AU2017202313B2

AU2017202313B2 - Method for producing beta-santalene

Info

Publication number: AU2017202313B2
Application number: AU2017202313A
Authority: AU
Inventors: Michel Schalk
Original assignee: Firmenich SA
Current assignee: Firmenich SA
Priority date: 2008-12-11
Filing date: 2017-04-07
Publication date: 2018-07-05
Anticipated expiration: 2029-12-09
Also published as: AU2015201051A1; AU2017202313A1; AU2015201051B2

Abstract

METHOD FOR PRODUCING BETA-SANTALENE The present invention provides a method of producing B-santalene, said method comprising contacting at least one polypeptide with farnesyl pyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce B-santalene, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing B-santalene is also an object of the present invention.

Description

The present invention provides a method of producing B-santalene, said method comprising contacting at least one polypeptide with farnesyl pyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce B-santalene, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing B-santalene is also an object of the present invention.

2017202313 07 Apr 2017

METHOD FOR PRODUCING BETA-SANTALENE

Cross-Reference to Related Applications

The present application is a divisional application of Australian Patent Application No. 2015201051, which is a divisional application of Australian Patent Application No. 2009325879 (PCT/IB2009/055589) which claims priority from EP 08171298.6 filed on 11 December 2008 and PCT/IB2009/053623 filed on 17 August 2009, the content of each of which is incorporated herein by reference in its entirety.

Technical field

The present invention provides a method of producing β-santalene, said method comprising contacting at least one polypeptide with famesyl pyrophosphate (FPP). In particular, said method may be carried out in vitro or in vivo to produce β-santalene, a very useful compound in the fields of perfumery and flavoring. The present invention also provides the amino acid sequence of a polypeptide useful in the method of the invention. A nucleic acid encoding the polypeptide of the invention and an expression vector containing said nucleic acid are also part of the present invention. A non-human host organism or a cell transformed to be used in the method of producing β-santalene is also an object of the present invention.

Prior art

Terpenes are found in most organisms (microorganisms, animals and plants). These compounds are made up of five carbon units called isoprene units and are classified by the number of these units present in their structure. Thus monoterpenes, sesquiterpenes and diterpenes are terpenes containing 10, 15 and 20 carbon atoms respectively. Sesquiterpenes, for example, are widely found in the plant kingdom. Many sesquiterpene molecules are known for their flavor and fragrance properties and their cosmetic, medicinal and antimicrobial effects. Over 300 sesquiterpene hydrocarbons and 3000 sesquiterpenoids have been identified (Joulain, D., and Konig, W.A., The Atlas of Spectral Data of Sesquiterpene Hydrocarbons, EB Verlag, Hamburg, 1998; Connolly, J.D., Hill R.A., Dictionary of Terpenoids, Vol 1, Chapman and Hall (publisher), 1991), and many new structures are identified each year. Plant extracts obtained by different means such as steam distillation or solvent extraction are used as source of terpenes. Terpene molecules are often used as such, but in some cases chemical reactions are used to transform the terpenes into other high value #12886836 la

2017202313 07 Apr 2017 molecules.

Biosynthetic production of terpenes involves enzymes called terpene synthases. There is virtually an infinity of sesquiterpene synthases present in the plant kingdom, all using the same substrate (farnesyl pyrophosphate, FPP) but having different product profiles. Genes and cDNAs encoding sesquiterpene synthases have been cloned and the #12886836

2017202313 07 Apr 2017 corresponding recombinant enzymes characterized. The biosynthesis of terpenes in plants and other organisms has been extensively studied and is not further detailed in here, but reference is made to Dewick, Nat. Prod. Rep., 2002, 19, 181-222, which reviews the state of the art of terpene biosynthetic pathways.

β-Santalene is a naturally occurring sesquiterpene molecule that can be used as starting material for the chemical synthesis or the biosynthesis of β-santalol (as represented in Figure 2B), which is a major constituent of sandalwood oil. Sandalwood oil is an important perfumery ingredient obtained by distillation of the heartwood of Santalum species. Sandalwood is also largely used for incenses and traditional medicine. The oil contains 90% of sesquiterpene alcohols. Among the different isomers of santalol, βsantalol is the principal contributor to the typical sweet-woody and balsamic odour of sandalwood oil. Other constituents such as epi^-santalol and α-santalol may also contribute to the sandalwood note.

Generally, the price and availability of plant natural extracts are dependent on the abundance, oil yield and geographical origin of the plants. In addition, the availability and quality of natural extracts is very much dependent on climate and other local conditions leading to variability from year to year, rendering the use of such ingredients in high quality perfumery very difficult or even impossible some years. Due to over-exploitation of the natural resources, difficulties of cultivation, slow growth of the Santalum plants, the availabilities of sandalwood raw material has dramatically decreased during the past decades. Therefore, it would be an advantage to provide a source of β-santalol, which is less subjected to fluctuations in availability and quality. A chemical synthesis of the sandalwood sesquiterpene constituents is so far not available. A biochemical pathway leading to the synthesis of β-santalene, which could then be used to produce β-santalol, would therefore be of great interest.

Santalane type sesquiterpene, and particularly sesquiterpenes with the β-santalane skeleton, were identified in several plant species. Though, no sesquiterpene synthase capable of producing β-santalene, has yet been described.

A sesquiterpene synthase capable of synthesizing at least one bi-cyclic and/or tri30 cyclic sesquiterpene having a santalane carbon skeleton, the corresponding nucleic acid and a method for producing such compound having a santalane carbon skeleton are disclosed in the International patent application WO 2006/134523. Nevertheless, no trace

2017202313 07 Apr 2017 of β-santalene was detected as product of the sesquiterpene synthases disclosed in the examples. The only product with a santalane skeleton was epi-beta-santalene. The properties of epi-beta-santalene are very different from those of β-santalene. In particular, it is of no interest in the synthesis of β-santalol. Moreover, the sesquiterpene synthase disclosed in WO 2006/134523 shares only 27% of identity with the sequence of the invention.

The percentage of identity between sesquiterpene synthases known from the databases and the polypeptides of the invention is very low. The closest protein sequence to the β-santalene synthase of the invention is a monoterpene synthase from Santalum album (access No. ACF24767; Jones, C.G., Keeling, C.I., Ghisalberti, E.F., Barbour, E.F., Plummer, J.A. and Bohlmann, J. Arch. Biochem. Biophys., 2008, 477(1), 121-130) which shares 58% amino acid sequence identity with the β-santalene synthase of the invention. When contacted with FPP, this enzyme produces over 90% of β-bisabolene and no santalene isomer is formed.

In addition to the difference between the sequences themselves, it also has to be pointed out that the structure and the properties of the products synthesized by the abovementioned enzyme are very different from those of the sesquiterpene β-santalene. In particular the monoterpenes produced by this enzyme, i.e. alpha-terpineol, limonene, geraniol, myrcene, linalool and some other minor products are not suitable as a starting material for the synthesis of β-santalol, which is a very useful ingredient in the field of perfumery.

Despite extensive studies of terpene cyclization, the isolation and characterization of the terpene synthases is still difficult, particularly in plants, due to their low abundance, their often transient expression patterns, and the complexity of purifying them from the mixtures of resins and phenolic compounds in tissues where they are expressed.

It is an objective of the present invention to provide methods for making β-santalene in an economic way, as indicated above. Accordingly, the present invention has the objective to produce β-santalene while having little waste, a more energy and resource efficient process and while reducing dependency on fossil fuels. It is a further objective to provide enzymes capable of synthesizing β-santalene, which is useful as perfumery and/or aroma ingredients.

2017202313 07 Apr 2017

	Abbreviations used
	ACC	1 -aminocyclopropanecarboxylic acid
	bp	base pair
	kb	kilo base
5	BSA	bovine serum albumin
	2,4D	2,4-dichlorophenoxyacetic acid
	DNA	deoxyribonucleic acid
	cDNA	complementary DNA
	dNTP	deoxy nucleotide triphosphate
10	DTT	dithiothreitol
	EDTA	ethylene-diamine-tetraacetic acid
	FPP	famesyl pyrophosphate
	GC	gaseous chromatograph
	IPTG	isopropyl-D-thio galacto -pyranoside
15	Kin	kinetin
	FB	lysogeny broth
	MS	mass spectrometer
	PCR	polymerase chain reaction
	RMCE	recombinase-mediated cassette exchange
20	3’-/5’-RACE	3’ and 5’ rapid amplification of cDNA ends
	RNA	ribonucleic acid
	mRNA	messenger ribonucleic acid
	RNAse	Ribonuclease
25	Description of the invention

The present invention provides a method to biosynthetically produce β-santalene in an economic, reliable and reproducible way, using a polypeptide having a β-santalene synthase activity. The present invention is particularly useful because no such polypeptide was known in the prior art and because no such biosynthesis of β-santalene has been described. This solves the very important problem of the supply of β-santalene, a compound which is very useful for the perfumery industry. The present invention also provides a nucleic acid sequence that encodes the polypeptides used in the method of the

2017202313 07 Apr 2017 invention, thus being intimately linked to said polypeptide. The polypeptide and the nucleic acid are very important tools, which are both necessary to carry out the method of the invention. The same is true for vectors and for organisms modified with the nucleic acid of the invention to heterologously express the polypeptide of the invention.

A “sesquiterpene synthase” or a “polypeptide having a sesquiterpene synthase activity” is intended for the purpose of the present application as a polypeptide capable of catalyzing the synthesis of a sesquiterpene molecule or of a mixture of sesquiterpene molecules from the acyclic terpene precursor FPP.

As a “β-santaiene synthase” or as a “polypeptide having a β-santalene synthase 10 activity”, we mean here a polypeptide capable of catalyzing the synthesis of β-santalene, in the form of any of its stereoisomers or a mixture thereof, starting from FPP. B-Santalene may be the only product or may be part of a mixture of sesquiterpenes.

B-Santalene is defined by the way of its structure, as represented in Figure 2A.

The ability of a polypeptide to catalyze the synthesis of a particular sesquiterpene 15 (for example β-santalene) can be simply confirmed by performing the enzyme assay as detailed in Example 3.

According to the present invention, polypeptides are also meant to include truncated polypeptides provided that they keep their sesquiterpene synthase activity as defined in any of the above embodiments and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO: 15.

As intended herein below, a nucleotide sequence obtained by modifying SEQ ID

NO: 14 or 2 encompasses any sequence that has been obtained by changing the sequence of SEQ ID NO: 14 or of SEQ ID NO:2 using any method known in the art, for example by introducing any type of mutations such as deletion, insertion or substitution mutations. Examples of such methods are cited in the part of the description relative to the variant polypeptides and the methods to prepare them.According to the present invention, polypeptides are also meant to include truncated polypeptides provided that they keep their sesquiterpene synthase activity as defined in any of the above embodiments and that they share at least the defined percentage of identity with the corresponding fragment of

SEQ ID NO: 1.

As intended herein below, “a nucleotide sequence obtained by modifying SEQ ID NO:2, 4 or the complement thereof’ encompasses any sequence that has been obtained by changing the sequence of SEQ ID NO:2, of SEQ ID NO:4 or of the complement thereof

AH26(9643524_1):CCG

2017202313 15 May 2018 using any method known in the art, for example by introducing any type of mutations such as deletion, insertion or substitution mutations. Examples of such methods are cited in the part of the description relative to the variant polypeptides and the methods to prepare them.

The percentage of identity between two peptidic or nucleotidic sequences is a function of the number of amino acids or nucleotide residues that are identical in the two sequences when an alignment of these two sequences has been generated. Identical residues are defined as residues that are the same in the two sequences in a given position of the alignment. The percentage of sequence identity, as used herein, is calculated from the optimal alignment by taking the number of residues identical between two sequences dividing it by the total number of residues in the shortest sequence and multiplying by 100. The optimal alignment is the alignment in which the percentage of identity is the highest possible. Gaps may be introduced into one or both sequences in one or more positions of the alignment to obtain the optimal alignment. These gaps are then taken into account as non-identical residues for the calculation of the percentage of sequence identity.

Alignment for the purpose of determining the percentage of amino acid or nucleic acid sequence identity can be achieved in various ways using computer programs and for instance publicly available computer programs available on the world wide web. Preferably, the BLAST program (Tatiana et al, FEMS Microbiol Lett., 1999, 174:247-250, 1999) set to the default parameters, available from the National Center for Biotechnology Information (NCBI) at http://www.ncbi.nlm.nih.gov/BLAST/bl2seq/wblast2.cgi, can be used to obtain an optimal alignment of peptidic or nucleotidic sequences and to calculate the percentage of sequence identity.

According to a first aspect, the present invention provides a method for producing βsantalene and derivatives thereof comprising

a) contacting FPP with at least one polypeptide having a β-santalene synthase activity and comprising an amino acid sequence at least 6% identical to SEQ ID NO: 1, SEQ ID NO: 3 or SEQ ID NO: 15, or comprising the amino acid sequence of SEQ ID NO: 27;

b) producing β-santalene; and

c) processing the β-santalene produced to β-santalene derivative using a chemical or biochemical synthesis or a combination of both.

According to a second aspect, the present invention provides a β-santalene or a derivative thereof produced according to the method of the first aspect.

(14692150_l):GGG

2017202313 15 May 2018

According to a preferred embodiment, β-santalene represents at least 20%, preferably at least 30%, preferably at least 35% of the products produced by the method of the invention.

The method can be carried out in vitro as well as in vivo, provided that methods involving only the natural metabolism of the plant, without any transformation, are not encompassed by the methods of the present invention, as will be explained in details further on.

The polypeptide to be contacted with FPP in vitro can be obtained by extraction from any organism expressing it, using standard protein or enzyme extraction technologies. If the host organism is a unicellular organism or cell releasing the polypeptide of the invention into the culture medium, the polypeptide may simply be collected from the culture medium, for example by centrifugation, optionally followed by washing steps and re-suspension in suitable buffer solutions. If the organism or cell accumulates the polypeptide within its cells, the polypeptide may be obtained by disruption or lysis of the cells and further extraction of the polypeptide from the cell lysate.

The polypeptide having a β-santalene synthase activity, either in an isolated form or together with other proteins, for example in a crude protein extract obtained from cultured cells or microorganisms, may then be suspended in a buffer solution at optimal pH. If adequate, salts, DTT, BSA and other kinds of enzymatic co-factors, may be added in order to optimize enzyme activity. The concentration of these co-factors can be adjusted in order to achieve an optimized yield. For example, lowering the concentration of Mg²⁺ ions in the polypeptide suspension is particularly advantageous to increase the yield of β-santalene. The optimal concentration of Mg²⁺ ions is comprised between 2 and 0.75 mM. Appropriate conditions are described in more details in the Examples further on.

The precursor FPP is added to the polypeptide suspension, which is then incubated at optimal temperature, for example between 15 and 40°C, preferably between 25 and 35°C, more preferably at 30°C. After incubation, the β-santalene produced may be isolated from the incubated solution by standard isolation procedures, such as solvent extraction and distillation, optionally after removal of polypeptides from the solution.

According to another preferred embodiment, the method of any of the abovedescribed embodiments is carried out in vivo. In this case, step a) comprises cultivating a non-human host organism or cell capable of producing FPP and transformed to express at (14692150_l):GGG

2017202313 07 Apr 2017 least one polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 3 and having a β-santalene synthase activity, under conditions conducive to the production of β -santalene.

According to a more preferred embodiment, the method further comprises, prior to 5 step a), transforming a non human organism or cell capable of producing FPP with at least one nucleic acid encoding a polypeptide comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 3 and having a β-santalene synthase activity, so that said organism expresses said polypeptide.

According to another preferred embodiment, the method of any of the 10 abovedescribed embodiments is carried out in vivo. In this case, step a) comprises cultivating a non-human host organism or cell capable of producing FPP and transformed to express at least one polypeptide comprising an amino acid sequence at least 60% identical to SEQ ID NO: 15 and having a β-santalene synthase activity, under conditions conducive to the production of β-santalene.

According to a more preferred embodiment, the method further comprises, prior to step a), transforming a non human organism or cell capable of producing FPP with at least one nucleic acid encoding a polypeptide comprising an amino acid sequence at least 60% identical to SEQ ID NO: 15 and having a β-santalene synthase activity, so that said organism expresses said polypeptide.

These embodiments of the invention are particularly advantageous since it is possible to carry out the method in vivo without previously isolating the polypeptide. The reaction occurs directly within the organism or cell transformed to express said polypeptide.

According to a particular embodiment of the invention, the at least one nucleic acid encoding the β-santalene synthase comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, 4 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO:2, 4 or the complement thereof.

In another preferred embodiment, the nucleic acid consists of a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably

AH26(9643524_1):CCG

2017202313 07 Apr 2017 at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, 4 or the complement thereof. In an even more preferred embodiment, said nucleic acid consists of SEQ ID NO:2, 4 or the complement thereof.

According to a more preferred embodiment the at least one nucleic acid used in any of the above embodiments comprises a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 3 or the complement thereof. According to an even more preferred embodiment, said at least one nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 3 or the complement thereof, preferably

SEQ ID NO:3 or the complement thereof.

According to a particular embodiment of the invention, the at least one nucleic acid encoding the β-santalene synthase comprises a nucleotide sequence at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO: 14, 2 or the complement thereof, and preferably to SEQ ID NO:2 or the complement thereof. According to a more preferred embodiment, said nucleic acid comprises the nucleotide sequence SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID NO:2 or the complement thereof. In an even more preferred embodiment, said nucleic acid consists of SEQ ID NO: 14, 2 or the complement thereof, preferably of SEQ ID NO:2 or the complement thereof.

According to a more preferred embodiment the at least one nucleic acid used in any of the above embodiments comprises a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof. According to an even more preferred embodiment, said at least one nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID NO:2 or the complement thereof.

According to another embodiment, the at least one nucleic acid is isolated from a plant of the Santalum species, preferably from Santalum album.

The organism or cell is meant to “express” a polypeptide, provided that the organism or cell is transformed to harbor a nucleic acid encoding said polypeptide, this nucleic acid is transcribed to mRNA and the polypeptide is found in the host organism or cell. The term “express” encompasses “heterologously express” and “over-express”, the latter referring to levels of mRNA, polypeptide and/or enzyme activity over and above what is measured in a non-transformed organism or cell. A more detailed description of

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2017202313 07 Apr 2017 suitable methods to transform a non-human host organism or cell will be described later on in the part of the specification that is dedicated to such transformed non-human host organisms or cells as specific objects of the present invention and in the examples.

A particular organism or cell is meant to be “capable of producing FPP” when it 5 produces FPP naturally or when it does not produce FPP naturally but is transformed to produce FPP, either prior to the transformation with a nucleic acid as described herein or together with said nucleic acid. Organisms or cells transformed to produce a higher amount of FPP than the naturally occurring organism or cell are also encompassed by the “organisms or cells capable of producing FPP”. Methods to transform organisms, for example microorganisms, so that they produce FPP are already known in the art. Such methods can for example be found in the literature, for example in the following publications: Martin, V.J., Pitera, D.J., Withers, S.T., Newman, J.D., and Keasling, J.D. Nat Biotechnol., 2003, 21(7), 796-802 (transformation of E. coli); Wu, S., Schalk, M., Clark, A., Miles, R.B., Coates, R., and Chappell, J., Nat Biotechnol., 2006, 24(11), 144115 1447 (transformation of plants); Takahashi, S., Yeo, Y., Greenhagen, B. T., McMullin, T.,

Song, L., Maurina-Brunker, J., Rosson, R., Noel, J., Chappell, J, Biotechnology and Bioengineering, 2007, 97(1), 170-181 (transformation of yeast).

To carry out the invention in vivo, the host organism or cell is cultivated under conditions conducive to the production of β-santalene. Accordingly, if the host is a transgenic plant, optimal growth conditions are provided, such as optimal light, water and nutrient conditions, for example. If the host is a unicellular organism, conditions conducive to the production of β-santalene may comprise addition of suitable cofactors to the culture medium of the host. In addition, a culture medium may be selected, so as to maximize β-santalene synthesis. Optimal culture conditions are described in a more detailed manner in the following Examples.

Non-human host organisms suitable to carry out the method of the invention in vivo may be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human host organism used to carry out the invention in vivo is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus can be used. Particularly useful plants are those that naturally produce high amounts of terpenes. In a more preferred embodiment, the plant is selected from the family of Solanaceae, Poaceae,

Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant is selected from the genera Nicotiana, Solanum, Sorghum, Arabidopsis, Brassica (rape),

Medicago (alfalfa), Gossypium (cotton), Artemisia, Salvia and Mentha. Preferably, the

AH26(9643524_1):CCG

2017202313 07 Apr 2017 plant belongs to the species of Nicotiana tabacum.

In a more preferred embodiment the non-human host organism used to carry out the method of the invention in vivo is a microorganism. Any microorganism can be used but according to an even more preferred embodiment said microorganism is a bacteria or yeast. Most preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

Some of these organisms do not produce FPP naturally. To be suitable to carry out the method of the invention, these organisms have to be transformed to produce said precursor. They can be so transformed either before the modification with the nucleic acid described according to any of the above embodiments or simultaneously, as explained above.

Isolated higher eukaryotic cells can also be used, instead of complete organisms, as hosts to carry out the method of the invention in vivo. Suitable eukaryotic cells may be any non-human cell, but are preferably plant or fungal cells.

According to a preferred embodiment, the at least one polypeptide having a β15 santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to

SEQ ID NO:1 or 3. According to a more preferred embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO :1 or 3.

In another preferred embodiment, the polypeptide consists of an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:1 or 3. In an even more preferred embodiment, said polypeptide consists of SEQ ID NO:1 or 3.

According to another preferred embodiment, the at least one polypeptide having a β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino acid sequence that is a variant of SEQ ID NO:1 or 3 obtained by genetic engineering, provided that said variant keeps its β-santalene synthase activity, as defined above and has the required percentage of identity to SEQ ID NO:1 or 3. In other terms, said polypeptide preferably comprises an amino acid sequence encoded by a nucleotide sequence that has

AH26(9643524_1):CCG

2017202313 07 Apr 2017 been obtained by modifying SEQ ID NO:2, 4 or the complement thereof. According to a more preferred embodiment, the at least one polypeptide having a β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments consists of an amino acid sequence that is a variant of SEQ ID NO:1 or 3 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 4 or the complement thereof.

According to another preferred embodiment, the at least one polypeptide having a β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments is a variant of SEQ ID NO:1 or 3 that can be found naturally in other organisms, such as other plant species, provided that it keeps its β-santalene synthase activity as defined above and has the required percentage of identity to SEQ ID NO:1 or 3.

As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they keep their β-santalene synthase activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO: 1 or 3.

According to a preferred embodiment, the at least one polypeptide having a β20 santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino acid sequence at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO: 15. According to a more preferred embodiment, said polypeptide comprises the amino acid sequence SEQ ID NO: 15. In an even more preferred embodiment, said polypeptide consists of SEQ ID NO:15.

According to another preferred embodiment, the at least one polypeptide having a β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments comprises an amino acid sequence that is a variant of SEQ ID NO: 15 obtained by genetic engineering, provided that said variant keeps its β-santalene synthase activity, as defined above and has the required percentage of identity to SEQ ID NO: 15. In other terms, said polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been

AH26(9643524_1):CCG

2017202313 07 Apr 2017 obtained by modifying SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID NO:2 or the complement thereof. According to a more preferred embodiment, the at least one polypeptide having a β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments consists of an amino acid sequence that is a variant of SEQ ID NO: 15 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID N0:2 or the complement thereof.

According to another preferred embodiment, the at least one polypeptide having a 10 β-santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments is a variant of SEQ ID NO: 15 that can be found naturally in other organisms, such as other plant species, provided that it keeps its β-santalene synthase activity as defined above and has the required percentage of identity to SEQ ID NO: 15.

As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequences identified herein, as well as truncated or variant polypeptides, provided that they keep their activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO:15.

Examples of variant polypeptides are naturally occurring proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of the invention.

Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention.

Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends can also be used in the methods of the invention. In particular such a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Accordingly, the present invention encompasses methods using variant polypeptides, such as those obtained by fusion with other oligo- or

AH26(9643524_1):CCG

2017202313 07 Apr 2017 polypeptides and/or those which are linked to signal peptides. Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway, can also be advantageously be used in the methods of the invention.

According to another embodiment, the at least one polypeptide having a β5 santalene synthase activity used in any of the above-described embodiments or encoded by the nucleic acid used in any of the above-described embodiments is isolated from a plant of the Santalum species, preferably from Santalum album.

An important tool to carry out the method of the invention is the polypeptide itself. 10 A polypeptide having a β-santalene synthase activity and comprising an amino acid sequence at least 50% identical to SEQ ID NO:1 or 3 is therefore another object of the present invention.

An important tool to carry out the method of the invention is the polypeptide itself. A polypeptide having a β-santalene synthase activity and comprising an amino acid sequence at least 60% identical to SEQ ID NO: 15 is therefore another object of the present invention.

According to a preferred embodiment, the polypeptide is capable of producing a mixture of sesquiterpenes wherein β-santalene represents at least 20%, preferably at least 30%, preferably at least 35%, of the sesquiterpenes produced.

According to a preferred embodiment, the polypeptide comprises an amino acid sequence at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:1 or 3. According to a more preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO :1 or 3.

According to another preferred embodiment, the polypeptide consists of an amino acid sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:1 or 3. According to an even more preferred embodiment, the polypeptide consists of SEQ ID NO:1 or 3.

The at least one polypeptide comprises an amino acid sequence that is a variant of

SEQ ID NO:1 or 3, either obtained by genetic engineering or found naturally in Santalum plants or in other plant species. In other terms, when the variant polypeptide is obtained by

AH26(9643524_1):CCG

2017202313 07 Apr 2017 genetic engineering, said polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 4 or the complement thereof. According to a more preferred embodiment, the at least one polypeptide having a β-santalene synthase activity consists of an amino acid sequence that is a variant of SEQ ID NO:1 or 3 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 4 or the complement thereof.

According to a preferred embodiment, the polypeptide comprises an amino acid sequence at least 65%, preferably at least 70%, preferably at least 75%, preferably at least

80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO: 15. According to a more preferred embodiment, the polypeptide comprises the amino acid sequence SEQ ID NO: 15. According to an even more preferred embodiment, the polypeptide consists of SEQ ID NO: 15.

SEQ ID NO: 15, either obtained by genetic engineering or found naturally in Santalum plants or in other plant species. In other terms, when the variant polypeptide is obtained by genetic engineering, said polypeptide comprises an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof. According to a more preferred embodiment, the at least one polypeptide having a β-santalene synthase activity consists of an amino acid sequence that is a variant of SEQ ID NO: 15 obtained by genetic engineering, i.e. an amino acid sequence encoded by a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof.

According to another embodiment, the polypeptide is isolated from a plant of the

Santalum species, preferably from Santalum album.

As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequence identified herein, as well as truncated or variant polypeptides, provided that they keep their activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO:1 or 3.

As used herein, the polypeptide is intended as a polypeptide or peptide fragment that encompasses the amino acid sequence identified herein, as well as truncated or variant

AH26(9643524_1):CCG

15a

2017202313 07 Apr 2017 polypeptides, provided that they keep their activity as defined above and that they share at least the defined percentage of identity with the corresponding fragment of SEQ ID NO: 15.

Examples of variant polypeptides are naturally occurring proteins that result from 5 alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C- termini upon expression in different types of host cells, due to proteolytic removal of one or more terminal amino acids from the polypeptides of the invention. Polypeptides encoded by a nucleic acid obtained by natural or artificial mutation of a nucleic acid of the invention, as described thereafter, are also encompassed by the invention.

Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends are also encompassed by the polypeptides of the invention. In particular such a fusion can enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Accordingly, the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides.

Polypeptides resulting from a fusion with another functional protein, such as another protein from the terpene biosynthesis pathway, are also encompassed by the polypeptides of the invention.

As mentioned above, the nucleic acid encoding the polypeptide of the invention is a useful tool to modify non-human host organisms or cells intended to be used when the method is carried out in vivo.

A nucleic acid encoding a polypeptide according to any of the above-described embodiments is therefore also an object of the present invention.

According to a preferred embodiment, the nucleic acid comprises a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, 4 or the complement thereof. According to a more preferred embodiment, the nucleic acid comprises the nucleotide

AH26(9643524_1):CCG

15b

2017202313 07 Apr 2017 sequence SEQ ID NO:2, 4 or the complement thereof.

According to another preferred embodiment, the nucleic acid consists of a nucleotide sequence at least 50%, preferably at least 55%, preferably at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO:2, 4 or the complement thereof. According to an even more preferred embodiment, the nucleic acid consists of SEQ ID NO:2, 4 or the complement thereof.

According to a preferred embodiment, the nucleic acid comprises a nucleotide 10 sequence at least 60%, preferably at least 65%, preferably at least 70%, preferably at least 75%, preferably at least 80%, preferably at least 85%, preferably at least 90%, more preferably at least 95% and even more preferably at least 98% identical to SEQ ID NO: 14, 2 or the complement thereof, preferably to SEQ ID NO:2 or the complement thereof. According to a more preferred embodiment, the nucleic acid comprises the nucleotide sequence SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID NO:2 or the complement thereof. According to an even more preferred embodiment, the nucleic acid consists of SEQ ID NO: 14, 2 or the complement thereof, preferably of SEQ ID NO:2 or the complement thereof.

According to another embodiment, the nucleic acid is isolated from a plant of the

Santalum species, preferably from Santalum album.

The nucleic acid of the invention can be defined as including deoxyribonucleotide or ribonucleotide polymers in either single- or double-stranded form (DNA and/or RNA). The terms nucleotide sequence should also be understood as comprising a polynucleotide molecule or an oligonucleotide molecule in the form of a separate fragment or as a component of a larger nucleic acid. Nucleic acids of the invention also encompass certain isolated nucleotide sequences including those that are substantially free from contaminating endogenous material. The nucleic acid of the invention may be truncated, provided that it encodes a polypeptide encompassed by the present invention, as described above.

The nucleic acid of the invention can be either present naturally in plants of the santalum species or other species, or be obtained by modifying SEQ ID NO:2, 4 or the complement thereof. Preferably said nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO:2, 4 or the complement thereof.

AH26(9643524_1):CCG

15c

2017202313 07 Apr 2017

The nucleic acids comprising a sequence obtained by mutation of SEQ ID NO:2, 4 or the complement thereof are encompassed by the invention, provided that the sequences they comprise share at least the defined percentage of identity with the corresponding fragments of SEQ ID NO:2, 4 or the complement thereof and provided that they encode a polypeptide having a β-santalene synthase activity, as defined in any of the above embodiments. Mutations may be any kind of mutations of these nucleic acids, such as point mutations, deletion mutations, insertion mutations and/or frame shift mutations. A variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by a preferred codon. Due to the degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, multiple DNA sequences can code for the same polypeptide, all these DNA sequences being encompassed by the invention.

The nucleic acid of the invention can be either present naturally in plants of the santalum species or other species, or be obtained by modifying SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID NO:2 or the complement thereof. Preferably said nucleic acid consists of a nucleotide sequence that has been obtained by modifying SEQ ID NO: 14, 2 or the complement thereof, preferably SEQ ID N0:2 or the complement thereof.

The nucleic acids comprising a sequence obtained by mutation of SEQ ID NO: 14, or the complement thereof are encompassed by the invention, provided that the sequences they comprise share at least the defined percentage of identity with the corresponding fragments of SEQ ID NO: 14, 2 or the complement thereof and provided that they encode a polypeptide having a β-santalene synthase activity, as defined in any of the above embodiments. Preferably, the sequence is obtained by mutation of SEQ ID NO:2 or the complement thereof. Mutations may be any kind of mutations of these nucleic acids, such as point mutations, deletion mutations, insertion mutations and/or frame shift mutations. A variant nucleic acid may be prepared in order to adapt its nucleotide sequence to a specific expression system. For example, bacterial expression systems are known to more efficiently express polypeptides if amino acids are encoded by a preferred codon. Due to the degeneracy of the genetic code, wherein more than one codon can encode the same amino acid, multiple DNA sequences can code for the same polypeptide, all these DNA sequences being encompassed by the invention.

Another important tool for transforming host organisms or cells suitable to carry

AH26(9643524_1):CCG

15d

2017202313 07 Apr 2017 out the method of the invention in vivo is an expression vector comprising a nucleic acid according to any embodiment of the invention. Such a vector is therefore also an object of the present invention.

An “expression vector” as used herein includes any linear or circular recombinant vector including but not limited to viral vectors, bacteriophages and plasmids. The skilled person is capable of selecting a suitable vector according to the expression system. In one embodiment, the expression vector includes the nucleic acid of the invention operably linked to at least one regulatory sequence, which controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosomal binding site and, optionally, including at least one selection marker. Nucleotide sequences are operably linked when the regulatory sequence functionally relates to the nucleic acid of the invention.

The expression vectors of the present invention may be used in the methods for preparing a genetically transformed host organism and/or cell, in host organisms and/or

AH26(9643524_1):CCG

2017202313 07 Apr 2017 cells harboring the nucleic acids of the invention and in the methods for making polypeptides having a β-santalene synthase activity, as disclosed further below.

Recombinant non-human host organisms and cells transformed to harbor at least 5 one nucleic acid of the invention so that it heterologously expresses or over-expresses at least one polypeptide of the invention are also very useful tools to carry out the method of the invention. Such non-human host organisms and cells are therefore another object of the present invention.

A nucleic acid according to any of the above-described embodiments can be used 10 to transform the non-human host organisms and cells and the expressed polypeptide can be any of the above-described polypeptides.

Non-human host organisms of the invention may be any non-human multicellular or unicellular organisms. In a preferred embodiment, the non-human host organism is a plant, a prokaryote or a fungus. Any plant, prokaryote or fungus is suitable to be transformed according to the present invention. Particularly useful plants are those that naturally produce high amounts of terpenes. In a more preferred embodiment, the plant is selected from the family of Solanaceae, Poaceae, Brassicaceae, Fabaceae, Malvaceae, Asteraceae or Lamiaceae. For example, the plant is selected from the genera Nicotiana, Solarium, Sorghum, Arabidopsis, Brassica (rape), Medicago (alfalfa), Gossypium (cotton),

Artemisia, Salvia and Mentha. Preferably, the plant belongs to the species of Nicotiana tabacum.

In a more preferred embodiment the non-human host organism is a microorganism. Any microorganism is suitable for the present invention, but according to an even more preferred embodiment said microorganism is a bacteria or yeast. Most preferably, said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

Isolated higher eukaryotic cells can also be transformed, instead of complete organisms. As higher eukaryotic cells, we mean here any non-human eukaryotic cell except yeast cells. Preferred higher eukaryotic cells are plant cells or fungal cells.

The term “transformed” refers to the fact that the host was subjected to genetic engineering to comprise one, two or more copies of each of the nucleic acids required in any of the above-described embodiment. Preferably the term “transformed” relates to hosts heterologously expressing the polypeptides encoded by the nucleic acid with which they

2017202313 07 Apr 2017 are transformed, as well as over-expressing said polypeptides. Accordingly, in an embodiment, the present invention provides a transformed organism, in which the polypeptides are expressed in higher quantity than in the same organism not so transformed.

There are several methods known in the art for the creation of transgenic host organisms or cells such as plants, fungi, prokaryotes, or cultures of higher eukaryotic cells. Appropriate cloning and expression vectors for use with bacterial, fungal, yeast, plant and mammalian cellular hosts are described, for example, in Pouwels et al., Cloning Vectors: A Laboratory Manual, 1985, Elsevier, New York and Sambrook et al., Molecular Cloning:

A Laboratory Manual, 2^nd edition, 1989, Cold Spring Harbor Laboratory Press. Cloning and expression vectors for higher plants and/or plant cells in particular are available to the skilled person. See for example Schardl et al. Gene 61: 1-11, 1987.

Methods for transforming host organisms or cells to harbor transgenic nucleic acids are familiar to the skilled person. For the creation of transgenic plants, for example, current methods include: electroporation of plant protoplasts, liposome-mediated transformation, agrobacterium-mediated transformation, polyethylene-glycol-mediated transformation, particle bombardement, microinjection of plant cells, and transformation using viruses.

In one embodiment, transformed DNA is integrated into a chromosome of a non20 human host organism and/or cell such that a stable recombinant system results. Any chromosomal integration method known in the art may be used in the practice of the invention, including but not limited to recombinase-mediated cassette exchange (RMCE), viral site-specific chromosomal insertion, adenovirus and pronuclear injection.

In order to carry out the method for producing β-santalene in vitro, as exposed herein above, it is very advantageous to provide a method of making at least one polypeptide having a β-santalene synthase activity as described in any embodiment of the invention. Therefore, the invention provides a method for producing at least one polypeptide according to any embodiment of the invention comprising

a) culturing a non-human host organism or cell transformed with the expression vector of the invention, so that it harbors a nucleic acid according to the invention and expresses or over-expresses a polypeptide of the invention;

2017202313 07 Apr 2017

b) isolating the polypeptide from the non-human host organism or cell cultured in step a).

According to a preferred embodiment, said method further comprises, prior to step a), transforming a non-human host organism or cell with the expression vector of the invention, so that it harbors a nucleic acid according to the invention and expresses or over-expresses the polypeptide of the invention.

A nucleic acid according to any of the above-described embodiments can be used.

Transforming and culturing of the non-human host organism or cell can be carried out as described above for the method of producing β-santalene in vivo. Step b) may be performed using any technique well known in the art to isolate a particular polypeptide from an organism or cell.

A “polypeptide variant as referred to herein means a polypeptide having a β-santalene synthase activity and being substantially homologous to the polypeptide according to any of the above embodiments, but having an amino acid sequence different from that encoded by any of the nucleic acid sequences of the invention because of one or more deletions, insertions or substitutions.

Variants can comprise conservatively substituted sequences, meaning that a given amino acid residue is replaced by a residue having similar physiochemical characteristics.

Examples of conservative substitutions include substitution of one aliphatic residue for another, such as Ile, Val, Leu, or Ala for one another, or substitutions of one polar residue for another, such as between Lys and Arg; Glu and Asp; or Gln and Asn. See Zubay, Biochemistry, 1983, Addison-Wesley Pub. Co. The effects of such substitutions can be calculated using substitution score matrices such a PAM-120, PAM-200, and PAM-250 as discussed in Altschul, J. Mol. Biol., 1991, 219, 555-565. Other such conservative substitutions, for example substitutions of entire regions having similar hydrophobicity characteristics, are well known.

Naturally occurring peptide variants are also encompassed by the invention. Examples of such variants are proteins that result from alternate mRNA splicing events or from proteolytic cleavage of the polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N- or C-termini upon expression in

2017202313 07 Apr 2017 different types of host cells, due to proteolytic removal of one or more terminal amino acid from the polypeptides encoded by the sequences of the invention.

Variants of the polypeptides of the invention may be used to attain for example desired enhanced or reduced enzymatic activity, modified regiochemistry or stereochemistry, or altered substrate utilization or product distribution, increased affinity for the substrate, improved specificity for the production of one or more desired compounds, increased velocity of the enzyme reaction, higher activity or stability in a specific environment (pH, temperature, solvent, etc), or improved expression level in a desired expression system. A variant or site directed mutant may be made by any method known in the art. Variants and derivatives of native polypeptides can be obtained by isolating naturally-occurring variants, or the nucleotide sequence of variants, of other or same plant lines or species, for examples plants from the Santalum species, or by artificially programming mutations of nucleotide sequences coding for the polypeptides of the invention. Alterations of the native amino acid sequence can be accomplished by any of a number of conventional methods.

Polypeptide variants resulting from a fusion of additional peptide sequences at the amino and carboxyl terminal ends of the polypeptides of the invention can be used to enhance expression of the polypeptides, be useful in the purification of the protein or improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences may be signal peptides, for example. Accordingly, the present invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligo- or polypeptides and/or those which are linked to signal peptides. Fusion polypeptide encompassed by the invention also comprise fusion polypeptides resulting from a fusion of other functional proteins, such as other proteins from the terpene biosynthesis pathway.

Therefore, in an embodiment, the present invention provides a method for preparing a variant polypeptide having a β-santalene synthase activity, as described in any of the above embodiments, and comprising the steps of:

(a) selecting a nucleic acid according to any of the embodiments exposed above;

(b) modifying the selected nucleic acid to obtain at least one mutant nucleic acid;

(c) transforming host cells or unicellular organisms with the mutant nucleic acid sequence to express a polypeptide encoded by the mutant nucleic acid sequence;

2017202313 07 Apr 2017 (d) screening the polypeptide for at least one modified property; and, (e) optionally, if the polypeptide has no desired variant β-santalene synthase activity, repeating the process steps (a) to (d) until a polypeptide with a desired variant β-santalene synthase activity is obtained;

(f) optionally, if a polypeptide having a desired variant β-santalene synthase activity was identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c).

According to a preferred embodiment, the variant polypeptide prepared is capable of producing a mixture of sesquiterpenes wherein β-santalene represents at least 20%, preferably at least 30%, preferably at least 35% of the sesquiterpenes produced.

In step (b), a large number of mutant nucleic acid sequences may be created, for example by random mutagenesis, site-specific mutagenesis, or DNA shuffling. The detailed procedures of gene shuffling are found in Stemmer, DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl

Acad Sci USA., 1994, 91(22): 10747-1075. In short, DNA shuffling refers to a process of random recombination of known sequences in vitro, involving at least two nucleic acids selected for recombination. For example mutations can be introduced at particular loci by synthesizing oligonucleotides containing a mutant sequence, flanked by restriction sites enabling ligation to fragments of the native sequence. Following ligation, the resulting reconstructed sequence encodes an analog having the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-specific mutagenesis procedures can be employed to provide an altered gene wherein predetermined codons can be altered by substitution, deletion or insertion.

Accordingly, the polypeptide comprising SEQ ID NO :1 may be recombined with any other sesquiterpene synthase encoding nucleic acids, for example isolated from an organism other than Santalum album. Thus, mutant nucleic acids may be obtained and separated, which may be used for transforming a host cell according to standard procedures, for example such as disclosed in the present examples.

In step (d), the polypeptide obtained in step (c) is screened for at least one modified property, for example a desired modified enzymatic activity. Examples of desired enzymatic activities, for which an expressed polypeptide may be screened, include enhanced or reduced enzymatic activity, as measured by K_M or V_max value, modified regio21

2017202313 07 Apr 2017 chemistry or stereochemistry and altered substrate utilization or product distribution. The screening of enzymatic activity can be performed according to procedures familiar to the skilled person and those disclosed in the present examples.

Step (e) provides for repetition of process steps (a)-(d), which may preferably be 5 performed in parallel. Accordingly, by creating a significant number of mutant nucleic acids, many host cells may be transformed with different mutant nucleic acids at the same time, allowing for the subsequent screening of an elevated number of polypeptides. The chances of obtaining a desired variant polypeptide may thus be increased at the discretion of the skilled person.

All the publications mentioned in this application are incorporated by reference to disclose and describe the methods and/or materials in connection with which the publications are cited.

Description of the drawings

Figure 1: GC-MS analysis of the sesquiterpene produced by the recombinant santalene synthase from Santalum album (SaSantS).

Part 1: Total ion chromatogram. 1, oc-santalene; 2, trans-cc-bergamotene; 3, epi-$santalene; 4, β-santalene; 5, β-famesene

Parts 2 to 4: Mass spectra of the peaks identified as sesquiterpenes.

Figure 2: Molecular structure of β-santalene and β-santalol.

Specific embodiments of the invention or Examples

The invention will now be described in further detail by way of the following 25 Examples.

2017202313 07 Apr 2017

Example 1

DNA library construction, sequencing and extraction of terpene synthase related sequences

Young hypocotyls segments obtained from aseptically germinated seeds of Santalum album L. (5 weeks old) were used to induce callus formation. The seeds of S. album were obtained from B&T World Seeds (Aigues-Vives, France) and from Sandeman Seeds (Lalongue, France). The seeds were first surface sterilised in 2.5% HC1O for

120 minutes, and rinsed three times in sterile ultrapure water. The seeds were then shelled and placed on MS basal medium (Murashige & Skoog, 1962, Physiologia Plantarum 15, 473-497) supplemented with 15 g/L sucrose and 7.8 g/L agar, pH 5.7. Germination was typically observed after 9 to 18 days with a yield of approximately 40%. The plantlets were allowed to grow in-vitro for 2 to 3 months in a cultivation room at a temperature of 27°C, with cool, white fluorescent light and with a 16 hours photoperiod. To induce the formation of green callus, the hypocotyls segments were cut into 3-4 mm transverse segments which were placed on Gbg basal medium (Gamborg & al, 1968, Exp Cell Res. 50(1), 151-158) supplemented with 0.5 μΜ 2,4D (2,4-Dichlorophenoxyacetic acid, Sigma-Aldrich Co.) and 10 μΜ Kin (Kinetin, Sigma-Aldrich Co.) in Petri dishes. The growth of the callus was perpetuated by transferring the tissue every four weeks to fresh medium in Petri dishes. All callus cultures were performed in a growth chamber in the same conditions as above.

Callus obtained after one month of culture in Gbg medium containing 5 μΜ Kin and 2 mM ACC were used for the RNA extraction and cDNA library construction. Total

RNA were extracted following the protocol described by Lefort and Douglas (Ann. For. Sci. 56 (1999), 259-263) except that the RNase treatment was omitted. The pellet was resuspended in 200 μΐ RNase-free water and centrifuged twice for 10 minutes at 20000 g to remove the polysaccharides. Approximately 125 pg total RNA were obtained from

2.2 g of cells. The mRNAs were purified using the FastTrack® 2.0 mRNA Isolation Kit (Invitrogen) and a cDNA library was made using the SMART® PCR cDNA Synthesis Kit (Clontech Laboratories, Inc.) following the manufacturer’s instructions.

2017202313 07 Apr 2017

The technology of massive parallel sequencing of small DNA fragments developed by Illumina (San Diego, California) was used to sequence the whole cDNA library. The preparation of the DNA for sequencing, the sequencing and the assembling of the reads were performed by Fastens SA (Plan-les-Ouates, Switzerland). The cDNA library was treated following the Genomic Sample Prep Kit (Illumina) and sequenced on the Genome Analyzer system (Illumina). A total of 4.03 millions of 35 bp sequences (reads) were obtained. These reads were assembled using EDENA 2.1.1, a software finding overlaps between the reads and assembling de novo contigs (Hernandez et al, De novo bacterial genome sequencing: Millions of very short reads assembled on a desktop computer.

Genome Res. 2008;18:802-809). The assembling was run with minimum matches of 26 to 20 bases. After eliminating contigs shorter than 100 bases, 1983 to 3473 unique contigs were obtained with a maximum length of 1331 to 1914 depending of the parameters selected for the assembling. Another assembling was performed using the Velvet 1.0 program (Zerbino and Birney (2008), Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res. 18(5), 821-829), providing 5905 unique contigs of length between 100 and 1616 bases.

All the contigs generated were compared against a protein sequences data base (non-redundant protein sequences, NCBI, http://www.ncbi.nlm.nih.gov) using the Blastx algorithm (Altschul et al, J. Mol. Biol. 215, 403-410, 1990;

http://www.ncbi.nlm.nih.gov/blast/Blast.cgi). The contigs showing significant sequence homology with plant sesquiterpene synthases were retained. A total of 46 contigs with a length of 100 to 621 bases were thus selected. These contigs were then processed using the CAP program (Huang, Genomics 14(1), 18-25, 1992) to assemble them and generate longer sequences. Five unique contigs of length of 445 to 1064 were thus assembled. The deduced amino acid sequences showed significant homology with plant terpene synthases and especially with sequences described or annotated as monoterpene synthases. Alignment of these amino acid sequences showed that at least two distinct cDNAs were present (two sequences were found in most of the positions across the alignment). This alignment showed also that at least one N-terminal and one C-terminal sequence was present. To obtain the full length sequences and to assign the exact 5’-end and 3’-end sequences to each cDNA, a rapid amplification of cDNA ends experiment (RACE) was employed.

2017202313 07 Apr 2017

Example 2

Amplification of the full-length sequences of a terpene synthase cDNA

For the RACE experiments, a set of primers was designed from one out of the five contigs obtained as described above. Thus the forward primers SCH5-Ct58-Rl (SEQ ID NO:6) and SCH5-Ct58-R2 (SEQ ID NO:7) and the reverse primers SCH5-Ct58-F3 (SEQ ID NO:8) and SCH5-Ct58-F4 (SEQ ID NO:9) were deduced from SCH5-contig-5 (SEQ ID NO:5).

The PCR were performed with the Universal Primer A Mix (UPM) (SMART™

RACE cDNA Amplification Kit, Clontech Laboratories, Inc.) in 50 μΐ final volume containing 200 μΜ dNTPs mix, 5 μΐ cDNA library (Example 1), 0.2 μΜ gene-specific primer, 0.2 μΜ UPM Primer Mix (Clontech Laboratories, Inc.), 1 μΐ Advantage 2 Polymerase Mix (Clontech Laboratories, Inc.) and 5 μΐ lOx cDNA PCR Reaction Buffer (Clontech Laboratories, Inc.). The thermal cycling conditions were as follows: 3 minutes at 94°C; 5 cycles of 30 sec at 94°C and 3 minutes at 72°C; 5 cycles of 30 sec at 94°C and 3 minutes at 70°C; 5 cycles of 30 sec at 94°C and 3 minutes at 68°C; 3 minutes at 72°C. With the 5’RACE, a 610 bp DNA fragment (SCH5-Ct58_RRl, SEQ ID NO:10) including the 5’end of the cDNA was obtained. With the 3’RACE a 1049 bp fragment (SCH5-Ct5820 RF4, SEQ ID NO:11) was obtained and the combination of the two RACE products with the SCH5-contig-5 sequence (SEQ ID NO:5) allowed the reconstitution of a new fulllength cDNA (SCH5-Ct58, SEQ ID NO: 12). The 2157 bp SCH5-Ct58 cDNA encoded for a 569 amino acid protein (SEQ ID NO: 13) showing homology with plant terpene synthases sequences and containing motifs characteristic of terpene synthases such as the

DDxxD motif present in all monoterpene and sesquiterpene synthases. Interestingly the amino acid sequence showed higher similarity to monoterpene synthases than to sesquiterpene synthases. However the presence of chloroplast peptide signal, a common feature in plant monoterpene synthases, was not predicted from the analysis of the Nterminal sequence (Emanuelsson, 0., Nielsen, N., and von Heijne, G. 1999. ChloroP, a neural network-based method for predicting chloroplast transit peptides and their cleavage sites. Protein Science 8, 978-984).

2017202313 07 Apr 2017

Example 3

Heterologous expression and in-vitro enzymatic activity of SCH5-Ct58

We decided to modify the DNA sequence of SCH5-Ct58 (SEQ ID NO: 12) and to redesign the sequence for optimal heterologous expression in E coli cells. To start with the true amino acid sequence, the exact nucleotidic sequence of SCH5-Ct58 in the cDNA library had first to be established. The Eland Software (Illumina) was used to retrieve all reads matching with the SCH5-Q58 sequence (SEQ ID NO: 12) with a maximum of 2 mismatches. A total of 5224 reads were recovered and were aligned using the CAP program (Huang, Genomics 14(1), 18-25, 1992) with the SCH5-Ct58 DNA sequence (SEQ ID NO: 12) as a reference. The average coverage over the whole sequence was above 100X allowing for the unambiguous deduction of the new cDNA sequence SCH5Ct94 (SEQ ID NO: 14). In this new sequence 5 bases were corrected compared to the SCH5-Ct58 sequence (SEQ ID NO: 12) deduced from the RACE results and those corrections resulted in a new amino acid sequence (SCH5-Ct94, SEQ ID NO: 15) with a two-residues difference. For heterologous expression, the DNA sequence of SCH5-Ct94 (SEQ ID NO: 14) was modified to remove the first 23 codons and replace by the ATGGCT sequence and the codon usage was changed to optimize the sequence for E coli expression (DNA 2.0, Menlo Park, CA, USA). The cDNA thus designed (SCH5-Ct94-opt, SEQ ID

NO:2) was synthesized (DNA 2.0, Menlo Park, CA, USA) and sub-cloned into the NdelKpnl sites of the pETDuet-1 plasmid providing the plasmid Ct94-pETDuet. This optimized cDNA sequence encoded for the polypeptide SCH5-Ct94-opt (SEQ ID NO:1).

Heterologous expression of Ct94 was performed in E coli BL21(DE3) cells using the plasmid Ct94-pETDuet. In-vitro enzyme assays were performed with FPP as substrate in the conditions described above and sesquiterpene synthase activity was observed with formation of a mixture of five sesquiterpenes. The identity of these sesquiterpenes was confirmed by GC-MS as being the sesquiterpene characteristic of santalum album: ocsantalene, /ram-cx-bcrgamotcnc, epz'^-santalene, β-santalene and β-famesene (Figure 1). At pH 7.0 and in the presence of 15 mM MgC’f, the relative proportion of the recombinant sequiterpene products was 38.0% of oc-santalene, 18.2% of /ram-cx-bcrgamotcnc, 5.7% of cyv^-santalcnc, 36.7% of β-santalene and 1.3% of β-famesene. Thus the SCH-Ct98-opt

2017202313 07 Apr 2017 cDNA encoded for a β-santalene synthase. The ratio of the products was very similar to the proportion observed in Santalum album oil for the hydroxylated products of these sesquiterpenes. No activity was detected when MgCT was omitted and the medium supplemented with 2.5 mM EDTA (to chelate residual cations) showing the strict requirement for divalent cations. The nature and concentration of the divalent cation present in the assay had an effect on the product profile (Table 1). For instance, lowering the concentration of Mg²⁺ had a benefit effect for β-santalene, the latest becoming the major product of the enzyme. Moreover, the addition of Mn²⁺ had a negative effect on the formation of β-santalene since the proportion of the santalene sesquiterpene products decreased and the proportion of /ram-cx-bcrgamotcnc and β-famesene increased, trans-Ubergamotene being the major product of the enzyme in the presence of 1 mM MgCT.

Table 1: Effect of the concentration of Mg²⁺ and Mn²⁺ ions on the composition of the mixture of sesquiterpenes obtained by contacting SEQ ID NO :1 with FPP

	Percentage, relative to the whole product mixture
15mMMgCl₂	2 mM MgCl₂	0.75 mM MgCT	0.75 mM MgCT + 1 mM MnCl₂
oc-santalene	38.0	33.0	36.5	24.5
trans-a- bergamotene	18.2	11.8	12.6	35.4
ep/^-santalcnc	5.7	6.4	5.6	4.1
β-santalene	36.7	47.5	44.1	33.3
β-famesene	1.3	1.3	1.1	2.75

2017202313 07 Apr 2017

Example 4

In-vivo production of sesquiterpenes in E coli using the Ct94 cDNA

The use of the S. album santalene synthase for the in-vivo production of sesquiterpenes in E coli cells was evaluated by coexpressing the enzymes of a five step biosynthetic pathway converting mevalonic acid to FPP.

The yeast FPP synthase gene was amplified from S. cerevisiae genomic DNA using the primers FPPyNcoI (SEQ ID NO: 16) AND fppY-Eco (SEQ ID NO: 17). The amplified DNA was ligated as Ndel-Ecorl fragment in the first multi cloning site (MCS1) of the pACYCDuet-1 plasmid providing the plasmid FPPs-pACYCDuet harbouring the FPPs gene under the control of the T7 promoter. An operon including the genes encoding for a mevalonate kinase (mvaKl), a phosphomevalonate kinase (mvaK2), a mevalonate diphosphate decarboxylase (MvaD) and a isopentenyl diphospahte isomerase (idi) was amplified from genomic DNA of Streptococcus pneumoniae (ATCC BAA-334) with the primers MVA-upl-start (SEQ ID NO:18) and MVA-up2-stop (SEQ ID NO: 19). The PCR was performed using the PfuUltra™ II Fusion HS DNA polymerase (Stratagen). The composition of the PCR mix was according to the manufacturer instructions. The thermal cycling condition were 2 minutes at 95°C; 30 cycles of 20 sec at 95°C, 20 sec at 58°C and

90 sec at 72°C; and 3 minutes at 72°C. The 3.8 Kb fragment was purified on an agarose gel and ligated using the In-Fusion™ Dry-Down PCR Cloning Kit (clontech) into the second MCS of the FPPs-pACYCDuet plasmid digested with Ndel and Xhol providing the plasmid pACYCDuet-4506. The sequences of the two inserts were fully sequenced to exclude any mutation.

BL21 Star™(DE3) E. coli cells (Invitrogen) were co-transformed with the plasmids pACYCDuet-4506 and Ct94-pETDuet and transformed cells were selected on carbenicillin (50 pg/ml) chloramphenicol (34 pg/ml) LB-agarose plates. Single colonies were used to inoculate 5 mL LB medium with 50 pg/ml carbenicilin and 34 pg/ml chloramphenicol. The culture was incubated overnight at 37°C. The next day 2 mL of TB medium supplemented with the same antibiotics were inoculated with 0.2 mL of the overnight culture. After 6 hours incubation at 37°C, the culture was cooled down to 28°C and 1 mM IPTG, 2 mg/mL mevalonate (prepared by dissolving mevalonolactone (Sigma) in 0.5N NaOH at a

2017202313 07 Apr 2017 concentration of 1 g/mL and incubating the solution for 30 minutes at 37°C) and 0.2 mL decane were added to each tube. The cultures were incubated for 48 hours at 28°C. The cultures were then extracted twice with 2 volumes of ethyl acetate, the organic phase was concentrated to 500 pL and analyzed by GC-MS as described above in Example 3. In these conditions sesquiterpene production above 200 mg/L was routinely achieved. Betasantalene was produced.

Example 5

In -vivo production of sesquiterpenes in S. cerevisiae using the Ct94 cDNA

For in-vivo production of sesquiterpenes in yeast cells, a saccharomyces cerevisiae strain YNP5 in which the ERG9 gene (coding for the squalene synthase, the enzyme converting FPP to squalene) has been down-regulated by replacing the native ERG9 promoter with the regulable MET3 promoter. In previous work with plant sesquiterpene synthases, this strategy led to a reduced ergosterol biosynthesis in the cells and an accumulation of FPP available for sesquiterpene synthases (Asadollahi, Biotechnology and Bioengineering, 99(3), 666-677, 2008).

The SCH5-Ct94-opt cDNA (SEQ ID NO:2) was amplified from the Ct94pETDuet with the primers Ct94_BamHI (SEQ ID NO:20) and T7term (SEQ ID NO:21).

The PCR was performed with the Pfu DNA Polymerase (Promega) using the following thermal cycling condition: 90 sec at 94°C; 35 cycles of 45 sec at 94°C, 45 sec at 55°C, 4 minutes at 72°C; and 10 minutes at 72°C. The amplified cDNA was digested with the BamHi and Xhol restriction sites and ligated in the corresponding sites of the pESC-URA plasmid (Stratagen) providing the plasmid Ct94-pESC-ura. S.c. The YNP5 cells were transformed using the S.c. EasyComp™ Transformation Kit (Invitrogen).

One single colony of transformed yeast strains were used to inciluate 20 ml of

YNB medium (5 g/L (NH₄)₂SO₄; 3 g/L KH₂PO₄; 0.5 g/L MgSO₄.7 H₂O; 1 mL/L trace metal solution) supplemented with 2% glucose. The culture was incubated for 24 hours at 28°C. The cells were recovered by centrifugation and resuspended in 20 mL of YNB medium supplemented with 2% galacoste. After on 1 hour culture, methionine at 0.5 mM final concentration and 2 mL decane were added to the culture. After 24 hours incubation at 28°C, the cultures were extracted with ethyl acetate and analysed by GC-MS as

2017202313 07 Apr 2017 described in Example 4. The total quantity of sesquiterpenes produced by the yeast cells in these conditions was estimated at 50 mg/L.

Example 6

Isolation of a santalene synthase from santalum album roots

Seedlings of Santalum album obtained from aseptically germinated seeds were transferred to soil 5 to 10 weeks after germination. Since santalum species are root hemiparasites, the soil adaptation was made in close contact with 6-months to 1-year old citrus (Citrus sinensis) plants. The roots of the santalum plants were harvested, 2-3 years after the transfer to the soils and separated from the host plant roots. GC-MS analysis of an extract of these roots showed the presence of the sandalwood oil characteristic sesquiterpenes. Total RNA was extracted from the roots using the Concert™ Plant RNA Reagent (Invitrogen). From 12 g of tissue, 640 pg of total RNA were isolated. The mRNA were purified using the FastTrack® 2.0 mRNA Isolation Kit (Invitrogen) and a cDNA library was made using the Marathon™ cDNA Amplification Kit (Clontech Faboratories, Inc.) following the manufacturer instructions.

An amount of 1 pg of cDNA was used for sequencing using the Genome Analyzer System (Illumina). A total of 10.3 millions of 35bp-length reads were obtained. These reads were assembled using in parallel the Edena (Hernandez et al, 2008, Genome Res. 18, 802-809) and the Velvet (Zerbinoa and Birney, 2008, Genome Res. 18: 821-829) assembler softwares resulting in 18’937 and 22’414 unique contigs with an average range of 242 and 211 bp. The reads were searched using the tBlastn program (Altschul et al, 1990, J. Mol. Biol. 215, 403-410) with the SCH5-CT94 amino acid sequence (SEQ ID

NO: 15) as query sequence. Fifteen contigs were selected showing significant homology of their deduced amino acid sequences with plant sesquiterpene synthases. These selected contigs were reassembled into two distinct sequences, of which SCH10-Ct8201 (SEQ ID NO:22) was 383 bp in length and showed the highest homology with SCH5-CT94 DNA sequence (SEQ ID NO: 14). The forward primer SCH10-Ctg8201-F2 (SEQ ID NO:23) was designed from the SCH10-Ct8201 sequence and successfully used for 3’RACE using the Marathon™ cDNA Amplification Kit (Clontech Faboratories, Inc.). From the sequence of the 3’RACE product thus obtained, two reverse primers (SCH10-CU9779-R3

2017202313 07 Apr 2017 (SEQ ID NO:24) and SCH10-Ctl9779-R4 (SEQ ID NO:25)) were designed and successfully used for the amplification by 5’RACE of the 5’end of the corresponding cDNA. From the sequences of the 3’RACE and 5’RACE a full-length sequence of a new terpene synthase was thus reconstituted. In order to verify the sequence, the MAQ program (Li et al, 2008, Genome Res. 18(11), 1851-1858) was used to search and map all the reads with a maximum of 2 mismatches. This approach provided a 1725 bp-length DNA sequence (SEQ ID NO:26) encoding for a 570 amino acid-length protein (SEQ ID NO:27) having 91.9 % identity with the amino acid sequence of SCH5-O94 (SEQ ID NO:15).

For heterologous expression in E coli, an optimized cDNA was designed by deleting the 21 first codons, adding the sequence ATGGCTACC as the first 3 codons and optimizing the codon usage for E coli. This optimized sequence (SCH10-Ct8201-opt, SEQ ID NO:4) encoding for the N-terminal modified protein SCH10-Tps8201-opt (SEQ ID NOG) was synthesized (DNA 2.0; Menlo Park, CA, USA) and sub-cloned in the Ndel15 Kpnl sites of the pETDuet-1 expression plasmid (Novagen). Heterologous expression and enzymatic characterization of SCH10-Tps8201-opt (SEQ ID NOG) was performed as described in Example 3. The recombinant protein showed sesquiterpene synthase activity and produced from FPP the same mixture of sesquiterpenes as the SCH5-CT94-opt recombinant protein (SEQ ID NO:1, Example 3) with the same relative proportions.

2017202313 15 May 2018

Claims

1. A method for producing β-santalene and derivatives thereof comprising

a) contacting FPP with at least one polypeptide having a β-santalene synthase activity and comprising an amino acid sequence at least 60% identical to SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 15, or comprising the amino acid sequence of SEQ ID NO: 27;

b) producing β-santalene; and

c) processing the β-santalene produced to a β-santalene derivative using a chemical or biochemical synthesis or a combination of both.

2. The method of claim 1, wherein the β-santalene derivative comprises a β-santalol.

3. The method of claim 1 or 2, wherein step a) comprises cultivating a non-human host organism or cell capable of producing FPP and transformed to express at least one polypeptide comprising an amino acid sequence at least 60% identical to SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 15, or comprising the amino acid sequence of SEQ ID NO: 27 and having a βsantalene synthase activity, under conditions conducive to the production of β-santalene.

4. The method of claim 3, wherein the method further comprises, prior to step a), transforming a non-human host organism or cell capable of producing FPP with at least one nucleic acid encoding a polypeptide comprising an amino acid sequence at least 60% identical to SEQ ID NO:1, SEQ ID NO: 3 or SEQ ID NO: 15, or comprising the amino acid sequence of SEQ ID NO: 27 and having a β-santalene synthase activity, so that said organism expresses said polypeptide.

5. The method of claim 4, wherein the at least one nucleic acid encoding the β-santalene synthase comprises a nucleotide sequence at least 60% identical to SEQ ID NO:2, 14 or the complement thereof.

6. The method of any one of claims 3 to 5, wherein the non-human host organism is a plant, a

2017202313 15 May 2018 prokaryote or a fungus.

7. The method of claim 6, wherein the non-human host organism is a microorganism.

8. The method of claim 7, wherein the microorganism is a bacteria or yeast.

9. The method of claim 8, wherein said bacteria is E. coli and said yeast is Saccharomyces cerevisiae.

10. β-santalene or a derivative thereof produced according to the method of any one of claims 1 to 9.

Firmenich SA

Patent Attorneys for the Applicant/Nominated Person

SPRUSON & FERGUSON

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Figure 1, part 1

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Figure 1, part 2

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Figure 1, part 3

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Figure 1, part 4

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Figure 2A

Beta Santalene

Beta Santalol

Figure 2B

2017202313 07 Apr 2017

SEQUENCE LISTING <110> Firmern'ch SA <120> Method for producing beta-santalene <130> 7530PRIOR <160> 27 <170> PatentIn version 3.5 <210> 1 <211> 548 <212> PRT <213> Santalum album <400> 1

Met 1 Al a Thr Asp Asn 5 Asp Ser Ser Glu Asn Arg Arg Met Gly Asn Tyr 10 15 Lys Pro Ser Ile T rp Asn Tyr Asp Phe Le u Gln Ser Le u Al a Thr Arg 20 25 30 Hi s Asn Ile Met Glu Glu Arg Hi s Le u Lys Le u Al a Glu Lys Le u Lys 35 40 45 Gly Gln Val Lys Phe Met Phe Gly Al a Pro Met Glu Pro Le u Al a Lys 50 55 60 Le u Glu Le u Val Asp Val Val Gln Arg Le u Gly Le u Asn Hi s Arg Phe 65 70 75 80 Glu Thr Glu Ile Lys Glu Al a Le u Phe Ser Ile Tyr Lys Asp Glu Ser 85 90 95 Asn Gly T rp T rp Phe Gly Hi s Le u Hi s Al a Thr Ser Le u Arg Phe Arg 100 105 110 Le u Le u Arg Gln cys Gly Le u Phe Ile Pro Gln Asp Val Phe Lys Thr 115 120 125 Phe Gln Ser Lys Thr Gly Gl u Phe Asp Met Lys Le u cys Asp Asn Val 130 135 140 Lys Gly Le u Le u Ser Le u Tyr Glu Al a Ser Phe Le u Gly T rp Arg Asp 145 150 155 160 Glu Asn Ile Le u Asp Glu Al a Lys Al a Phe Al a Thr Lys Tyr Le u Lys 165 170 175 Asn Al a T rp Glu Asn Ile Ser Gln Lys T rp Le u Al a Lys Arg Val Lys 180 185 190 Hi s Al a Le u Al a Le u Pro Le u Hi s T rp Arg Val Pro Arg Ile Glu Al a 195 200 205

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Arg T rp Phe Val Glu Al a Tyr Gly Glu Glu Glu Asn Met Asn Pro Thr 210 215 220 Le u Le u Lys Le u Al a Lys Le u Asp Phe Asn Met Val Gln Ser Ile Hi s 225 230 235 240 Gln Lys Glu Ile Gly Glu Le u Al a Arg T rp T rp Val Thr Thr Gly Le u 245 250 255 Asp Lys Le u Al a Phe Al a Arg Asn Asn Le u Le u Gln Ser Tyr Met T rp 260 265 270 Ser cys Al a Ile Al a Ser Asp Pro Lys Phe Lys Le u Al a Arg Glu Thr 275 280 285 Ile Val Glu Ile Gly Ser Val Le u Thr Val Val Asp Asp Al a Tyr Asp 290 295 300 Val Tyr Gly Ser Met Asp Gl u Le u Asp Le u Tyr Thr Asn Ser Val Glu 305 310 315 320 Arg T rp Ser cys Thr Glu Ile Asp Lys Le u Pro Asn Thr Le u Lys Le u 325 330 335 Ile Phe Met Al a Met Phe Asn Lys Thr Asn Glu Val Gly Le u Arg Val 340 345 350 Gln Hi s Glu Arg Gly Tyr Ser Gly Ile Thr Thr Phe Ile Lys Al a T rp 355 360 365 Val Glu Gln cys Lys Ser Tyr Gln Lys Glu Al a Arg T rp Tyr Hi s Gly 370 375 380 Gly Hi s Thr Pro Pro Le u Gl u Glu Tyr Ser Le u Asn Gly Le u Val Ser 385 390 395 400 Ile Gly Phe Pro Le u Le u Le u Ile Thr Gly Tyr Val Al a Ile Al a Glu 405 410 415 Asn Glu Al a Al a Le u Asp Lys Val Hi s Pro Le u Pro Asp Le u Le u Hi s 420 425 430 Tyr Ser Ser Le u Le u Ser Arg Le u Ile Asn Asp Met Gly Thr Ser Ser 435 440 445 Asp Glu Le u Glu Arg Gly Asp Asn Le u Lys Ser Ile Gln cys Tyr Met 450 455 460 Asn Gln Thr Gly Al a Ser Gl u Lys Val Al a Arg Glu Hi s Ile Lys Gly 465 470 475 480

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Ile Ile Glu Glu Asn T rp Lys Ile Le u Asn Glu cys cys Phe Asp Gln 485 490 495 Ser Gln Phe Gln Glu Pro Phe Val Thr Phe Asn Le u Asn Ser Val Arg 500 505 510 Gly Ser Hi s Phe Phe Tyr Gl u Phe Gly Asp Gly Phe Gly Val Thr Asn 515 520 525 Ser T rp Thr Lys Val Asp Met Lys Ser Val Le u Ile Asp Pro Ile Pro 530 535 540 Le u Asp Glu Glu

545 <210> 2 <211> 1650 <212> DNA <213> Santalum album <400> 2 atggctaccg ataatgacag ctctgaaaac cgtcgtatgg gtaattacaa gccgtccatc tggaactacg acttcctgca gtccctggct acccgccaca atatcatgga agagcgccac ttgaaactgg cggagaaact gaaaggccag gtgaagttta tgtttggtgc cccgatggag ccgctggcca aactggagct ggttgatgtt gttcagcgcc tgggtctgaa tcatcgcttc gagacggaga ttaaggaggc cctgttcagc atctacaagg atgagagcaa cggttggtgg tttggccacc tgcatgccac cagcctgcgt tttcgcctgc tgcgccagtg tggtctgttc attccgcaag acgttttcaa gacgttccaa agcaagaccg gcgagttcga catgaaactg tgcgacaacg tcaagggttt gctgagcctg tacgaggctt cctttctggg ctggcgtgac gaaaatatcc tggacgaagc gaaagctttt gccacgaagt acctgaagaa cgcatgggaa aacattagcc agaagtggct ggcgaaacgc gtgaagcatg cgttggcact gccgttgcac tggcgtgtgc ctcgtattga agcacgctgg tttgttgagg cgtacggcga ggaggaaaat atgaatccga ccttgctgaa gctggctaag ttggatttta acatggtgca atctattcac caaaaggaaa tcggtgaatt ggcacgttgg tgggtcacca ccggtctgga caaactggca ttcgcgcgca ataatttgct gcaaagctac atgtggagct gcgcgatcgc atctgacccg aagtttaagc tggctcgcga aaccatcgtg gagatcggtt ccgtgctgac tgttgtggat gacgcctacg atgtttacgg tagcatggac gaactggact tgtataccaa tagcgtggag cgttggagct gtacggaaat cgataagctg ccgaatacgc tgaaactgat ttttatggct atgtttaaca agaccaatga agttggtctg cgtgttcagc acgagcgtgg ttactccggc atcaccacct tcattaaggc atgggtcgaa cagtgtaaga gctatcaaaa agaagcgcgc tggtatcatg gtggtcacac gcctccgctg gaagagtact ccttgaatgg cttggtgagc attggtttcc cgctgctgct gattaccggc tacgtcgcca ttgccgaaaa cgaagcagcg

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ctggacaaag tgcatccgct gccggatctg ctgcactata gctctctgct gagccgcctg 1320 atcaacgaca tgggtacgag cagcgacgag ctggagcgcg gcgataatct gaaaagcatc 1380 caatgctata tgaatcagac cggcgcgagc gagaaggtgg cgcgcgagca catcaagggc 1440 atcattgagg agaattggaa gattctgaac gaatgttgct tcgaccaaag ccaatttcaa 1500 gagccgttcg tgacgttcaa cctgaacagc gttcgtggtt cccatttctt ttacgagttt 1560 ggtgacggtt tcggtgtgac gaatagctgg accaaggttg acatgaagag cgtcctgatt 1620 gatccgattc cactggatga agaataatga 1650

<210> 3 <211> 551 <212> PRT <213> Artificial sequence <220>

<223> Polypeptide encoded by the optimized sequence SCH10-Ctg8201-opt for expression in E. coli.

<400> 3

Met Ala Thr 1 Le u Lys 5 Thr Asp Thr Asp Ala Ser Glu Asn Arg Arg Met 10 15 Gly Asn Tyr Lys Pro Ser Ile T rp Asn Tyr Asp Phe Le u Gln Ser Le u 20 25 30 Al a Thr Hi s Hi s Asn Ile Val Glu Glu Arg Hi s Le u Lys Le u Al a Glu 35 40 45 Lys Le u Lys Gly Gln Val Lys Phe Met Phe Gly Al a Pro Met Glu Pro 50 55 60 Le u Al a Lys Le u Glu Le u Val Asp Val Val Gln Arg Le u Gly Le u Asn 65 70 75 80 Hi s Le u Phe Glu Thr Glu Ile Lys Glu Al a Le u Phe Ser Ile Tyr Lys 85 90 95 Asp Gly Ser Asn Gly T rp Trp Phe Gly Hi s Le u Hi s Al a Thr Ser Le u 100 105 110 Arg Phe Arg Le u Le u Arg Gl n Cys Gly Le u Phe Ile Pro Gln Asp Val 115 120 125 Phe Lys Thr Phe Gln Asn Lys Thr Gly Glu Phe Asp Met Lys Le u T rp 130 135 140 Asp Asn Val Lys Gly Le u Le u Ser Le u Tyr Glu Al a Ser Tyr Le u Gly 145 150 155 160 T rp Lys Gly Glu Asn Ile Le u Asp Glu Al a Lys Al a Phe Thr Thr Lys 165 170 175

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cys Le u Lys Ser Aia 180 Trp Gi u Asn lie Ser 185 Giu Lys T rp Le u 190 Ai a Lys Arg Vai Lys Hi s Ai a Le u Ai a Le u Pro Le u Hi s T rp Arg Vai Pro Arg 195 200 205 lie Giu Ai a Arg T rp Phe lie Giu Vai Tyr Giu Gin Giu Ai a Asn Met 210 215 220 Asn Pro Thr Le u Le u Lys Le u Ai a Lys Le u Asp Phe Asn Met Vai Gin 225 230 235 240 Ser lie Hi s Gin Lys Giu lie Giy Giu Le u Ai a Arg T rp T rp Vai Thr 245 250 255 Thr Giy Le u Asp Lys Le u Asp Phe Ai a Arg Asn Asn Le u Le u Gin Ser 260 265 270 Tyr Met T rp Ser cys Ai a lie Ai a Ser Asp Pro Lys Phe Lys Le u Ai a 275 280 285 Arg Giu Thr lie Vai Giu lie Giy Ser Vai Le u Thr Vai Vai Asp Asp 290 295 300 Giy Tyr Asp Vai Tyr Giy Ser Met Asp Giu Le u Asp Le u Tyr Thr Ser 305 310 315 320 Ser Vai Giu Arg T rp Ser cys Vai Lys lie Asp Lys Le u Pro Asn Thr 325 330 335 Le u Lys Le u lie Phe Met Ser Met Phe Asn Lys Thr Asn Giu Vai Giy 340 345 350 Le u Arg Vai Gin Hi s Giu Arg Giy Tyr Asn Ser lie Pro Thr Phe lie 355 360 365 Lys Ai a T rp Vai Giu Gin cys Lys Ser Tyr Gin Lys Giu Ai a Arg T rp 370 375 380 Phe Hi s Giy Giy Hi s Thr Pro Pro Le u Giu Giu Tyr Ser Le u Asn Giy 385 390 395 400 Le u Vai Ser lie Giy Phe Pro Le u Le u Le u lie Thr Giy Tyr Vai Ai a 405 410 415 lie Ai a Giu Asn Giu Ai a Ai a Le u Asp Lys Vai Hi s Pro Le u Pro Asp 420 425 430 Le u Le u Hi s Tyr Ser Ser Le u Le u Ser Arg Le u lie Asn Asp lie Giy 435 440 445

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Thr Ser Pro Asp Glu Met Al a Arg Gly Asp Asn Le u Lys Ser Ile Hi s 450 455 460 cys Tyr Met Asn Glu Thr Gly Al a Ser Glu Glu Val Al a Arg Glu Hi s 465 470 475 480 Ile Lys Gly Val Ile Glu Gl u Asn T rp Lys Ile Le u Asn Gln cys cys 485 490 495 Phe Asp Gln Ser Gln Phe Gl n Glu Pro Phe Ile Thr Phe Asn Le u Asn 500 505 510 Ser Val Arg Gly Ser Hi s Phe Phe Tyr Glu Phe Gly Asp Gly Phe Gly 515 520 525 Val Thr Asp Ser T rp Thr Lys Val Asp Met Lys Ser Val Le u Ile Asp 530 535 540 Pro Ile Pro Le u Gly Glu Gl u

545

550 <210> 4 <211> 1656 <212> DNA <213> Artificial sequence <220>

<223> Optimized sequence for expression in E. coli.

<400> 4 atggcaacct tgaagactga caccgacgct agcgagaatc gtcgcatggg caactataaa ccgagcattt ggaactacga tttcctgcaa agcctggcta cccaccacaa tatcgtggag gagcgtcacc tgaaactggc agaaaaattg aaaggccaag tgaaattcat gttcggcgca ccgatggaac cgctggcgaa actggagctg gtcgacgtgg tccaacgcct gggtctgaat cacctgtttg aaaccgaaat taaagaggca ctgttcagca tctataagga cggttcgaac ggttggtggt tcggtcacct gcatgcaacc agcctgcgtt ttcgtctgct gcgtcagtgt ggcctgttca ttccgcagga cgtctttaaa acctttcaga acaaaaccgg cgagtttgac atgaagctgt gggacaatgt gaaaggcctg ttgagcctgt atgaggcgag ctacctgggt tggaagggtg aaaacatcct ggatgaagca aaggcattta ccaccaagtg tctgaagagc gcgtgggaaa atatctctga gaaatggttg gcgaaacgtg tgaagcacgc gctggcgctg ccgctgcact ggcgcgttcc gcgcatcgaa gcgcgctggt ttatcgaagt ttatgaacag gaagctaata tgaacccgac cctgctgaag ctggcgaagc tggatttcaa catggttcaa agcattcatc aaaaggagat cggcgagctg gcccgctggt gggtgaccac gggtttggac aagctggact ttgcacgtaa taatctgttg caaagctaca tgtggagctg cgctatcgca tccgacccga aatttaagtt ggcacgtgaa accatcgttg aaattggtag cgtgctgact gtggtggatg acggttacga tgtttacggt agcatggacg aactggacct gtacacgtcg

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2017202313 07 Apr 2017 agcgtcgagc gctggagctg tgtcaaaatt gataagctgc cgaacacgct gaaactgatc 1020 ttcatgagca tgttcaacaa aaccaacgaa gtgggcctgc gcgtgcagca cgaacgtggc 1080 tataatagca ttccgacgtt tatcaaggca tgggtggagc aatgtaaaag ctaccaaaaa 1140 gaggcccgtt ggtttcatgg cggccatacc ccgcctctgg aggaatatag cctgaacggc 1200 ctggtgtcca ttggttttcc gctgctgctg atcaccggct acgtggcaat cgcggaaaat 1260 gaagccgcgc tggacaaggt ccatccactg ccggacctgt tgcattatag ctctctgctg 1320 agccgtctga tcaatgatat cggtacgagc ccggacgaga tggctcgtgg tgacaacctg 1380 aaaagcatcc attgttatat gaacgagacg ggtgcgtccg aagaggtcgc ccgcgagcat 1440 atcaagggcg ttattgagga gaactggaaa atcctgaatc aatgttgctt cgatcaaagc 1500 cagttccaag agccgttcat cacgttcaat ctgaacagcg ttcgcggtag ccactttttc 1560 tacgaatttg gcgacggttt tggcgttacg gacagctgga ccaaagttga tatgaaatcc 1620 gttctgatcg acccgatccc gttgggtgaa gagtag 1656 <210> 5 <211> 1064 <212> DNA <213> Santalum album <400> 5

cccccgccat gagagctcca ttcattgatc atactgatca tgtgaatctc agaactgata 60 acgattcctc agagaatcga aggatgggga attataaacc cagtatttgg aactatgatt 120 ttttgcaatc gcttgcgact cgccacaata ttatggaaga gaggcatcta aagctagctg 180 agaagctgaa gggccaagtg aagtttatgt ttggggcacc aatggagccg ttagcaaagc 240 tggagcttgt ggatgtggtt caaaggctcg ggctaaacca ccgatttgag acagagatca 300 aggaagcgct atttagtatt tataaggatg agagcaatgg atggtggttt ggccacctcc 360 atgcgacatc tctccgattt aggctgctac gacagtgtgg gctttttatc ccccaggatg 420 tgtttaaaac atttcagagc aaaactggtg aatttgatat gaaactgtgt gacaatgtaa 480 aaggattgct gagcttgtat gaagcttcat tcttggggtg gagggatgaa aacatcttag 540 atgaagccaa agccttcgcc accaagtact tgaaaaatgc atgggaaaac atatcccaaa 600 agtggcttgc caaaagagtg aagcatgcac tggctttgcc tttgcactgg agagtcccta 660 gaatcgaagc tagatggttc gttgaggcat atggggaaga agagaatatg aacccaacac 720 ttctcaaact tgcaaaattg gactttaaca tggtgcaatc aattcatcag aaagagattg 780 gggaattagc gaggtggtgg gtgactacgg ggttggataa gttagcgttt gctaggaata 840 atttactgca aagctatatg tggagctgcg cgattgcttc cgacccaaag ttcaaacttg 900 ctagagaaac tattgttgaa atcggaagtg tactcacagt tgttgacgat gcatatgacg 960 tctatggttc aatggatgaa cttgatctct acacgaactc cgttgaaagg tggagctgta 1020 cagaaattga caagttacca aacacattaa aattgatttt tatg 1064

<210>

Page 7

2017202313 07 Apr 2017 <211> 29 <212> DNA <213> Artificial <220>

<223> Primer synthesized on the basi s of SCH5-Contig5. <400> ₆ cttcactctt ttggcaagcc acttttggg 29 <210> 7 <211> 30 <212> DNA <213> Arti fi ci al <220> <223> Primer synthesized on the basi s of SCH5-Contig5, <400> 7 gtggcgaagg ctttggcttc atctaagatg 30

<210> 8 <211> 31 <212> DNA <213> Artificial <220>

<223> Primer synthesized on the basis of SCH5-Contig5 <400> 8 gcatatgacg tctatggttc aatggatgaa c 31 <210> 9 <211> 30 <212> DNA <213> Artificial <220>

<223> Primer synthesized on the basis of SCH5-Contig5 <400> 9 gttgaaaggt ggagctgtac agaaattgac 30 <210> 10 <211> 610 <212> DNA <213> Santalum album <400> 10 acaaaataaa tctcttgttc tgttctttgg atctcgtttt cttcccctca gctctctcac 60 taatggattc ttccaccgcc accgccatga gagctccatt cattgatcat actgatcatg 120 tgaatctcag aactgataac gattcctcag agaatcgaag gatggggaat tataaaccca 180 gtatttggaa ctatgatttt ttgcaatcgc ttgcgactcg ccacaatatt atggaagaga 240 ggcatctaaa gctagctgag aagctgaagg gccaagtgaa gtttatgttt ggggcaccaa 300 tggagccgtt agcaaagctg gagcttgtgg atgtggttca aaggctcggg ctaaaccacc 360 gatttgagac agagatcaag gaagcgctat ttagtattta taaggatgag agcaatggat 420 ggtggtttgg ccacctccat gcgacatctc tccgatttag gctgctacga cagtgtgggc 480

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2017202313 07 Apr 2017

tttttatccc ccaggatgtg tttaaaacat ttcagagcaa aactggtgaa tttgatatga 540 aactgtgtga caatgtaaaa ggattgctga gcttgtatga agcttcattc ttggggtgga 600 gggatgaaaa 610 <210> 11 <211> 1049 <212> DNA <213> Santalum album <400> 11 caagttacca aacacattaa aattgatttt tatgtctatg tttaacaaga ccaatgaagt 60 tggccttcga gtccagcatg agcgaggcta cagtggcatc actactttta tcaaagcgtg 120 ggttgaacag tgtaaatcgt accagaaaga agcaagatgg taccatgggg gacacacgcc 180 tccactggaa gaatatagct tgaatggact ggtttccata ggattccctc tcttgttgat 240 cacaggctac gtggcaatcg ctgagaacga ggctgcactg gataaagtgc acccccttcc 300 tgatcttctg cactactcct ccctccttag tcgcctcatc aatgatatgg gaacctcttc 360 ggacgagttg gaaaggggag ataatctgaa gtcaattcaa tgttacatga accaaactgg 420 ggcttctgag aaagttgctc gtgagcacat aaagggaata atcgaggaaa actggaaaat 480 actgaatgag tgttgctttg atcaatctca gtttcaggag ccttttgtaa cattcaattt 540 gaactctgtt cgagggtctc atttcttcta cgaatttgga gatggctttg gggtgacgga 600 tagctggaca aaggttgata tgaagtctgt tttgatcgat cctattcctc tcgacgagga 660 gtagaaaact caaagcttgt gcttggttta cggtaatagt gattcagtat aaatataaaa 720 atcggacgaa cttgaggaat atgtgaggca taactatttt taatgatcat gagttaaata 780 attaagaaat atctattcgg ctcatgattc ttgagtatat attattcctt atgcgttata 840 tttccatcaa ataattagtc cgctcctgta agtcgactgt aacattactc taagggtcgc 900 tattggtttt atgttatatt aagtctacta gtttgaagtg atggaataaa tgtttgtttt 960 taagggggtt atgcactatg ttctcggttg ccttttacta ataaattttt tatgaaactc 1020 aaaaaaaaaa aaaaaaaaaa aaaaaaaaa 1049

<210> 12 <211> 2157 <212> DNA <213> Santalum album <400> 12

acaaaataaa tctcttgttc tgttctttgg atctcgtttt cttcccctca gctctctcac 60 taatggattc ttccaccgcc accgccatga gagctccatt cattgatcat actgatcatg 120 tgaatctcag aactgataac gattcctcag agaatcgaag gatggggaat tataaaccca 180 gtatttggaa ctatgatttt ttgcaatcgc ttgcgactcg ccacaatatt atggaagaga 240 ggcatctaaa gctagctgag aagctgaagg gccaagtgaa gtttatgttt ggggcaccaa 300 tggagccgtt agcaaagctg gagcttgtgg atgtggttca aaggctcggg ctaaaccacc 360 gatttgagac agagatcaag gaagcgctat ttagtattta Page 9 taaggatgag agcaatggat 420

2017202313 07 Apr 2017

ggtggtttgg ccacctccat gcgacatctc tccgatttag gctgctacga cagtgtgggc 480 tttttatccc ccaggatgtg tttaaaacat ttcagagcaa aactggtgaa tttgatatga 540 aactgtgtga caatgtaaaa ggattgctga gcttgtatga agcttcattc ttggggtgga 600 gggatgaaaa catcttagat gaagccaaag ccttcgccac caagtacttg aaaaatgcat 660 gggaaaacat atcccaaaag tggcttgcca aaagagtgaa gcatgcactg gctttgcctt 720 tgcactggag agtccctaga atcgaagcta gatggttcgt tgaggcatat ggggaagaag 780 agaatatgaa cccaacactt ctcaaacttg caaaattgga ctttaacatg gtgcaatcaa 840 ttcatcagaa agagattggg gaattagcga ggtggtgggt gactacgggg ttggataagt 900 tagcgtttgc taggaataat ttactgcaaa gctatatgtg gagctgcgcg attgcttccg 960 acccaaagtt caaacttgct agagaaacta ttgttgaaat cggaagtgta ctcacagttg 1020 ttgacgatgc atatgacgtc tatggttcaa tggatgaact tgatctctac acgaactccg 1080 ttgaaaggtg gagctgtaca gaaattgaca agttaccaaa cacattaaaa ttgattttta 1140 tgtctatgtt taacaagacc aatgaagttg gccttcgagt ccagcatgag cgaggctaca 1200 gtggcatcac tacttttatc aaagcgtggg ttgaacagtg taaatcgtac cagaaagaag 1260 caagatggta ccatggggga cacacgcctc cactggaaga atatagcttg aatggactgg 1320 tttccatagg attccctctc ttgttgatca caggctacgt ggcaatcgct gagaacgagg 1380 ctgcactgga taaagtgcac ccccttcctg atcttctgca ctactcctcc ctccttagtc 1440 gcctcatcaa tgatatggga acctcttcgg acgagttgga aaggggagat aatctgaagt 1500 caattcaatg ttacatgaac caaactgggg cttctgagaa agttgctcgt gagcacataa 1560 agggaataat cgaggaaaac tggaaaatac tgaatgagtg ttgctttgat caatctcagt 1620 ttcaggagcc ttttgtaaca ttcaatttga actctgttcg agggtctcat ttcttctacg 1680 aatttggaga tggctttggg gtgacggata gctggacaaa ggttgatatg aagtctgttt 1740 tgatcgatcc tattcctctc gacgaggagt agaaaactca aagcttgtgc ttggtttacg 1800 gtaatagtga ttcagtataa atataaaaat cggacgaact tgaggaatat gtgaggcata 1860 actattttta atgatcatga gttaaataat taagaaatat ctattcggct catgattctt 1920 gagtatatat tattccttat gcgttatatt tccatcaaat aattagtccg ctcctgtaag 1980 tcgactgtaa cattactcta agggtcgcta ttggttttat gttatattaa gtctactagt 2040 ttgaagtgat ggaataaatg tttgttttta agggggttat gcactatgtt ctcggttgcc 2100 ttttactaat aaatttttta tgaaactcaa aaaaaaaaaa aaaaaaaaaa aaaaaaa 2157

<210> 13 <211> 569 <212> PRT <213> Santalum album <400> 13

Met Asp Ser Ser Thr Ala Thr Ala Met Arg Ala Pro Phe Ile Asp His 15 10 15

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2017202313 07 Apr 2017

Thr Asp Hi s Val 20 Asn Le u Arg Thr Asp Asn Asp Ser Ser Glu Asn Arg 25 30 Arg Met Gly Asn Tyr Lys Pro Ser Ile T rp Asn Tyr Asp Phe Le u Gln 35 40 45 Ser Le u Al a Thr Arg Hi s Asn Ile Met Glu Glu Arg Hi s Le u Lys Le u 50 55 60 Al a Glu Lys Le u Lys Gly Gl n Val Lys Phe Met Phe Gly Al a Pro Met 65 70 75 80 Glu Pro Le u Al a Lys Le u Gl u Le u Val Asp Val Val Gln Arg Le u Gly 85 90 95 Le u Asn Hi s Arg Phe Glu Thr Glu Ile Lys Glu Al a Le u Phe Ser Ile 100 105 110 Tyr Lys Asp Glu Ser Asn Gly T rp T rp Phe Gly Hi s Le u Hi s Al a Thr 115 120 125 Ser Le u Arg Phe Arg Le u Le u Arg Gln cys Gly Le u Phe Ile Pro Gln 130 135 140 Asp Val Phe Lys Thr Phe Gl n Ser Lys Thr Gly Glu Phe Asp Met Lys 145 150 155 160 Le u cys Asp Asn Val Lys Gly Le u Le u Ser Le u Tyr Glu Al a Ser Phe 165 170 175 Le u Gly T rp Arg Asp Glu Asn Ile Le u Asp Glu Al a Lys Al a Phe Al a 180 185 190 Thr Lys Tyr Le u Lys Asn Al a T rp Glu Asn Ile Ser Gln Lys T rp Le u 195 200 205 Al a Lys Arg Val Lys Hi s Al a Le u Al a Le u Pro Le u Hi s T rp Arg Val 210 215 220 Pro Arg Ile Glu Al a Arg Trp Phe Val Glu Al a Tyr Gly Glu Glu Glu 225 230 235 240 Asn Met Asn Pro Thr Le u Le u Lys Le u Al a Lys Le u Asp Phe Asn Met 245 250 255 Val Gln Ser Ile Hi s Gln Lys Glu Ile Gly Glu Le u Al a Arg T rp T rp 260 265 270 Val Thr Thr Gly Le u Asp Lys Le u Al a Phe Al a Arg Asn Asn Le u Le u 275 280 285

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2017202313 07 Apr 2017

Gln Ser Tyr 290 Met Trp Ser cys Ala Ile Ala Ser Asp Pro Lys Phe Lys 295 300 Le u Al a Arg Glu Thr Ile Val Glu Ile Gly Ser Val Le u Thr Val Val 305 310 315 320 Asp Asp Al a Tyr Asp Val Tyr Gly Ser Met Asp Glu Le u Asp Le u Tyr 325 330 335 Thr Asn Ser Val Glu Arg Trp Ser cys Thr Glu Ile Asp Lys Le u Pro 340 345 350 Asn Thr Le u Lys Le u Ile Phe Met Ser Met Phe Asn Lys Thr Asn Glu 355 360 365 Val Gly Le u Arg Val Gln Hi s Glu Arg Gly Tyr Ser Gly Ile Thr Thr 370 375 380 Phe Ile Lys Al a T rp Val Gl u Gln cys Lys Ser Tyr Gln Lys Glu Al a 385 390 395 400 Arg T rp Tyr Hi s Gly Gly Hi s Thr Pro Pro Le u Glu Glu Tyr Ser Le u 405 410 415 Asn Gly Le u Val Ser Ile Gly Phe Pro Le u Le u Le u Ile Thr Gly Tyr 420 425 430 Val Al a Ile Al a Glu Asn Gl u Al a Al a Le u Asp Lys Val Hi s Pro Le u 435 440 445 Pro Asp Le u Le u Hi s Tyr Ser Ser Le u Le u Ser Arg Le u Ile Asn Asp 450 455 460 Met Gly Thr Ser Ser Asp Gl u Le u Glu Arg Gly Asp Asn Le u Lys Ser 465 470 475 480 Ile Gln cys Tyr Met Asn Gl n Thr Gly Al a Ser Glu Lys Val Al a Arg 485 490 495 Glu Hi s Ile Lys Gly Ile Ile Glu Glu Asn T rp Lys Ile Le u Asn Glu 500 505 510 cys cys Phe Asp Gln Ser Gl n Phe Gln Glu Pro Phe Val Thr Phe Asn 515 520 525 Le u Asn Ser Val Arg Gly Ser Hi s Phe Phe Tyr Glu Phe Gly Asp Gly 530 535 540 Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Le u 545 550 555 560

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2017202313 07 Apr 2017

Ile Asp Pro Ile Pro Leu Asp Glu Glu 565 <210> 14 <211> 1710 <212> DNA <213> Santalum album <400> 14

atggattctt ccaccgccac cgccatgaga gctccattca ttgatcatac tgatcatgtg 60 aatctcagaa ctgataacga ttcctcagag aatcgaagga tggggaatta taaacccagt 120 atttggaact atgatttttt gcaatcgctt gcgactcgcc acaatattat ggaagagagg 180 catctaaagc tagctgagaa gctgaagggc caagtgaagt ttatgtttgg ggcaccaatg 240 gagccgttag caaagctgga gcttgtggat gtggttcaaa ggctcgggct aaaccaccga 300 tttgagacag agatcaagga agcgctattt agtatttata aggatgagag caatggatgg 360 tggtttggcc acctccatgc gacatctctc cgatttaggc tgctacgaca gtgtgggctt 420 tttatccccc aggatgtgtt taaaacattt cagagcaaaa ctggtgaatt tgatatgaaa 480 ctgtgtgaca atgtaaaagg attgctgagc ttgtatgaag cttcattctt ggggtggagg 540 gatgaaaaca tcttagatga agccaaagcc ttcgccacca agtacttgaa aaatgcatgg 600 gaaaacatat cccaaaagtg gcttgccaaa agagtgaagc atgcactggc tttgcctttg 660 cactggagag tccctagaat cgaagctaga tggttcgttg aggcatatgg ggaagaagag 720 aatatgaacc caacacttct caaacttgca aaattggact ttaacatggt gcaatcaatt 780 catcagaaag agattgggga attagcgagg tggtgggtga ctacggggtt ggataagtta 840 gcgtttgcta ggaataattt actgcaaagc tatatgtgga gctgcgcgat tgcttccgac 900 ccaaagttca aacttgctag agaaactatt gttgaaatcg gaagtgtact cacagttgtt 960 gacgatgcat atgacgtcta tggttcaatg gatgaacttg atctctacac gaactccgtt 1020 gaaaggtgga gctgtacaga aattgacaag ttaccaaaca cattaaaatt gatttttatg 1080 gctatgttta acaagaccaa tgaagttggc cttcgagtcc agcatgagcg aggctacagc 1140 ggcatcacta cttttatcaa agcatgggtt gaacagtgta aatcgtacca gaaagaagca 1200 agatggtacc atgggggaca cacgcctcca ctggaagaat atagcttgaa tggacttgtt 1260 tccataggat tccctctctt gttgatcaca ggctacgtgg caatcgctga gaacgaggct 1320 gcactggata aagtgcaccc ccttcctgat cttctgcact actcctccct ccttagtcgc 1380 ctcatcaatg atatgggaac ctcttcggac gagttggaaa ggggagataa tctgaagtca 1440 attcaatgtt acatgaacca aactggggct tctgagaaag ttgctcgtga gcacataaag 1500 ggaataatcg aggaaaactg gaaaatactg aatgagtgtt gctttgatca atctcagttt 1560 caggagcctt ttgtaacatt caatttgaac tctgttcgag ggtctcattt cttctacgaa 1620 tttggagatg gctttggggt gacgaatagc tggacaaagg ttgatatgaa gtctgttttg 1680 atcgatccta ttcctctcga cgaggagtag 1710

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2017202313 07 Apr 2017 <210> 15 <211> 569 <212> PRT <213> Santa!um album <400> 15

Met 1 Asp Ser Ser Thr 5 Ala Thr Ala Met Arg Ala Pro 10 Phe Ile Asp 15 Hi s Thr Asp Hi s Val Asn Le u Arg Thr Asp Asn Asp Ser Ser Glu Asn Arg 20 25 30 Arg Met Gly Asn Tyr Lys Pro Ser Ile T rp Asn Tyr Asp Phe Le u Gln 35 40 45 Ser Le u Al a Thr Arg Hi s Asn Ile Met Glu Glu Arg Hi s Le u Lys Le u 50 55 60 Al a Glu Lys Le u Lys Gly Gl n Val Lys Phe Met Phe Gly Al a Pro Met 65 70 75 80 Glu Pro Le u Al a Lys Le u Gl u Le u Val Asp Val Val Gln Arg Le u Gly 85 90 95 Le u Asn Hi s Arg Phe Glu Thr Glu Ile Lys Glu Al a Le u Phe Ser Ile 100 105 110 Tyr Lys Asp Glu Ser Asn Gly T rp T rp Phe Gly Hi s Le u Hi s Al a Thr 115 120 125 Ser Le u Arg Phe Arg Le u Le u Arg Gln cys Gly Le u Phe Ile Pro Gln 130 135 140 Asp Val Phe Lys Thr Phe Gl n Ser Lys Thr Gly Glu Phe Asp Met Lys 145 150 155 160 Le u cys Asp Asn Val Lys Gly Le u Le u Ser Le u Tyr Glu Al a Ser Phe 165 170 175 Le u Gly T rp Arg Asp Glu Asn Ile Le u Asp Glu Al a Lys Al a Phe Al a 180 185 190 Thr Lys Tyr Le u Lys Asn Al a T rp Glu Asn Ile Ser Gln Lys T rp Le u 195 200 205 Al a Lys Arg Val Lys Hi s Al a Le u Al a Le u Pro Le u Hi s T rp Arg Val 210 215 220 Pro Arg Ile Glu Al a Arg Trp Phe Val Glu Al a Tyr Gly Glu Glu Glu 225 230 235 240 Asn Met Asn Pro Thr Le u Le u Lys Le u Al a Lys Le u Asp Phe Asn Met 245 250 255

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2017202313 07 Apr 2017

Val Gln Ser Ile 260 Hi s Gln Lys Glu Ile Gly 265 Glu Le u Al a Arg 270 T rp T rp Val Thr Thr Gly Le u Asp Lys Le u Al a Phe Al a Arg Asn Asn Le u Le u 275 280 285 Gln Ser Tyr Met T rp Ser cys Al a Ile Al a Ser Asp Pro Lys Phe Lys 290 295 300 Le u Al a Arg Glu Thr Ile Val Glu Ile Gly Ser Val Le u Thr Val Val 305 310 315 320 Asp Asp Al a Tyr Asp Val Tyr Gly Ser Met Asp Glu Le u Asp Le u Tyr 325 330 335 Thr Asn Ser Val Glu Arg Trp Ser cys Thr Glu Ile Asp Lys Le u Pro 340 345 350 Asn Thr Le u Lys Le u Ile Phe Met Al a Met Phe Asn Lys Thr Asn Glu 355 360 365 Val Gly Le u Arg Val Gln Hi s Glu Arg Gly Tyr Ser Gly Ile Thr Thr 370 375 380 Phe Ile Lys Al a T rp Val Gl u Gln cys Lys Ser Tyr Gln Lys Glu Al a 385 390 395 400 Arg T rp Tyr Hi s Gly Gly Hi s Thr Pro Pro Le u Glu Glu Tyr Ser Le u 405 410 415 Asn Gly Le u Val Ser Ile Gly Phe Pro Le u Le u Le u Ile Thr Gly Tyr 420 425 430 Val Al a Ile Al a Glu Asn Gl u Al a Al a Le u Asp Lys Val Hi s Pro Le u 435 440 445 Pro Asp Le u Le u Hi s Tyr Ser Ser Le u Le u Ser Arg Le u Ile Asn Asp 450 455 460 Met Gly Thr Ser Ser Asp Gl u Le u Glu Arg Gly Asp Asn Le u Lys Ser 465 470 475 480 Ile Gln cys Tyr Met Asn Gl n Thr Gly Al a Ser Glu Lys Val Al a Arg 485 490 495 Glu Hi s Ile Lys Gly Ile Ile Glu Glu Asn T rp Lys Ile Le u Asn Glu 500 505 510 cys cys Phe Asp Gln Ser Gl n Phe Gln Glu Pro Phe Val Thr Phe Asn

515 520 525

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2017202313 07 Apr 2017

Le u Asn Ser Val Arg Gly Ser Hi s Phe Phe Tyr Glu Phe Gly Asp Gly 530 535 540 Phe Gly Val Thr Asn Ser Trp Thr Lys Val Asp Met Lys Ser Val Le u 545 550 555 560 Ile Asp Pro Ile Pro Le u Asp Glu Glu 565

<210> 16 <211> 34 <212> DNA <213> Artificial <220>

<223> Primer <400> 16

ctagccatgg cttcagaaaa agaaattagg agag 34 <210> 17 <211> 40 <212> DNA <213> Arti fi ci al <220> <223> Pri mer <400> 17 ccggaattcc tatttgcttc tcttgtaaac tttgttcaag 40 <210> 18 <211> 42 <212> DNA <213> Arti fi ci al <220> <223> Pri mer <400> 18 aaggagatat acatatgaca aaaaaagttg gtgtcggtca gg 42 <210> 19 <211> 43 <212> DNA <213> Arti fi ci al <220> <223> Pri mer <400> 19 ctttaccaga ctcgagttac gcctttttca tctgatcctt tgc 43 <210> 20 <211> 35 <212> DNA <213> Pri mer <400> 20 cccgggggat ccatggctac cgataatgac agctc 35

Page 16

2017202313 07 Apr 2017 <210> 21 <211> 23 <212> DNA <213> Artificial <220>

<223> Primer <400> 21 caccgctgag caataactag cat 23 <210> 22 <211> 383 <212> DNA <213> Santalum album <400> 22 gatcaaggaa gcgctgttta gtatttacaa ggatgggagc aatggatggt ggtttggcca 60 ccttcatgcg acatctctcc gatttaggct gctacgacag tgtgggcttt ttattcccca 120 agatgtgttt aaaacgttcc aaaacaaaac tggggaattt gatatgaaac tgtgggacaa 180 cgtaaaaggg ctgctgagct tatatgaagc ttcatacttg ggatggaagg gtgaaaacat 240 cctagatgaa gccaaggcct tcaccaccaa gtgcttgaaa agtgcatggg aaaatatatc 300 cgaaaagtgg ttagccaaaa gagtgaagca tgcattggct ttgcctttgc attggagagt 360 ccctcgaatc gaagctagat ggt 383 <210> 23 <211> 26 <212> DNA <213> Artificial sequence <220>

<223> Primer <400> 23 ccgaaaagtg gttagccaaa agagtg 26 <210> 24 <211> 23 <212> DNA <213> Artificial sequence <220>

<223> Primer <400> 24 cgggtcggaa gcaatcgcgc agc 23 <210> 25 <211> 26 <212> DNA <213> Artificial sequence <220>

<223> Primer <400> 25 ccgatttcga caatagtttc tctagc 26

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2017202313 07 Apr 2017 <210> 26 <211> 1725 <212> DNA <213> Santalum album <400> 26

actaatggat tcttccaccg ccaccgccat gacagctcca ttcattgatc ctactgatca 60 tgtgaatctc aaaactgata ctgatgcctc agagaatcga aggatgggaa attataaacc 120 cagcatttgg aattatgatt ttttacaatc acttgcaact catcacaata ttgtggaaga 180 gaggcatcta aagctagctg agaagctgaa gggccaagtg aagtttatgt ttggggcacc 240 aatggagccg ttagcaaagc tggagcttgt ggatgtggtt caaaggcttg ggctaaacca 300 cctatttgag acagagatca aggaagcgct gtttagtatt tacaaggatg ggagcaatgg 360 atggtggttt ggccaccttc atgcgacatc tctccgattt aggctgctac gacagtgtgg 420 gctttttatt ccccaagatg tgtttaaaac gttccaaaac aaaactgggg aatttgatat 480 gaaactgtgg gacaacgtaa aagggctgct gagcttatat gaagcttcat acttgggatg 540 gaagggtgaa aacatcctag atgaagccaa ggccttcacc accaagtgct tgaaaagtgc 600 atgggaaaat atatccgaaa agtggttagc caaaagagtg aagcatgcat tggctttgcc 660 tttgcattgg agagtccctc gaatcgaagc tagatggttc attgaggtat atgagcaaga 720 agcgaatatg aacccaacac tactcaaact cgcaaaatta gactttaata tggtgcaatc 780 aattcatcag aaagagattg gggaattagc aaggtggtgg gtgactactg gcttggataa 840 gttagacttt gctaggaata atttactgca gagctatatg tggagctgcc cgattgcttc 900 cgacccgaag ttcaaacttg ctagagaaac tattgtcgaa atcggaagtg tactcacagt 960 tgttgacgat ggatatgacg tctatggttc aatggacgaa cttgatctct acacaagctc 1020 cgttgaaagg tggagctgtg tgaaaattga caagttgcca aacacgttaa aattaatttt 1080 tatgtctatg ttcaacaaga ccaatgaggt tggtcttcga gtccagcatg agcgaggcta 1140 caatagcatc cctactttta tcaaagcgtg ggttgaacag tgtaaatcat accagaaaga 1200 agcaagatgg ttccacgggg gacacacgcc tccattggaa gaatatagct tgaatggact 1260 tgtttccata ggattccctc tcttgttaat cacaggctac gtggcaatcg ctgagaacga 1320 ggctgcactg gataaagtgc acccccttcc tgatcttctg cactactcct ccctccttag 1380 tcgcctcatc aatgatatag gaacgtctcc ggatgagatg gcaagaggcg ataatctgaa 1440 gtcaatccat tgttacatga acgaaactgg ggcttccgag gaagttgctc gtgagcacat 1500 aaagggagta atcgaggaga attggaaaat actgaatcag tgctgctttg atcaatctca 1560 gtttcaggag ccttttataa ccttcaattt gaactctgtt cgagggtctc atttcttcta 1620 tgaatttggg gatggctttg gggtgacgga tagctggaca aaggttgata tgaagtccgt 1680 tttgatcgac cctattcctc tcggcgagga gtagtaagct cgaag 1725

<210> 27 <211> 569 <212> PRT

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2017202313 07 Apr 2017

<213> Santa!um Al b u m <400> 27 Met Asp Ser Ser Thr Al a Thr Al a Met Thr Al a Pro Phe Ile Asp Pro 1 5 10 15 Thr Asp Hi s Val Asn Le u Lys Thr Asp Thr Asp Al a Ser Glu Asn Arg 20 25 30 Arg Met Gly Asn Tyr Lys Pro Ser Ile T rp Asn Tyr Asp Phe Le u Gln 35 40 45 Ser Le u Al a Thr Hi s Hi s Asn Ile Val Glu Glu Arg Hi s Le u Lys Le u 50 55 60 Al a Glu Lys Le u Lys Gly Gl n Val Lys Phe Met Phe Gly Al a Pro Met 65 70 75 80 Glu Pro Le u Al a Lys Le u Gl u Le u Val Asp Val Val Gln Arg Le u Gly 85 90 95 Le u Asn Hi s Le u Phe Glu Thr Glu Ile Lys Glu Al a Le u Phe Ser Ile 100 105 110 Tyr Lys Asp Gly Ser Asn Gly T rp T rp Phe Gly Hi s Le u Hi s Al a Thr 115 120 125 Ser Le u Arg Phe Arg Le u Le u Arg Gln cys Gly Le u Phe Ile Pro Gln 130 135 140 Asp Val Phe Lys Thr Phe Gl n Asn Lys Thr Gly Glu Phe Asp Met Lys 145 150 155 160 Le u T rp Asp Asn Val Lys Gly Le u Le u Ser Le u Tyr Glu Al a Ser Tyr 165 170 175 Le u Gly T rp Lys Gly Glu Asn Ile Le u Asp Glu Al a Lys Al a Phe Thr 180 185 190 Thr Lys cys Le u Lys Ser Al a T rp Glu Asn Ile Ser Glu Lys T rp Le u 195 200 205 Al a Lys Arg Val Lys Hi s Al a Le u Al a Le u Pro Le u Hi s T rp Arg Val 210 215 220 Pro Arg Ile Glu Al a Arg Trp Phe Ile Glu Val Tyr Glu Gln Glu Al a 225 230 235 240 Asn Met Asn Pro Thr Le u Le u Lys Le u Al a Lys Le u Asp Phe Asn Met 245 250 255 Val Gln Ser Ile Hi s Gln Lys Glu Ile Gly Glu Le u Al a Arg T rp T rp

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2017202313 07 Apr 2017

260 265 270 Vai Thr Thr Giy Le u Asp Lys Le u Asp Phe Ai a Arg Asn Asn Le u Le u 275 280 285 Gin Ser Tyr Met T rp Ser cys Pro lie Ai a Ser Asp Pro Lys Phe Lys 290 295 300 Le u Ai a Arg Giu Thr lie Vai Giu lie Giy Ser Vai Le u Thr Vai Vai 305 310 315 320 Asp Asp Giy Tyr Asp Vai Tyr Giy Ser Met Asp Giu Le u Asp Le u Tyr 325 330 335 Thr Ser Ser Vai Giu Arg Trp Ser cys Vai Lys lie Asp Lys Le u Pro 340 345 350 Asn Thr Le u Lys Le u lie Phe Met Ser Met Phe Asn Lys Thr Asn Giu 355 360 365 Vai Giy Le u Arg Vai Gin Hi s Giu Arg Giy Tyr Asn Ser lie Pro Thr 370 375 380 Phe lie Lys Ai a T rp Vai Gi u Gin cys Lys Ser Tyr Gin Lys Giu Ai a 385 390 395 400 Arg T rp Phe Hi s Giy Giy Hi s Thr Pro Pro Le u Giu Giu Tyr Ser Le u 405 410 415 Asn Giy Le u Vai Ser lie Giy Phe Pro Le u Le u Le u lie Thr Giy Tyr 420 425 430 Vai Ai a lie Ai a Giu Asn Gi u Ai a Ai a Le u Asp Lys Vai Hi s Pro Le u 435 440 445 Pro Asp Le u Le u Hi s Tyr Ser Ser Le u Le u Ser Arg Le u lie Asn Asp 450 455 460 lie Giy Thr Ser Pro Asp Gi u Met Ai a Arg Giy Asp Asn Le u Lys Ser 465 470 475 480 lie Hi s cys Tyr Met Asn Gi u Thr Giy Ai a Ser Giu Giu Vai Ai a Arg 485 490 495 Giu Hi s lie Lys Giy Vai lie Giu Giu Asn T rp Lys lie Le u Asn Gin 500 505 510 cys cys Phe Asp Gin Ser Gi n Phe Gin Giu Pro Phe lie Thr Phe Asn 515 520 525 Le u Asn Ser Vai Arg Giy Ser Hi s Phe Phe Tyr Giu Phe Giy Asp Giy 530 535 540

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2017202313 07 Apr 2017

Phe Gly Val Thr Asp Ser Trp Thr Lys Val Asp Met Lys Ser Val Leu 545 550 555 560

Ile Asp Pro Ile Pro Leu Gly Glu Glu 565

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