSTTNL NVKESISSNTL

GRLVKHALNLPLHWSAARYEARWFIDEYEPvEENVIPN

LLKYAKLDFNVVOSIHOKELGNLARWWVETGLD

KLGFVRNTLMONFMWGCAMAFEPOYGKVRDAAVKL

GSLITMVDDVYDVYGTLEELEIFTDIVDRWDINGI

DKLPRNISMIVLTMFNTANOISYDLLRDRGFNSIPHIAE

AWATLCKTYLKEAKWYHSGYKPTLEEYLENG

LVSISFVLSLVTAYLOTERLENLTYESAAYVNSVPPLV

RYSGLLNRLYNDLGTSSAEIARGDTLKSIOCY

MTOTGATEEVAREHIKGLVHEAWKGMNRCLFEOTPL

AEPFVGFNVNTVRGSOFFYOHGDGYAVTESWTKD

LSLSVLIHPIPLNEED

Bisabolol synthase ATGTCTCTTACAGAAGAAAAACCTATTCGCCCCATT from Artemisia GCCAACTTTTCTCCAAGCATTTGGGGAGATCAGT

TTCTTATCTATGACAATCAAGTAGAGCAAGGGGTGG annua AACAGATAGTGAAAGATTTAAAAAAAGAAGTGCG GCAGCTACTAAAAGAAGCTTTGGATATTCCTATGAA

ACATGCCAATTTGTTAAAGCTGGTTGATGAAATA

CAACGCCTTGGAATATCGTATCTCTTTGAACAGGAG

ATTGATCATGCATTGCAACATATCTATGAAACAT

ATGGTGATAACTGGAGTGGTGACCGCTCTTCCTTAT

GGTTCCGTCTTATGCGAAAACAAGGATATTTTGT

TACATGTGATGTTTTCAATAACCATAAAGACGAAAG

CGGAGTGTTCAAGCAATCGTTAAAGAATCATGTT GAAGGTTTGTTAGAGTTGTACGAAGCAACGTCTATG

AGGGTACCAGGGGAGATTATATTAGAAGATGCTC TTGTTTTTACACAATCTCATCTTAGCATTATAGCAAA

AGACACTCTTTCGATCAACCCTGCTCTTTCTAC

CGAAATACAACGGGCATTAAAGAAACCCCTTTGGA

AAAGGTTGCCAAGAATAGAGGCGGTGCAATACATT

CCTTTCTATGAACAACAAGATTCTCATAACAAGACT

TTAATTAAACTTGCTAAATTGGAGTTCAATTTGC

TTCAGTCATTGCATAGGGAAGAGCTCAGCCAACTGT

CCAAATGGTGGAAAGCTTTCGATGTCAAGAATAA

CGCACCTTATTCAAGAGATAGAATTGTTGAATGCTA

CTTTTGGGCACTAGCTTCACGCTTTGAGCCACAA

TATTCTCGGGCTAGAATTTTCTTGGCAAAAGTTATT

GCACTTGTAACTCTTATAGATGACATTTATGATG

CGTATGGTACTTATGAAGAACTTAAGATCTTTACTG

AAGCAATTGAAAGGTGGTCAATTACATGCTTAGA

CATGATTCCAGAATACATGAAACCGATATACAAATT

ATTCATGGATACGTACACCGAAATGGAAGAAATT

CTTGCAAAGGAGGGAAAAACAAATATATTTAACTG

TGGCAAAGAATTTGTGAAAGATTTTGTTAGAGTCC

TGATGGTTGAAGCACAATGGTTAAATGAGGGACAC

ATACCAACCACTGAAGAGCTTGATTCAATTGCAGT

CAATCTTGGCGGTGCTAACCTGCTTACAACAACTTG

TTATCTTGGCATGAGTGATATAGTCACAAAAGAG

GCTTTCGAATGGGCTGTCTCTGAACCTCCTCTTTTAA

GATACAAAGGTATACTTGGTCGACGCCTAAATG ATCTTGCGGGCCACAAGGAGGAGCAAGAAAGAAAG

CATGTTTCATCGAGCGTTGAAAGTTACATGAAGGA ATATAATGTCAGTGAGGAGTATGCCAAAAACTTGTT

GTACAAGCAAGTAGAAGATCTGTGGAAAGATATA

AACCGAGAGTACCTCATAACTAAAACCATTCCAAG

GCCACTATTGGTGGCTGTGATCAATTTGGTACATT

TTCTGGATGTTCTGTATGCAGAAAAGGATAACTTCA

CACGTATGGGAGAAGAATACAAAAATCTCGTAAA

GTCTCTACTCGTTTATCCTATGAGTATATGA Bisabolol synthase MSLTEEKPIRPIANFSPSIWGDOFLIYDNOVEOGVEOIV from Artemisia KDLKKEVROLLKEALDIPMKHANLLKLVDEI annua ORLGISYLFEOEIDHALOHIYETYGDNWSGDRSSLWFR

LMRKOGYFVTCDVF NHKDESGVFKOSLK HV

EGLLELYEATSMRVPGEIILEDALVFTOSHLSIIAKDTL

SINPALSTEIORALKKPLWKRLPRIEAVOYI

PFYEOODSHNKTLIKLAKLEFNLLOSLHREELSOLSKW

WKAFDVK NAPYSRDRIVECYFWALASRFEPO

YSRARIFLAKVIALVTLIDDIYDAYGTYEELKIFTEAIER

WSITCLDMIPEYMKPIYKLFMDTYTEMEEI

LAKEGKTNIFNCGKEFVKDFVRVLMVEAOWLNEGHIP

TTEELDSIAVNLGGANLLTTTCYLGMSDIVTKE

AFEWAVSEPPLLRYKGILGRRLNDLAGHKEEOERKHV

S S S VE S YMKE YN VSEE Y AK LL YKO VEDL WKDI

NREYLITKTIPRPLLVAVINLVHFLDVLYAEKDNFTRM

GEEYK LVKSLLVYPMSI

Valencene ATGGCTGAAATGTTTAATGGAAATTCCAGCAATGAT Synthase from GGAAGTTCTTGCATGCCCGTGAAGGACGCCCTTC Callitropis GTCGGACTGGAAATCATCATCCTAACTTGTGGACTG

ATGATTTCATACAGTCCCTCAATTCTCCATATTC

nootkatensis

GGATTCTTCATACCATAAACATAGGGAAATACTAAT

TGATGAGATTCGTGATATGTTTTCTAATGGAGAA

GGCGATGAGTTCGGTGTACTTGAAAATATTTGGTTT

GTTGATGTTGTACAACGTTTGGGAATAGATCGAC

ATTTTCAAGAGGAAATCAAAACTGCACTTGATTATA

TCTACAAGTTCTGGAATCATGATAGTATTTTTGG

CGATCTCAACATGGTGGCTCTAGGATTTCGGATACT

ACGACTGAATAGATATGTCGCTTCTTCAGATGTT

TTTAAAAAGTTCAAAGGTGAAGAAGGACAATTCTCT

GGTTTTGAATCTAGCGATCAAGATGCAAAATTAG

AAATGATGTTAAATTTATATAAAGCTTCAGAATTAG

ATTTTCCTGATGAAGATATCTTAAAAGAAGCAAG

AGCGTTTGCTTCTATGTACCTGAAACATGTTATCAA

AGAATATGGTGACATACAAGAATCAAAAAATCCA

CTTCTAATGGAGATAGAGTACACTTTTAAATATCCT

TGGAGATGTAGGCTTCCAAGGTTGGAGGCTTGGA

ACTTTATTCATATAATGAGACAACAAGATTGCAATA

TATCACTTGCCAATAACCTTTATAAAATTCCAAA

AATATATATGAAAAAGATATTGGAACTAGCAATACT

GGACTTCAATATTTTGCAGTCACAACATCAACAT

GAAATGAAATTAATATCCACATGGTGGAAAAATTC

AAGTGCAATTCAATTGGATTTCTTTCGGCATCGTC

ACATAGAAAGTTATTTTTGGTGGGCTAGTCCATTAT

TTGAACCTGAGTTCAGTACATGTAGAATTAATTG TACCAAATTATCTACAAAAATGTTCCTCCTTGACGA

TATTTATGACACATATGGGACTGTTGAGGAATTG

AAACCATTCACAACAACATTAACAAGATGGGATGTT

TCCACAGTTGATAATCATCCAGACTACATGAAAA

TTGCTTTCAATTTTTCATATGAGATATATAAGGAAA

TTGCAAGTGAAGCCGAAAGAAAGCATGGTCCCTT

TGTTTACAAATACCTTCAATCTTGCTGGAAGAGTTA

TATCGAGGCTTATATGCAAGAAGCAGAATGGATA

GCTTCTAATCATATACCAGGTTTTGATGAATACTTG

ATGAATGGAGTAAAAAGTAGCGGCATGCGAATTC

TAATGATACATGCACTAATACTAATGGATACTCCTT

TATCTGATGAAATTTTGGAGCAACTTGATATCCC

ATCATCCAAGTCGCAAGCTCTTCTATCATTAATTACT

CGACTAGTGGATGATGTCAAAGACTTTGAGGAT

GAACAAGCTCATGGGGAGATGGCATCAAGTATAGA

GTGCTACATGAAAGACAACCATGGTTCTACAAGGG

AAGATGCTTTGAATTATCTCAAAATTCGTATAGAGA

GTTGTGTGCAAGAGTTAAATAAGGAGCTTCTCGA

GCCTTCAAATATGCATGGATCTTTTAGAAACCTATA

TCTCAATGTTGGCATGCGAGTAATATTTTTTATG

CTCAATGATGGTGATCTCTTTACACACTCCAATAGA

AAAGAGATACAAGATGCAATAACAAAATTTTTTG

TGGAACCAATCATTCCATAG

Valencene MAEMFNGNS SNDGS SCMP VKD ALRRTGNHHPNL WT Synthase from DDFIOSLNSPYSDSSYHKHPvEILIDEIPvDMFSNGE Callitropis GDEFGVLENIWFVDVVOPvLGIDPvHFOEEIKTALDYIYK

FWNHDSIFGDLNMVALGFRILRLNRYVASSDV

nootkatensis

FKKFKGEEGOFSGFESSDODAKLEMMLNLYKASELDF

PDEDILKEARAFASMYLKHVIKEYGDIOESK P

LLMEIEYTFKYPWRCRLPRLEAWNFIHIMROODCNISL

A NL YKIPKI YMK ILEL AILDFNILO S OHOH

EMKLISTWWK SSAIOLDFFRHRHIESYFWWASPLFEP

EFSTCRINCTKLSTKMFLLDDIYDTYGTVEEL

KPFTTTLTRWDVSTVDNHPDYMKIAFNFSYEIYKEIAS

EAERKHGPFVYKYLOSCWKSYIEAYMOEAEWI

ASNHIPGFDEYLMNGVKSSGMRILMIHALILMDTPLSD

EILEOLDIP S SKS 0 ALL SLITRL VDD VKDFED

EOAHGEMASSIECYMKDNHGSTREDALNYLKIRIESC

VOELNKELLEPSNMHGSFRNLYLNVGMRVIFFM

LNDGDLFTHSNRKEIODAITKFFVEPIIP

Valencene ATGTC GTCTGG AG AAAC ATTTC GTC CT ACTGC AG AT Synthase from TTCCATCCTAGTTTATGGAGAAACCATTTCCTCA

Citrus sinensis AAGGTGCTTCTGATTTCAAGACAGTTGATCATACTG

CAACTCAAGAACGACACGAGGCACTGAAAGAAGA GGTAAGGAGAATGATAACAGATGCTGAAGATAAGC

CTGTTCAGAAGTTACGCTTGATTGATGAAGTACAA

CGCCTGGGGGTGGCTTATCACTTTGAGAAAGAAATA

GGAGATGCAATACAAAAATTATGTCCAATCTATA

TTGACAGTAATAGAGCTGATCTCCACACCGTTTCCC

TTCATTTTCGGTTGCTTAGGCAGCAAGGAATCAA

GATTTCATGTGATGTGTTTGAGAAGTTCAAAGATGA

TGAGGGTAGATTCAAGTCATCGTTGATAAACGAT

GTTCAAGGGATGTTAAGTTTGTACGAGGCAGCATAC

ATGGCAGTTCGCGGAGAACATATATTAGATGAAG

CCATTGCTTTCACTACCACTCACCTGAAGTCATTGGT

AGCTCAGGATCATGTAACCCCTAAGCTTGCGGA

ACAGATAAATCATGCTTTATACCGTCCTCTTCGTAA

AACCCTACCAAGATTAGAGGCGAGGTATTTTATG

TCCATGATCAATTCAACAAGTGATCATTTATGCAAT

AAAACTCTGCTGAATTTTGCAAAGTTAGATTTTA

ACATATTGCTAGAGCTGCACAAGGAGGAACTCAAT

GAATTAACAAAGTGGTGGAAAGATTTAGACTTCAC

TACAAAACTACCTTATGCAAGAGACAGATTAGTGG

AGTTATATTTTTGGGATTTAGGGACATACTTCGAG

CCTCAATATGCATTTGGGAGAAAGATAATGACCCAA

TTAAATTACATATTATCCATCATAGATGATACTT

ATGATGCGTATGGTACACTTGAAGAACTCAGCCTCT

TTACTGAAGCAGTTCAAAGATGGAATATTGAGGC

CGTAGATATGCTTCCAGAATACATGAAATTGATTTA

CAGGACACTCTTAGATGCTTTTAATGAAATTGAG

GAAGATATGGCCAAGCAAGGAAGATCACACTGCGT

ACGTTATGCAAAAGAGGAGAATCAAAAAGTAATTG

GAGCATACTCTGTTCAAGCCAAATGGTTCAGTGAAG

GTTACGTTCCAACAATTGAGGAGTATATGCCTAT

TGCACTAACAAGTTGTGCTTACACATTCGTCATAAC

AAATTCCTTCCTTGGCATGGGTGATTTTGCAACT

AAAGAGGTTTTTGAATGGATCTCCAATAACCCTAAG

GTTGTAAAAGCAGCATCAGTTATCTGCAGACTCA

TGGATGACATGCAAGGTCATGAGTTTGAGCAGAAG

AGAGGACATGTTGCGTCAGCTATTGAATGTTACAC

GAAGCAGCATGGTGTCTCTAAGGAAGAGGCAATTA

AAATGTTTGAAGAAGAAGTTGCAAATGCATGGAAA

GATATTAACGAGGAGTTGATGATGAAGCCAACCGT

CGTTGCCCGACCACTGCTCGGGACGATTCTTAATC

TTGCTCGTGCAATTGATTTTATTTACAAAGAGGACG

ACGGCTATACGCATTCTTACCTAATTAAAGATCA

AATTGCTTCTGTGCTAGGAGACCACGTTCCATTTTG

A 24 Valencene MSSGETFRPTADFHPSLWR HFLKGASDFKTVDHTAT Synthase from QERHEALKEEVRRMITDAEDKPVQKLRLIDEVQ

Citrus sinensis RLGVAYHFEKEIGDAIQKLCPIYIDSNRADLHTVSLHF

RLLRQQGIKISCDVFEKFKDDEGRFKSSLIND

VQGMLSLYEAAYMAVRGEHILDEAIAFTTTHLKSLVA

QDHVTPKLAEQINHALYRPLRKTLPRLEARYFM

SMINSTSDHLCNKTLLNFAKLDFNILLELHKEELNELT

KWWKDLDFTTKLPYARDRLVELYFWDLGTYFE

PQYAFGRKIMTQLNYILSIIDDTYDAYGTLEELSLFTEA

VQRWNIEAVDMLPEYMKLIYRTLLDAFNEIE

EDMAKQGRSHCVRYAKEENQKVIGAYSVQAKWFSEG

Y VPTIEE YMPI ALT S C AYTF VITN SFLGMGDF AT

KEVFEWIS NPKVVKAASVICRLMDDMQGHEFEQKR

GHVASAIECYTKQHGVSKEEAIKMFEEEVANAWK

DINEELMMKPTVVARPLLGTILNLARAIDFIYKEDDGY

THSYLIKDQIASVLGDHVPF

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 for farnesene

Figure 6 GC results, counts vs acquisition time for santalene

Figure 7 GC results, counts vs acquisition time for bisabolol

Figure 8 GC results, FID units pA vs acquisition time for valencene

Figure 9 Valencene production in 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 sesquiterpene a-farnesene.

The inventors have introduced a specific two-enzyme pathway into a cyanobacterial cell to produce a -famesene. a-farnesene (3,7,1 l-trimethyldodeca-l,3E,6E,10-tetraene) is one of the simplest acyclic sesquiterpenes. It can exist as four stereoisomers that differ about the geometry of two of its three internal double bonds (the stereoisomers of the third internal double bond are identical). (E, E)-a-Farnesene is the most common isomer found in nature and is hereafter referred to as famesene. Famesene is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by famesyl diphosphate synthase (FPPS) sequentially first to form geranyl diphosphate (GPP) and then another condensation between IPP and GPP to get Famesyl diphosphate (FPP). In the second step, the enzyme famesene Synthase (FS) catalyzes the conversion of FPP to famesene. To make famesene in Synechocystis, we chose the following two FPP synthases (1) The gene ispA from Escherichia coli (SEQ ID NO: 1, 2) and erg20 gene, the FPP synthase from Saccharomyces cerevisiae (SEQ ID NO: 3, 4). We chose the samesene synthase (FS) from Malus x domestica which is specific for (Ε,Ε)-α- famesene (SEQ ID NO: 9, 10). The ispA gene from Escherichia coli and the fs gene from Malus x domestica were codon optimized.

Example 2. Biochemical Background of a cyanobacterial cell producing famesene according to the present invention

The genes encoding the famesene synthase (FS) from Malus x domestica and the FPPS from Escherichia coli were codon-optimized for expression in Synechocystis and obtained through chemical synthesis. While the erg20 gene was amplified from Saccharomyces cerevisiae and. 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 making of 4 plasmids,

1. Integration plasmid with FS from Malus x domestica and the FPPS from Escherichia coli (H-ispA-FS)

2. Conjugative plasmid pVZ with FS from Malus x domestica and the FPPS from Escherichia coli (VZ-ispA-FS)

3. Integration plasmid with FS from Malus x domestica and the FPPS from Saccharomyces cerevisiae (H-erg20-FS)

4. Conjugative plasmid pVZ with FS from Malus x domestica and the FPPS from Saccharomyces cerevisiae (VZ-erg20-FS)

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

Example 3. Production of farnesene by a cyanobacterial cell

Mutant cultures obtained in example 2 were grown to an OD730 = 1 to 3 and were used for farnesene 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 then 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 a- Farnesene (mixture of isomers) to obtain the elution time for (E,E)-a-farnesene and make a standard curve. The wild-type culture was used as negative control. (E,E)-a-farnesene elutes at a retention time of 9.14-9.16 minutes. That the peaks observed represent (E,E)-a-farnesene, was also confirmed by the mass fragmentation pattern. All four strains: H-ispA-FS, VZ-ispA- FS, H-erg20-FS, VZ-erg20-FS obtained in example 2 were tested. Figure 5 shows that the all the tested strains produced farnesene in different yields, whereas the wild-type cell did not produce farnesene. It can be concluded that erg20 is the preferred FPPS gene and that yields are higher in strains carrying the heterologous genes on self-replicating plasmids as compared to the strains were genes are integrated into the genome.

Example 4. Enzymes for production of the sesquiterpene a -santalene.

The inventors have introduced a specific two-enzyme pathway into a cyanobacterial cell to produce a -santalene. a-Santalene (l,7-Dimethyl-7-(4-methyl-3-pentenyl)-tricyclo[2.2.1.0(2,6)]heptane) is the sesquiterpene precursor for sandalwood fragrance compound santalol. Santalene is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by farnesyl diphosphate synthase (FPPS) sequentially first to form geranyl diphosphate (GPP) and then another condensation between IPP and GPP to get farnesyl diphosphate (FPP). In the second step, the enzyme santalene synthase (SS) catalyzes the cyclization of FPP to santalene. To make santalene in Synechocystis, we chose the following two FPP synthases (1) The gene ispA from Escherichia coli (SEQ ID NO: 1, 2) and erg20 gene, the FPP synthase from Saccharomyces cerevisiae (SEQ ID NO: 3, 4). We chose the santalene synthase (SS) from Santalum album (SEQ ID NO: 13, 14). The ispA gene from Escherichia coli and the ss gene from Santalum album were codon optimized. Example 5. Biochemical Background of a cyanobacterial cell producing santalene according to the present invention

The genes encoding the santalene synthase (SS) from Santalum album and the FPPS from Escherichia coli were codon-optimized for expression in Synechocystis and obtained through chemical synthesis. While the erg20 gene was amplified from Saccharomyces cerevisiae and. 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 making of 4 plasmids,

1. Integration plasmid with SS from Santalum album and the FPPS from Escherichia coli (H-ispA-SS) 2. Conjugative plasmid pVZ with SS from Santalum album and the FPPS from Escherichia coli (VZ-ispA-SS)

3. Integration plasmid with SS from Santalum album and the FPPS from Saccharomyces cerevisiae (H-erg20-SS)

4. Conjugative plasmid pVZ with SS from Santalum album and the FPPS from

Saccharomyces cerevisiae (VZ-erg20-SS)

Each plasmid was transformed into Synechocystis PCC 6803 as described in patent application EP2563927. Example 6. Production of Santalene by a cyanobacterial cell

Mutant cultures obtained in example 5 were grown to an OD730 = 1 to 3 and were used for santalene measurements. 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 high light intensity (~250 μΕ), 30° C, and shaking for 2 hours. The culture were heated to 55 deg C for 20 minutes to terminate growth. 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. 2.5 ml of headspace air was injected into GC/MS and a program with longer ramp time was used to separate peaks better. The peak for santalene, which elutes at 14.7 minutes, was identified by comparing the fragmentation pattern to fragmentation pattern of standard santalene. All the four strains obtained in example 5: H-ispA-SS, VZ-ispA-SS, H-erg20-SS, VZ-erg20-SS, were tested. Figure 6 shows that the all the tested strains produced santalene in different yields, whereas the wild-type strain did not produce any santalene. The mutant strains also produced bergamotene (peak observed at 14.9 minutes also identified by analysis of mass fragmentation pattern) .

Example 7. Enzymes for production of the sesquiterpene a -bisabolol.

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

Bisabolol, or more formally a-(-)-bisabolol is a natural monocyclic sesquiterpene alcohol. It is a colorless viscous oil that is the primary constituent of the essential oil from German chamomile {Matricaria recutita). Bisabolol has a weak sweet floral aroma and is used in various fragrances. It is used in cosmetics because of its perceived skin healing and whitening properties. Bisabolol is also known to have anti-irritant, anti-inflammatory and anti-microbial properties and also demonstrated to enhance the percutaneous absorption of certain molecules Bisabolol is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by farnesyl diphosphate synthase (FPPS) sequentially first to form geranyl diphosphate (GPP) and then another condensation between IPP and GPP to get farnesyl diphosphate (FPP). In the second step, the enzyme bisabolol synthase (BS) catalyzes the cyclization of FPP to bisabolol. To make bisabolol in Synechocystis, we chose the FPP synthase from Saccharomyces cerevisiae (SEQ ID NO: 3, 4). We chose the bisabolol synthase (BS) either from Santalum spicatum (SEQ ID NO: 17, 18) or from Artemisia annua (SEQ ID NO: 19, 20). Both the bisabolol synthase genes were codon optimized.

Example 8. Biochemical Background of a cyanobacterial cell producing bisabolol according to the present invention

The genes encoding the bisabolol synthase (SS) from Artemisia annua and the FPPS (erg20 gene) was amplified from Saccharomyces cerevisiae. 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 plasmid was transformed into Synechocystis PCC 6803 as described in patent application EP2563927. Example 9. Production of Bisabolol by a cyanobacterial cell

The strain obtained in example 8 (H_20_aBS) was grown to an OD730 = 1 to 3 and used for bisabolol measurements. 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 high light intensity (~250 μΕ), 30° C, and shaking for 2 hours. The culture were heated to 55 deg C for 20 minutes to terminate growth. 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. 2.5 ml of headspace air was injected into GC/MS and a program with longer ramp time was used to separate peaks better. The peak for bisabolol, which elutes at 14.6 minutes, was identified by comparing the fragmentation pattern to fragmentation pattern of standard bisabolol as well as by using a known standard. The strain produced bisabolol as the major product. However as expected and based on literature, other sesquiterpenes also were seen in minor amounts between retention times 13-13.6 mins (Figure 7). Example 10. Enzymes for production of the sesquiterpene valencene.

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

Valencene is a sesquiterpene that is an aroma component of citrus fruit and citrus-derived odorants and used extensively in the beverage industry. It is a precursor to nootkatone, the main aroma and flavor component of grapefruit. Valencene is made in two steps from the isoprenoid precursors. In the first step IPP and DMAPP are condensed by farnesyl diphosphate synthase (FPPS) sequentially first to form geranyl diphosphate (GPP) and then another condensation between IPP and GPP to get Farnesyl diphosphate (FPP). In the second step, the enzyme Valencene Synthase (VS) catalyzes the conversion of FPP to valencene. To make valencene in Synechocystis, we chose the FPP synthase from Saccharomyces cerevisiae (SEQ ID NO: 3, 4). We chose the Valencene Synthase (VS) from Callitropis nootkatensis (CnVS) (SEQ ID NO: 21, 22) and Citrus sinensis (CsVS) (SEQ ID NO: 23, 24). Both the valencene synthase genes were codon optimized.

Example 11. Biochemical Background of a cyanobacterial cell producing valencene according to the present invention

The genes encoding the Valencene Synthase (VS) from Callitropis nootkatensis and Citrus sinensis were codon-optimized for expression in Synechocystis and synthesized. While the erg20 gene for FPPS was amplified from Saccharomyces cerevisiae. 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 either as operons, with both genes sharing the same trc promoter or 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 operons. This led to the provision of three plasmids,

1. Integration plasmid with VS from Callitropis nootkatensis and the FPPS from Saccharomyces cerevisiae as operon (H-erg20-CnVS_op)

2. Integration plasmid with VS from Citrus sinensis and the FPPS from Saccharomyces cerevisiae as cassette (H-erg20-CsVS_cas)

3. Conjugative plasmid pVZ with VS from Callitropis nootkatensis and the FPPS from Saccharomyces cerevisiae as operon (VZ-erg20-CnVS_op) Each plasmid was transformed into Synechocystis PCC 6803 as described in patent application EP2563927.

Example 12. Production of valencene by a cyanobacterial cell

Mutant cultures obtained in example 11 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 resin. The bound terpene was eluted with hexane and the eluate was analyzed by GC FID. Standard solution of valencene in hexane were used to obtain a calibration curve for quantitative determination. A wild- type culture was used a negative control. Valencene elutes at a retention time of around 11.1 minutes. All strains obtained in example 10: integrated and plasmid were tested. Figure 8 shows the FID units vs acquisition time plots obtained from GC analysis. From the figure, it is evident that all strains tested produce valencene while the wild-type strain did not produce any valencene. Figure 9 shows that valencene can be produced in continuously growing cultures and maximum production rates of about 200 μg/gDW/L/day were achieved.

Reference list

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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.

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

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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.

Claims

Claims

1. A cyanobacterial cell capable of expressing, preferably expressing, at least one functional enzyme selected from the group of enzymes consisting of a farnesyl diphosphate synthase (FPPS) and a sesquiterpene synthase (STS), said at least one functional enzyme preferably having ability to condense IPP and DMAPP to GPP and further to FPP

2. A cyanobacterial cell according to claim 1, wherein the at least one functional enzyme is a heterologous enzyme.

3. A cyanobacterial cell according to claim 1 or 2, wherein the at least one functional enzyme is selected from the group consisting of FPPS from E. coli, Methanothermobacter thermautotrophicus, Mentha x piperita and Saccharomyces cerevisiae, and/or

wherein the at least one functional enzyme is selected from the group consisting of sesquiterpene synthases from Mains domestica, Ricinus communis, Solanum habrochaites, Santalum spicatum, Artemisia annua, Callitropis nootkatensis, Citrus sinensis and Santalum album.

4. A cyanobacterial cell according to any of the preceding claims, wherein the at least one functional enzyme comprises or consists of a polypeptide that has an amino acid sequence with at least 30% 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 and SEQ ID NO: 24.

5. A cyanobacterial cell according to any of the preceding claims, wherein the at least one functional enzyme is encoded by a codon optimized polynucleotide.

6. A cyanobacterial cell according to any of the preceding claims, wherein the at least one functional enzyme is encoded by a polynucleotide that has an nucleic acid sequence with at least 30% 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: 1 1, SEQ ID NO: 13, SEQ ID NO: 15, SEQ ID NO: 17; SEQ ID NO: 19, SEQ ID NO: 21 and SEQ ID NO: 23.

7. A cyanobacterial cell according to any of the preceding claims, wherein the cyanobacterial cell is a Synechocystis, preferably a Synechocystis PCC 6803.

8. A cyanobacterial cell according to any of the preceding claims, 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.

9. A process for producing a sesquiterpene comprising culturing a cyanobacterial cell according to any one of claims 1-8 under conditions conducive to the production of a sesquiterpene and, optionally, isolating and/or purifying the sesquiterpene from the culture broth or headspace.

10. A process according to claim 9, wherein the culture conditions comprise feeding carbon dioxide to the culture and/or subjecting the culture to light.

11. A process according to any of claims 9 or 10, wherein the sesquiterpene is selected from the group consisting of: artemisinin, bisabolol, farnesene, valencene, santalene, bergamotene.

12. A process according to claim 11, wherein the sesquiterpene is farnesene, bisabolol, valencene or santalene, most preferably the sesquiterpene is valencene or santalene.

13. A sesquiterpene obtainable by a process according to any of claims 9 - 12.

14. A pharmaceutical composition, a fuel compostion, a flavor composition, a flagrance composition or a cosmetic composition comprising a sequiterpene obtainable by a process according to any of claims 9 - 12.