VECTOR EXPRESSION, MICROBIAL HOST CELL, MUSHROOM OR TRANSGENIC CULTURE, AND METHODS FOR PREPARING SANTALOL

Abstract

The invention is directed to a santalene synthase, a nucleic acid encoding said santalene synthase, an expression vector comprising said nucleic acid, a host cell comprising said expression vector, a method of preparing santalene, a method of preparing santalol, and a method of preparing a santalene synthase. The invention is further directed to an antibody specific for santalene synthase.

Description

1 / 120 Descriptive Report of the Invention Patent for: EXPRESSION VECTOR, MICROBIAL HOST CELL, TRANSGENIC MUSHROOM OR CULTURE, AND METHODS FOR PREPARING SANTALENE AND SANTALOL

[001] The invention is directed to a santalene synthase, to a nucleic acid encoding said santalene synthase, to an expression vector comprising said nucleic acid, to a host cell comprising said expression vector, to a method of preparing santalene, to a method of preparing santalol and to a method of preparing a santalene synthase.

[002] Many organisms have the ability to produce a wide array of terpenes and terpenoids. Terpenes are, in fact or conceptually, built from 2-methylbutane residues, commonly called isoprene units, which have the molecular formula CrHg. It is possible to consider the isoprene unit as one of nature's common building blocks. The basic molecular formulas of terpenes are multiples of such a formula: (C5H8)n, where n is the number of isoprene units linked. This is called the isoprene rule, as a result of which terpenes are also denoted as isoprenoids. Isoprene units can be connected head-to-tail to form linear chains or can be arranged to form rings. In their biosynthesis, terpenes are formed from the universal 5-carbon precursors isopentenyl diphosphate (IPP) and Petition 870260065022, dated 02 / 07 / 2026, p. 11 / 19 2 / 120 its isomer, dimethylallyl diphosphate (DMAPP). Consequently, a terpene carbon skeleton generally comprises a multiple of 5 carbon atoms. The most common are terpenes with 5, 10, 15, 20, and 30 carbons, which are called hemi-, mono-, sesqui-, di-, tri-, and tetraterpenes, respectively. In addition, head-to-tail connections, tri- and tetraterpenes also contain a tail-to-tail connection at their center. Terpenes may comprise additional functional groups such as alcohols and their glycosides, ethers, aldehydes, ketones, carboxylic acids, and esters. These functionalized terpenes are, in this document, called terpenoids. Like terpenes, terpenoids generally have a carbon skeleton that has a multiple of 5 carbon atoms.It should be noted that the total number of carbons in a terpenoid does not need to be a multiple of 5; for example, the functional group may be an ester group comprising an alkyl radical that has numerous carbon atoms.

[003] In addition to the definitions provided above, it is important to note that the terms terpene, terpenoid, and isoprenoid are often used interchangeably in the open literature, as well as patent literature.

[004] Santalene is a naturally occurring sesquiterpene produced in specific plants, such as the tree of Petition 870190082172, dated 08 / 22 / 2019, p. 9 / 248 3 / 120 Sandalwood. Santalene, and especially β-santalene, is useful as a starting material for the chemical synthesis or biosynthesis of santalol, and in particular for β-santalol, which is a major constituent of sandalwood oil. Sandalwood oil is an important perfumery ingredient obtained by steam distillation of the heartwood of several species of sandalwood tree (Santalum), for example, Santalum album and Santalum spicalum. Sandalwood oil is used in perfumes, cosmetics, and for flavoring. Sandalwood oil contains more than 90% sesquiterpene alcohols, of which 40 to 60% is α-santalol, while β-santalol comprises 15 to 25%. Although other constituents such as α-santalol, epi-β-santalol, and bergamotol may also contribute to the typical sandalwood oil sensory profile, β-santalol is considered the most important odor-defining molecule in sandalwood oil.The exact composition of the oil depends on the species of Santalum, the harvesting conditions, and the distillation process employed.

[005] Sandalwood trees have been overexploited for the production of sandalwood and sandalwood oil for a long period, leading to the endangered status of several sandalwood species (Teixeira da Silva et al., 2016). Consequently, the supply of sandalwood oil has decreased significantly over the last few years. Therefore, it is desirable to provide an alternative source of sandalwood oil terpenes, and Petition 870190082172, dated 08 / 22 / 2019, p. 10 / 248 4 / 120 especially α- and β-santhols, which are the key molecules in defining the sweet, warm, and woody odor of sandalwood oil (Baldovini et al, 2011) (Diaz-Chavez et al., 2013).

[006] The preparation of santalene (or santalol) microbiologically has been proposed, using genetically modified microorganisms by incorporating a gene that codes for a protein that has santalene synthase activity. A santalene synthase can be used for the preparation of santalene from FPP, a conversion that can be performed as an isolated reaction (in vitro) or as part of a longer metabolic pathway that eventually leads to the production of santalene from sugar (in vivo).

[007] Document WO / 2010 / 067309 describes a method for producing β-santalene using a santalene synthase from Santalum (Schalk, 2014). Patent no. US 8993284,

[008] WO201100026 and Jones et al. (2011) describe terpene synthases from three different species of Santalum (Santalum album, S. austrocaledonicum and S. spicatum), producing α-santhalene, α-trans-bergamotene, epi-e-santhalene and β-santhalene simultaneously (Zulak et al, 2016). The document WO2015153501 describes modified santalene synthase enzymes derived from Petition 870190082172, dated 08 / 22 / 2019, p. 11 / 248 5 / 120 santalene synthase from S. album with increased terpene synthase activity compared to native S. album santalene synthase. Document WO2012 / 110375 describes a synthesis pathway for an intermediate that can be used for the chemical synthesis of beta-santalol.

[009] The only terpene synthases known to form α-santalene, β-santalene, epi-p-santalene, and bergamotene, to date, have been identified in the genus Santalum. Other plants, however, have been described as producing some of the santalol-type sesquiterpenoids: Document WO2006 / 134523 describes a terpene synthase capable of synthesizing sesquiterpenes with a santalene backbone, such as epi-p-santalene and trans-α-bergamotene, however, no production of β-santalene and α-santalene is described (SCHALK, 2006). Epi-β-santalene cannot be used for the synthesis of the desired β-santalol. Document WO2009 / 109597 describes another terpene synthase capable of producing santalene-type terpenes (Schalk, 2016). However, the described synthase produces α-santhalene from E,E-farnesyl pyrophosphate, but without β-santhalene. Document WO 2008 / 142318 describes a santalene αsynthase from Solatium habrochailes.This enzyme uses Z,Z-farnesyl pyrophosphate as a substrate to produce α-santhalene. Again, the synthase described produces only α-santhalene and no β-santhalene. Petition 870190082172, dated 08 / 22 / 2019, page 12 / 248 6 / 120

[010] Essential oils derived from hydrodistillation of leaves, stem and bark of the camphor tree, Cinnamomum camphora, have been described as containing santalene-type terpenes, namely α-santhalene, cisα-bergamotene, epi-beta-santhalene and beta-santhalene (Pelissier & Bessiere, 1995), however, no corresponding terpene synthase has been identified.

[011] Currently known santalene synthases have a number of distinct disadvantages which are particularly undesirable when applied in an industrial santalene production process where santalene (or santalol and in particular β-santalol) is prepared from FPP, in an isolated reaction (in vitro), for example, using an isolated santalene synthase or whole (permeabilised) cells or otherwise, for example, in a fermentative process which is part of a longer metabolic pathway eventually leading to the production of β-santalol from sugar (in vivo).

[012] Thus, there is a need for an alternative santalene synthase that can be used in the preparation of santalene, in particular, β-santalene and / or β-santalol. In particular, there is a need for an alternative santalene synthase that exhibits enhanced expression, at least in selected host cells; a synthase of Petition 870190082172, dated 08 / 22 / 2019, page 13 / 248 7 / 120 an alternative santalene synthase that has high enzymatic activity at least under specific conditions, such as at a neutral or alkaline pH and / or intracellularly in the cell in which it was produced; and / or an alternative santalene synthase that is highly specific, in particular, that has enhanced specificity compared to the santalene synthase from Santalum album, with respect to catalyzing the conversion of FPP to β-santhalene, at least under specific conditions, such as at approximately neutral or alkaline pH and / or intracellularly in the cell in which it was produced.

[013] It has been found that a specific polypeptide, which was unknown until now, has santalene synthase activity and that this polypeptide can be used as a catalyst that can serve as an alternative to known santalene synthases.

[014] Consequently, the present invention relates to a santalene synthase comprising an amino acid sequence as shown in SEQ ID NO: 3 or a functional homolog thereof, wherein said functional homolog is a santalene synthase comprising an amino acid sequence having a sequence identity of at least 60% with SEQ ID NO: 3. Said homolog may, in particular, be a santalene synthase comprising an amino acid sequence having a sequence identity of at least 65%, at least 70%, Petition 870190082172, dated 08 / 22 / 2019, p. 14 / 248 8 / 120 at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% with SEQ ID NO: 3.

[015] Furthermore, the invention relates to an antibody that has binding affinity to a santalene synthase according to the invention. An antibody according to the invention, therefore, binds specifically to a santalene synthase according to the invention.

[016] The antibody, which can be monoclonal or polyclonal, can be produced by methods known in the art, for example, as described by Hudson et al. Practical Immunology, Third Edition (1989), Blackwell Scientific Publications.

[017] The invention further relates to a nucleic acid comprising a nucleic acid sequence encoding a santalene synthase according to the invention or comprising a nucleic acid sequence complementary to said encoding sequence. In particular, the nucleic acid may be selected from nucleic acids comprising a nucleic acid sequence as shown in SEQ ID NO: 1 or SEQ ID NO: 2, and other nucleic acid sequences encoding a santalene synthase according to the invention, wherein said other sequences comprise a nucleic acid sequence having a sequence identity of at least Petition 870190082172, dated 08 / 22 / 2019, p. 15 / 248 9 / 120 60%, in particular, at least 65%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% with the nucleic acid sequence shown in SEQ ID NO: 1 or SEQ ID NO: 2 or respectively complementary nucleic acids thereto. Said other nucleic acid sequence encoding a santalene synthase according to the invention may hereinafter be referred to as a functional analogue.

[018] A santalene or nucleic acid synthase according to the invention may be a natural compound or fragment of a compound isolated from its natural source (e.g., Cinnamomum camphora), be a compound or fragment synthesized chemically or enzymatically from a compound, or a compound or fragment of a compound produced in a recombinant cell, the recombinant cell in which it may be present, or the cell from which it may have been isolated.

[019] The invention further relates to an expression vector comprising a nucleic acid according to the invention.

[020] The invention relates to a host cell, which may be an organism itself or part of a multicellular organism, comprising an expression vector comprising a nucleic acid, preferably a nucleic acid heterologous to said host cell, according to the invention. The host cell is Petition 870190082172, dated 08 / 22 / 2019, page 16 / 248 10 / 120 preferably selected from the group of bacterial cells, fungal cells (including yeast) and plant cells.

[021] The invention further relates to a method for preparing santalene; comprising converting FPP into santalene in the presence of a santalene synthase according to the invention. Four different geometric isomers of FPP can exist, namely, 2E.6E-FPP, 2Z,GEFPP, 2E,6Z-FPP and 2Z,6Z-FPP. Good results have been obtained with 2E,6E-FPP, although, in principle, any other isomer of FPP could be a suitable substrate for an enzyme according to the invention.

[022] The invention is further directed to a method for producing a santalene synthase according to the invention, comprising growing a host cell according to the invention under conditions conducive to the production of santalene synthase and optionally recovering santalene synthase from the host cell.

[023] It has been found that a santalene synthase according to the invention is more specific with respect to santalene and in particular β-santhalene synthesis than a santalene synthase from S. album, in particular at neutral or approximately neutral pH in an in vitro assay or in a method in which santalene, and in particular β-santhalene, is synthesized intracellularly in a host cell. Petition 870190082172, dated 08 / 22 / 2019, page 17 / 248 11 / 120 genetically modified to produce a santalene synthase according to the invention and a santalene synthase from S. album, respectively. Initial results show that under identical conditions, the amount of major side product (bergamotene) formed with the innovative enzyme of the invention is significantly lower, namely, a molar ratio α-santhalene / bergamotene = 2:1 (0.5:1 for S. album) α-santhalene / bergamotene = 0.9:1 (0.3:1 for S. album) α+β-santhalene / bergamotene = 2.9:1 (0.8:1 for S. album)

[024] According to the invention, it has been found that it is possible to bring santalene synthase into expression with good yield in different organisms. For example, it has been found that santalene synthase is well expressed in E. coli, Rhodobacter sphaeroides and in Nicotiana benthamiana plants.

[025] Thus, in an advantageous embodiment, the present invention provides a santalene synthase with improved specificity for catalyzing santalene synthesis and an improved production rate for βsanthalene when used in a method for preparing santalene, in particular, compared with santalene synthase from S. album or other santalene synthase according to the prior art, mentioned in this document. Petition 870190082172, dated 08 / 22 / 2019, page 18 / 248 12 / 120

[026] In a preferred embodiment, a method for preparing santalene according to the invention is provided, wherein santalene is prepared in a host cell, a plant or plant culture, or a mushroom or mushroom culture, according to the invention, expressing said santalene synthase. Preferably, the method for preparing santalene according to the invention further comprises isolating santalene from said host cell, plant or plant culture or mushroom or mushroom culture. Preferably, the method for preparing santalene according to the invention results in an α-santhalene to α-bergamotene ratio that is greater than 1:1, more preferably greater than 1.5:1, more preferably greater than 1.7:1, more preferably greater than 1.9:1, most preferably about 2:1.Preferably, the method for preparing santalene according to the invention results in a ratio of β-santhalene to α-bergamotene greater than 0.5:1, more preferably greater than 0.6:1, more preferably greater than 0.7:1, more preferably greater than 0.8:1, most preferably about 0.9:1. Preferably, the method for preparing santalene according to the invention results in a ratio of santalenes (α- and β-santhalene) to α-bergamotene greater than 2:1, more preferably greater than 2.3:1, more preferably greater than 2.5:1, more. Petition 870190082172, dated 08 / 22 / 2019, p. 19 / 248 13 / 120 preferably greater than 2.7:1, more preferably greater than 2.8:1, with the highest preference being around 2.9:1.

[027] Without adhering to theory, it is believed that high specificity with respect to catalyzing santalene synthesis at neutral or mildly alkaline pH is considered particularly desirable for methods in which santalene is prepared intracellularly, since several host cells are believed to have a neutral or slightly alkaline intracellular pH, such as a pH of 7.0 to 8.5 (for intracellular pH values ​​of bacteria, see, for example: Booth, Microbiological Reviews (1985) 49: 359 to 378). When, for example, E. cells When coli were exposed to pH values ​​ranging from 5.5 to 8.0, the intracellular pH was between 7.1 and 7.9 (Olsen et al., Appl. Environ. Microbiol. (2002) 68: 4145 to 4147). This may explain an enhanced specificity with respect to santalene synthesis of a santalene synthase according to the invention, also intracellularly.

[028] The term or, as used in this document, is defined as and / or, unless otherwise specified.

[029] The term "a" or "an," as used in this document, is defined as "at least one" unless otherwise specified.

[030] When referring to a pronoun (for example, a Petition 870190082172, dated 08 / 22 / 2019, p. 20 / 248 14 / 120 compound, an additive, etc.) in the singular, the plural is intended to be included.

[031] The terms farnesyl diphosphate and farnesyl pyrophosphate (both abbreviated as FPP), as used interchangeably, in this document refer to the compound 3,7,11-trimethyl-2,6,10-dodecatrienyl-yl pyrophosphate and include all known isomers of this compound. The term recombinant, in relation to a recombinant cell, vector, nucleic acid or similar, as used in this document, refers to a cell, vector, nucleic acid or similar containing nucleic acid that is not naturally occurring in such cell, vector, nucleic acid or similar and / or not naturally occurring in the same location. Generally, said nucleic acid was introduced into such strain (cell) using recombinant DNA techniques.

[032] The term heterologous, when used in relation to a nucleic acid (DNA or RNA) or protein, refers to a nucleic acid or protein that does not occur naturally as part of the organism, cell, genome, or DNA or RNA sequence in which it is present, or that is found in a cell or location or locations in the genome or DNA or RNA sequence that differ from that in which it is found in nature. Heterologous nucleic acids or proteins are not endogenous to the cell in which they are found. Petition 870190082172, dated 08 / 22 / 2019, p. 21 / 248 15 / 120 introduced, but were obtained from another cell synthetically or recombinantly produced. Generally, though not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed.

[033] A gene that is endogenous to a particular host cell, but has been modified from its natural form, through, for example, the use of DNA scrambling, is also called heterologous. The term heterologous also includes multiple non-naturally occurring copies of a naturally occurring DNA sequence. Thus, the term heterologous can refer to a segment of DNA that is foreign or heterologous to the cell, or homologous to the cell, but in a position and / or number in the host cell's nucleic acid where the segment is not commonly found. Exogenous DNA segments are expressed to produce exogenous polypeptides. A homologous DNA sequence is a DNA sequence that is naturally associated with a host cell into which it is introduced.

[034] Any nucleic acid or protein that experts in the field would recognize as heterologous or foreign to the cell in which it is expressed is covered in this document by the term heterologous nucleic acid or protein. Petition 870190082172, dated 08 / 22 / 2019, page 22 / 248 16 / 120

[035] The term mutated or mutation, as used in this document with respect to proteins or polypeptides, means that at least one amino acid in the sequence of wild-type or naturally occurring polypeptides or proteins has been replaced by a different amino acid, or deleted from, or inserted into the sequence by means of mutagenesis of nucleic acids encoding those amino acids. Mutagenesis is a well-known method in the art and includes, for example, site-directed mutagenesis by means of PCR or by means of oligonucleotide-mediated mutagenesis as described in Sambrook, J. and Russell, DW Molecular Cloning: A Laboratory Manual. 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001). The term mutated or mutation, as used in this document with respect to genes, means that at least one nucleotide in the nucleotide sequence of such gene or a regulatory sequence thereof has been replaced by a different nucleotide, or has been deleted from or inserted into the sequence by means of mutagenesis.

[036] The terms open reading frame and ORF refer to the amino acid sequence encoded between the initiation and termination codons of a coding sequence. The terms initiation codon and termination codon refer to a unit of three adjacent nucleotides (codon) in a sequence of Petition 870190082172, dated 08 / 22 / 2019, p. 23 / 248 17 / 120 encoding that specifies the initiation and termination of the protein synthesis chain (mRNA translation), respectively.

[037] The term gene is used broadly to refer to any segment of nucleic acids associated with a biological function. Thus, genes include coding sequences and / or the regulatory sequences necessary for their expression. For example, a gene refers to a fragment of nucleic acid that expresses functional mRNA or RNA, or encodes a specific protein and includes regulatory sequences. Genes also include unexpressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesizing known or predicted sequence information, and may include sequences designed to have desired parameters.

[038] The term chimeric gene refers to any gene that contains 1) DNA sequences, including regulatory and coding sequences, that are not found together in nature, or 2) sequences that encode parts of proteins that are not naturally adjoined, or 3) parts of promoters that are not naturally adjoined. Consequently, a chimeric gene may comprise Petition 870190082172, dated 08 / 22 / 2019, p. 24 / 248 18 / 120 regulatory sequences and coding sequences that are derived from different sources, or comprising regulatory sequences and coding sequences derived from the same source, but arranged in a manner different from that found in nature.

[039] The term transgenic for a transgenic cell or organism as used in this document, refers to an organism or cell (where such cell may be an organism itself or a cell of a multicellular organism from which it has been isolated) containing a nucleic acid that does not occur naturally in such organism or cell and the nucleic acid which has been introduced into such organism or cell (i.e., has been introduced into the organism or cell itself or into an ancestor of the organism or an ancestral organism of an organism from which the cell has been isolated) using recombinant DNA techniques.

[040] A transgene refers to a gene that has been introduced into the genome by transformation and is preferably maintained in a stable manner. Transgenes may include, for example, genes that are heterologous or homologous to the genes of a particular plant being transformed. Additionally, transgenes may comprise native genes inserted into a non-native organism or chimeric genes. The term endogenous gene refers to a native gene and its natural location in the genome of an organism. Petition 870190082172, dated 08 / 22 / 2019, p. 25 / 248 19 / 120 A foreign gene refers to a gene not normally found in the host organism, but which is introduced through gene transfer.

[041] Transformation and transformation, as used in this document, refers to the introduction of a heterologous nucleotide sequence into a host cell, regardless of the method used for insertion, for example, direct uptake, transduction, conjugation, f-coupling or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, for example, a plasmid, or alternatively, it may be integrated into the host cell genome.

[042] The coding sequence refers to a DNA or RNA sequence that codes for a specific amino acid sequence and excludes non-coding sequences. It may constitute an uninterrupted coding sequence, i.e., devoid of an intron, as in a cDNA, or it may include one or more introns linked by appropriate splicing junctions. An intron is an RNA sequence that is contained in the primary transcript, but which is removed through cleavage and rejoining of the RNA within the cell to create the mature mRNA that can be translated into a protein.

[043] Regulatory sequences refer to nucleotide sequences located upstream Petition 870190082172, dated 08 / 22 / 2019, p. 26 / 248 Regulatory sequences are 20 / 120 (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence, and which influence the transcription, RNA processing, or stability or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translational leader sequences, introns, and polyadenylation signal sequences. These include both natural and synthetic sequences, as well as sequences that may be a combination of synthetic and natural sequences. As noted above, the term proper regulatory sequences is not limited to promoters.

[044] Examples of regulatory sequences include promoters (such as transcriptional promoters, constitutive promoters, inducible promoters), operators or enhancers, ribosomal mRNA binding sites, and suitable sequences that control the initiation and termination of transcription and translation. Nucleic acid sequences are operationally linked when the regulatory sequence functionally relates to the cDNA sequence of the invention.

[045] Each of the regulatory sequences can be independently selected from heterologous and homologous regulatory sequences.

[046] Prosecutor refers to a sequence of Petition 870190082172, dated 08 / 22 / 2019, p. 27 / 248 A promoter is a 21 / 120 nucleotide sequence, usually upstream (δ') of its coding sequence, that controls the expression of said coding sequence by providing recognition for RNA polymerase and other factors necessary for proper transcription. A promoter includes a minimal promoter, which is a short DNA sequence comprising a TATA box and other sequences that serve to specify the transcription initiation site, to which regulatory elements are added for expression control. A promoter also refers to a nucleotide sequence that includes a minimal promoter plus regulatory elements capable of controlling the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements; these elements are often called enhancers.Consequently, an enhancer is a DNA sequence that can stimulate promoter activity and can be an innate promoter element or a heterologous element inserted to enhance the level or tissue specificity of a promoter. It is capable of operating in both orientations (normal or flipped), and is able to function even when moved upstream or downstream of the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their activity. Petition 870190082172, dated 08 / 22 / 2019, p. 28 / 248 22 / 120 effects. Promoters may be derived entirely from a native gene, or be composed of different elements derived from different promoters found in nature, or even be comprised of synthetic DNA segments. A promoter may also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions. The term nucleic acid, as used in this document, includes reference to a deoxyribonucleotide or ribonucleotide polymer, i.e., a polynucleotide, in a single- or double-stranded form and, except as otherwise limited, encompasses known analogues that have the essential nature of natural nucleotides in the sense that they hybridize to single-stranded nucleic acids in a manner similar to naturally occurring nucleotides (e.g., peptide nucleic acids).A polynucleotide can be the full length or a subsequence of a native or heterologous gene, structural or regulatory. Unless otherwise indicated, the term includes reference to the specified sequence as well as its complementary sequence. Thus, DNAs or RNAs with backbones modified for stability or other reasons are polynucleotides, as intended in this document. Furthermore, DNAs or... Petition 870190082172, dated 08 / 22 / 2019, p. 29 / 248 23 / 120 RNAs comprising unusual bases, such as inosine, or modified bases, such as titrated bases, to mention just two examples, are polynucleotides as the term is used in this document. It will be observed that a wide variety of modifications have been made to DNA and RNA, serving many useful purposes known to experts in the field. The term polynucleotide, as employed in this document, encompasses such chemically, enzymatically, or metabolically modified forms of polynucleotides, as well as the chemical forms of DNA and RNA characteristic of viruses and cells, including, among other things, simple and complex cells.

[047] Every nucleic acid sequence in this document that codes for a polypeptide also, by reference to the code, describes every possible silent variation of the nucleic acid. The term conservatively modified variants applies to both amino acid and nucleic acid sequences. With respect to particular nucleic acid sequences, the term conservatively modified variants refers to those nucleic acids that encode conservatively modified or identical variants of the amino acid sequences due to genetic code degeneracy. The term genetic code degeneracy refers to the fact that a large number of functionally modified nucleic acids Petition 870190082172, dated 08 / 22 / 2019, p. 30 / 248 24 / 120 identical codons code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at any position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are silent variations and represent a type of conservatively modified variation. The terms polypeptide, peptide, and protein are used interchangeably in this document to refer to a polymer of amino acid residues. The terms apply to amino acid polymers in which one or more amino acid residues are an artificial chemical analogue of a corresponding naturally occurring amino acid, as well as to naturally occurring amino acid polymers.The essential nature of such naturally occurring amino acid analogs is that, when incorporated into a protein, that protein is specifically reactive to antibodies elicited against the same protein, but consisting entirely of naturally occurring amino acids. The terms polypeptide, peptide, and protein are also inclusive of modifications including, but not limited to, glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation, and ADP-ribosylation. Petition 870190082172, dated 08 / 22 / 2019, page 31 / 248 25 / 120

[048] Within the context of the present application, oligomers (such as oligonucleotides, oligopeptides) are considered a type of polymer group. Oligomers have a relatively small number of monomeric units, generally 2 to 100, in particular 6 to 100.

[049] The expression cassette, as used in this document, means a DNA sequence capable of directing the expression of a particular nucleotide sequence in a suitable host cell, comprising a promoter operationally linked to the nucleotide sequence of interest, which is operationally linked to termination signals. It also typically comprises sequences necessary for the proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest, but may also code for a functional RNA of interest, for example, antisense RNA or an untranslated RNA, in either sense or antisense. The expression cassette comprising the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous with respect to at least one of its other components.The expression cassette can also be one that is naturally occurring, but has been obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence in the cassette... Petition 870190082172, dated 08 / 22 / 2019, page 32 / 248 26 / 120 expression may be under the control of a constitutive promoter or an inducible promoter, which initiates transcription only when the host cell is exposed to some particular external stimulus. In the case of a multicellular organism, the promoter may also be specific to a particular tissue or organ or developmental stage.

[050] The term vector as used in this document refers to a construct comprising genetic material designed to direct the transformation of a target cell. A vector contains multiple genetic elements oriented in a positional and sequential manner, that is, operationally linked with other necessary elements, so that the nucleic acid in a nucleic acid cassette can be transcribed and, when necessary, translated in the transformed cells. In particular, the vector may be selected from the group of viral vectors, (bacterio)phages, cosmids or plasmids. The vector may also be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAG) or an Agrobacterium binary vector.The vector can be linear or circular in form, double or single-stranded, which may or may not be self-transmissible or mobilizable, and which can transform the prokaryotic or eukaryotic host through integration into the cellular genome or exist extrachromosomally (e.g., a self-replicating plasmid with an origin of...). Petition 870190082172, dated 08 / 22 / 2019, page 33 / 248 27 / 120 replication). Specifically included are transport vectors, which are said to be a vehicle for DNA capable, naturally or through design, of replication in two different host organisms, which may be selected from actinomycetes and related species, bacteria, and eukaryotes (e.g., higher plant, mammalian, yeast, or fungal cells). Preferably, the nucleic acid in the vector is under the control of, and operationally linked to, a suitable promoter or other regulatory elements for transcription in a host cell, such as a microbial cell, for example, bacterial or plant. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, it may contain its own promoter or other regulatory elements, and in the case of cDNA, it may be under the control of a suitable promoter or other regulatory elements for expression in the host cell.

[051] Vectors containing a polynucleic acid according to the invention can be prepared based on known methodologies in the art itself. For example, a cDNA sequence encoding the polypeptide according to the invention can be used, operationally linked to suitable regulatory elements, such as transcriptional nucleic acid regulatory sequences or Petition 870190082172, dated 08 / 22 / 2019, page 34 / 248 28 / 120 translational.

[052] The term vector as used in this document includes reference to a vector for standard cloning work (cloning vector) as well as a more specialized type of vector, such as an expression vector (autosomal) and a cloning vector used for integration into the host cell chromosome (integration vector).

[053] Cloning vectors typically contain one or a small number of restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in a determinable manner without loss of essential biological function of the vector, as well as a marker gene that is suitable for use in identifying and selecting cells transformed with the cloning vector.

[054] The term expression vector refers to a linear or circular DNA molecule comprising a segment encoding a polypeptide of interest under the control of (i.e., operationally linked to) additional nucleic acid segments that provide that transcription. Such additional segments may include promoter and terminator sequences, and may optionally include one or more origins of replication, one or more selectable markers such as an enhancer, a polyadenylation signal, and the like. Expression vectors are generally derived from plasmid or viral DNA, or they may Petition 870190082172, dated 08 / 22 / 2019, page 35 / 248 29 / 120 contain elements of both. In particular, an expression vector comprises a nucleotide sequence that is operationally linked in the 5' to 3' direction: (a) a transcription and translation initiation region that is recognized by the host organism, (b) a coding sequence for a polypeptide of interest, and (c) a transcription and translation termination region that is recognized by the host organism. Plasmid refers to autonomously replicating extrachromosomal DNA that is not integrated into a microorganism's genome and is generally circular in nature.

[055] An integration vector refers to a linear or circular DNA molecule that can be incorporated into a microorganism's genome and provides the stable inheritance of a gene encoding a polypeptide of interest. The integration vector typically comprises one or more segments containing a genetic sequence encoding a polypeptide of interest under the control of (i.e., operationally linked to) additional nucleic acid segments that provide its transcription. Such additional segments may include promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the target cell genome, usually by the process of homologous recombination. Typically, the integration vector will be one that can be Petition 870190082172, dated 08 / 22 / 2019, page 36 / 248 30 / 120 transferred to the target cell, however, which has a replicon that is non-functional in that organism. Integration of the segment comprising the gene of interest can be selected if a suitable marker is included in that segment.

[056] As used in this document, the term operationally linked or operatively connected refers to a juxtaposition, where the components thus described are in a relationship that allows them to function in their intended manner. An operationally linked control sequence to another control sequence and / or to a coding sequence is linked in such a way that such transcription and / or expression of the coding sequence is achieved under conditions compatible with the control sequence. In general, operationally linked means that the nucleic acid sequences that are linked are contiguous and, where it is necessary to join two protein coding regions, contiguous and in the same reading frame.

[057] The term santalene synthase is used in this document for polypeptides that have catalytic activity in the formation of santalene and santalene-like terpenes such as α-santhalene, β-santhalene, trans-αbergamotene and epi-p-santhalene of farnesyl diphosphate, and other chemical moieties comprising such Petition 870190082172, dated 08 / 22 / 2019, page 37 / 248 31 / 120 polypeptide. Examples of such other chemical moieties include complexes of said polypeptide with one or more other polypeptides, fusion proteins comprising a santalene synthase polypeptide fused to a peptide or protein tag sequence, other complexes of said polypeptides (e.g., metalloprotein complexes), macromolecular compounds comprising said polypeptide and another organic chemical moiety, said polypeptide bound to a carrier material, etc. Santalene synthase may be supplied in its natural environment, i.e., within a cell in which it was produced, or in the medium in which it was excreted by the cell, producing the same. It may also be supplied separately from the source that produced the polypeptide and may be manipulated by attachment to a carrier, labeled with a chemical labeling moiety and the like.The term functional homologue of a sequence, or simply homologue, as used in this document, refers to a polypeptide comprising said specific sequence provided that one or more amino acids are substituted, deleted, added and / or inserted, and the polypeptide which has (qualitatively) the same enzymatic functionality for substrate conversion in the case of the term functional homologue being used for an enzyme, i.e., a homologue of the sequence with SEQ ID NO: 3 that has catalytic activity in the formation of santalene a. Petition 870190082172, dated 08 / 22 / 2019, p. 38 / 248 32 / 120 from farnesyl diphosphate. In the examples, a test is described that is suitable for verifying whether a polypeptide or a chemical portion comprising a polypeptide is a santalene synthase (santalene synthase activity test). Furthermore, the person skilled in the art recognizes that the equivalent nucleotide sequences encompassed by this invention may also be defined by the ability to hybridize, under low, moderate and / or rigorous conditions, with the nucleotide sequences that are within the literal scope of the present claims.

[058] A preferred homologue for SEQ ID NO: 3 according to the invention has a specificity with respect to the catalysis of santalene formation, expressed as the molar ratio of santalene to bergamotene (a known byproduct formed in known santalene synthase-catalyzed reactions) of at least 1:1, in particular, at least 1.5:1, more particularly, at least 2:1, more particularly, at least 2.4:1, most particularly, at least 2.8:1, when determined at pH 7, using the santalene synthase activity test described herein below in the examples (such as using a purified polypeptide). Said ratio may be infinite (1:0; i.e., no detectable amount of bergamotene formed), or up to 100:1, or up to 10:1 or up to 5:1. Sequence identity or similarity is defined in Petition 870190082172, dated 08 / 22 / 2019, page 39 / 248 33 / 120 in this document as a relationship between two or more polypeptide sequences or two or more nucleic acid sequences, as determined by comparing such sequences. Generally, sequence identities or similarities are compared along the entire length of the sequences, however, they can also be compared only for a portion of the sequences that align with each other. In the art, identity or similarity also means the degree of sequence relationship between polypeptide sequences or nucleic acid sequences, as the case may be, as determined by the correspondence between such sequences. Sequence identity, as used in this document, is the value as determined by the EMBOSS Pairwise Needle Alignment Algorithm, for example, on the European Bioinformatics Institute server (http: / / www.ebi.ac.uk / Tools / emboss / alignfl).For amino acid sequence alignment, the predefined parameters are: Matrix = Blosum62; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5. For nucleic acid sequence alignment, the predefined parameters are: Matrix = DNAfull; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5.

[059] Discrepancies between a santalene synthase according to SEQ ID NO: 3 or a nucleic acid according to SEQ ID NO: 1 or SEQ ID NO: 2 available and a Petition 870190082172, dated 08 / 22 / 2019, p. 40 / 248 34 / 120 functional homologues of said santalene synthase may, in particular, be the result of modifications made, for example, to enhance a property of santalene synthase or polynucleic acid (e.g., enhanced expression) by a biological technique known to those skilled in the art, such as molecular evolution or rational design, or by using a mutagenesis technique known in the art (random mutagenesis, site-directed mutagenesis, directed evolution, gene recombination, etc.). The amino acid sequence or nucleic acid coding sequence of santalene synthase may be altered compared to the sequences of SEQ ID NO: 3 and SEQ ID NO: 1 or SEQ ID NO: 2, respectively, as a result of one or more naturally occurring variations.Examples of such natural modifications / variations are differences in glycosylation (more broadly defined as post-translational modifications), differences due to alternative splicing, and single nucleic acid polymorphisms (SNPs). The nucleic acid can be modified so that it encodes a polypeptide that differs in at least one amino acid from the polypeptide of SEQ ID NO: 3, so that it encodes a polypeptide comprising one or more amino acid substitutions, deletions, and / or insertions compared with SEQ ID NO: 3, wherein such polypeptide still has activity. Petition 870190082172, dated 08 / 22 / 2019, page 41 / 248 35 / 120 santalene synthase. Furthermore, codon optimization or codon pair optimization can be used. For example, based on a method as described in WO 2008 / 000632, or as offered by commercial DNA synthesis companies such as DNA2.0, Geneart, and GenScript. Examples of a codon-optimized sequence consist of SEQ ID NO: 2. One or more sequences encoding suitable signal peptides that are not naturally associated with the polypeptides of the invention can be incorporated into (expression) vectors. For example, a DNA sequence for a signal peptide leader can be fused in the frame to a nucleic acid sequence of the invention, so that the polypeptide of the invention is initially translated as a fusion protein comprising the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptide will be targeted differently.A secretory signal peptide that is functional in the intended host cells, for example, enhances the extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptides to certain organelles, such as chloroplasts, mitochondria, and peroxisomes. The signal peptide can be cleaved from the polypeptide by transporting it to the intended organelle or from the cell. It is possible to provide a fusion of a sequence of... Petition 870190082172, dated 08 / 22 / 2019, page 42 / 248 36 / 120 additional peptides at the amino or carboxyl terminal end of a polypeptide according to SEQ ID NO: 3 or homolog thereof.

[060] As mentioned above, the invention further refers to a host cell comprising a vector according to the invention. The term host cell means a cell that contains a vector and supports the replication and / or expression of the vector.

[061] The nucleic acid of the invention is heterologous to the host cell of the invention. The host cell can be a prokaryotic cell, a eukaryotic cell, or a cell of a member of the Archaea. The host cell can be from any organism, in particular, any non-human organism. In particular, the host cell can be selected from bacterial cells, fungal cells, archaea, protists, plant cells (including algae), cells originating from an animal (in particular, isolated from said animal). The host cell can form part of a multicellular organism, other than a human organism or the organism from which the enzyme naturally originates (such as Cinnamomum camphora in the case of santalene synthase of SEQ ID NO: 3). In a specific embodiment, the host cells of the invention are in a cell culture originating from a multicellular organism, but isolated from it. Petition 870190082172, dated 08 / 22 / 2019, page 43 / 248 37 / 120

[062] In general, the host cell is a single cell that comprises genes to express the enzymes to catalyze the reaction steps of the mevalonate pathway or another metabolic pathway (such as the deoxyxylulose-5-phosphate (DXP) pathway) enabling the production of C5 prenyl diphosphates, isopentenyl diphosphate (IPP), and dimethylallyl diphosphate (DMAPP), which are the universal isoprenoid building blocks. To the best of our knowledge, unless specific genes have been knocked out, all known organisms comprise such a pathway. Eukaryotes in general are naturally capable of preparing IPP via the mevalonate pathway. This IPP is then isomerized to DMAPP by the action of the enzyme isopentenyl diphosphate isomerase (ldi). The DXP pathway, which provides TPP and DMAPP in a 5:1 ratio, is common for prokaryotes, although several prokaryotes are naturally capable of preparing IPP via the mevalonate pathway.These pathways are known in the art, and have been described, for example, by Withers & Keasling in Appl. Microbiol. Biotechnol. (2007) 73: 980 to 990. The genes in these pathways can each be independently homologous or heterologous to the cell.

[063] The host cells will also comprise, endogenously or from heterologous sources, one or more genes to express enzymes with prenyl activity. Petition 870190082172, dated 08 / 22 / 2019, page 44 / 248 38 / 120 transferase catalyzes the head-to-tail condensation of C5 prenyl diphosphates, producing longer prenyl diphosphates. The universal sesquiterpene precursor farnesyl diphosphate (FPP), for example, is formed by the action of these enzymes through the successive head-to-tail addition of 2 IPP molecules to 1 DMAPP molecule.

[064] In one embodiment, the host cell is a bacterium. The bacterium may be gram-positive or gram-negative. Gram-positive bacteria may be selected from the genera Bacillus and Lactobacillus, in particular, from the species Bacillus subtilis and Lactobacillus casei.

[065] In a preferred embodiment, the bacterium is selected from the group of Gram-negative bacteria, in particular, from the group of Rhodobacter, Paracoccus and Escherichia, more particularly, from the group of Rhodobacter capsulatus, Rhodobacter sphoeroides, Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens and Escherichia coli. Rhodobacter sphoeroides is an example of an organism that naturally contains all the genes necessary to express enzymes that catalyze the various reaction interruptions in the DXP pathway, enabling the intracellular production of IPP and DMAPP. In a preferred embodiment, the host cell is a fungal cell, in Petition 870190082172, dated 08 / 22 / 2019, p. 45 / 248 39 / 120 particular, a fungal cell selected from the group of Aspergillus, Blakeslea, Penicillium, Phaffia (Xanthophyllomyces). Pichia, Saccharomyces and Yarrowia, more particularly, from the group of Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Blakeslea trispora, Penicillium chrysogenum, Phaffia rhodozyma (Xanthophyllomyces dendrorhous), Pichia pastoris, Saccharomyces cerevisiae and Yarrowia lipolytica. It is also possible to express the nucleic acids of the invention in cells derived from higher eukaryotic organisms, such as plant cells and animal cells, such as insect cells or mouse, rat or human cells. Said cells can be maintained in a cell or tissue culture and used for in vitro production of santalene synthase.

[066] A multicellular organism comprising the host cells according to the invention, in particular, may be selected from the group of multicellular mushroom plants (Basidiomycetes).

[067] Thus, in a specific embodiment, the invention relates to a transgenic plant or plant cell or tissue culture comprising transgenic plant cells, wherein said plant or culture comprises plant host cells according to the invention. The transgenic plant or cell culture Petition 870190082172, dated 08 / 22 / 2019, page 46 / 248 40 / 120 transgenic plants can, in particular, be selected from Nicotiana spp., Solarium spp., Cichomm infybus, Lactuca saliva, Mentha spp., Artemisia annua, tuber-forming plants such as Helianthus tuberosus, cassava and Beta vulgaris, oilseed crops such as Brassica spp., Elaeis spp. (oil palm), Helianthus annuus, Glycine max and

[068] Arachis hypogaea, liquid culture plants, such as duckweed Lemna spp., BY2 cells of tobacco and Physcomitrella patens, trees, such as pine and poplar, respectively, a cell culture or a tissue culture of any of said plants. In a specific embodiment, the tissue culture is a capillary root culture.

[069] In a further specific embodiment, the invention relates to a transgenic mushroom or culture comprising transgenic mushroom cells. The transgenic mushroom or culture comprising transgenic host cells, in particular, may be selected from the Schizophyllum, Agaricus and Pleurotus groups, more particularly from Schizophyllum commune, the common mushroom (Agaricus sporus), the oyster mushroom (Pleurotus ostreotus and Pleurotus sapidus), respectively, a culture comprising cells of any of the so-called mushrooms. Petition 870190082172, dated 08 / 22 / 2019, p. 47 / 248 41 / 120

[070] A host cell according to the invention can be produced based on predefined genetic and molecular biology techniques that are generally known in the art, for example, as described in Sambrook, J. and Russell, DW Molecular Cloning: A Laboratory Manual 3rd edition, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and FM Ausubel et al, editions, Current Protocols in Molecular Biology, John Wiley and Sons, Inc., New York (1987), and subsequent supplements thereto.

[071] Methods for transforming Basidiomycetes are known from, for example, Alves et al (Appl. Environ. Microbiol. (2004) 70: 6379 to 6384), Godio et al (Curr. Genet, (2004) 46: 287 to 294), Schuurs et al (Genetics (1997) 147: 589 to 596) and the WO document 06 / 096050. To achieve the expression of a suitable santalene synthase gene in basidiomycetes, its complete open reading frame is typically cloned into an expression vector suitable for basidiomycete transformation. The expression vector preferably also comprises nucleic acid sequences that regulate transcription initiation and termination. It is also preferable to incorporate at least one selectable marker gene to allow for transformant selection. The expression of a santalene synthase can be achieved Petition 870190082172, dated 08 / 22 / 2019, page 48 / 248 42 / 120 with the use of a basidiomycete promoter, for example, a constitutive promoter or an inducible promoter. An example of a strong constitutive promoter is the glyceraldehyde-3-phosphate dehydrogenase (gpdA) promoter. This promoter is preferred for constitutive expression when recombinant DNA material is expressed in a basidiomycete host. Other examples are the phosphoglycerate kinase (pgk) promoter, the pyruvate kinase (pki) promoter, TPI, the triose phosphate isomerase (tpi) promoter, the APC synthase g subunit (oliC) promoter, the sc3 promoter, and the acetamidase (amdS) promoter from a basidiomycete (WO 96 / 41882).

[072] If necessary, the primary nucleotide sequence of the santalene synthase gene can be adapted to use the basidiomycete host codon.

[073] Furthermore, the expression can be directed especially to the mycelium (monokaryotes) or to the fruiting bodies (dicakaryotes). In the latter case, the Fbhl promoter from Pleurotis is especially useful (Penas, MM et al, Mycologia (2004) 96: 75 to 82).

[074] The methodologies for constructing plant transformation constructs are described in the technique. Overexpression can be achieved by inserting one or more extra copies of the selected gene. This is not an unknown fact for plants or their progeny, Petition 870190082172, dated 08 / 22 / 2019, page 49 / 248 43 / 120 originally transformed with one or more extra copies of a nucleotide sequence to exhibit overexpression.

[075] Obtaining sufficient levels of transgenic expression in suitable plant tissues is an important aspect in the production of genetically modified crops. The expression of heterologous DNA sequences in a plant host is dependent on the presence of an operationally linked promoter, which is functional within the plant host. The choice of promoter sequence will determine when and where within the organism the heterologous DNA sequence is expressed. Although many dicotyledonous promoters have proven operational in monocotyledons and vice versa, ideally dicotyledonous promoters are selected for expression in dicotyledons, and monocotyledonous promoters for expression in monocotyledons. However, there is no restriction on the provenance of selected promoters; it is sufficient that they are operational in driving the expression of nucleotide sequences in the desired cell or tissue.In some cases, expression in multiple tissues is desirable, and constitutive promoters such as the 35S promoter series can be used for this purpose. However, in some embodiments of the present invention, it is preferable that expression in transgenic plants be specific with respect to... Petition 870190082172, dated 08 / 22 / 2019, page 50 / 248 44 / 120 leaf, more preferably, gene expression occurs in leaf plastids. The promoter of the isoprene synthase gene from Populus alba (PalspS) (Sasaki et al, FEBS Letters (2005) 579: 2514 to 2518) appears to drive plastid-specific expression. Therefore, this promoter is a very suitable promoter for use in an expression vector of the present invention.

[076] Other leaf-specific promoters suitable for this purpose include the rbcS (Rubisco) promoter (e.g., from coffee, see document WO 02 / 092822); from Brassica, see document US 7,115,733; from soybean, see Dhanker, O., et al, Nature Diotechnol. (2002) 20: 1140 to 1145), the cy-FBPase promoter (see document US 6,229,067), the light oil palm chlorophyll a / b binding protein promoter sequence (see document US 2006 / 0288409), the STP3 promoter of Arabidopsis thaliana (see Bilttner, M. et al., Plant Cell & Environ. (2001) 23: 175-184), the PAL2 gene promoter of beans (see Sablowski, R.W. et al., Proc. Natl. Acad. Sci. USA (1995) 92: 6901-6905), ST-LS1 promoter enhancer sequences of potatoes (see Stockhaus, J. et al., Proc. Natl. Acad. Sci. USA (1985) 84: 7,943 to 7,947), the wheat CAB1 promoter (see Gotor, C. et al, Plant J. (1993) 3: 509 to 518), the Petition 870190082172, dated 08 / 22 / 2019, page 51 / 248 45 / 120 specific to the stomata of the potato ADP-glucose-phosphorylase gene (see document in US 5,538,879), the LPSE1 element of the rice P(D540) gene (see CN 2007 / 10051443), and the stomata-specific promoter, pGC2(Atlg22690) from Arabidopsis thaliana (see Yang, Y. et al, Plant Methods (2008) 4: 6).

[077] Plant species, for example, can be transformed by DNA-mediated transformation of plant cell protoplasts and subsequent regeneration of the plant from the transformed protoplasts according to procedures well known in the art.

[078] Additional examples of methods for transforming plant cells include microinjection (Crossway et al., Mol. Gen. Genet. (1986) 202: 179 to 185), electroporation (Riggs, CD. and Bates, GW., Proc. Natl. Acad. Sci. USA (1986), 83: 5602 to 5606), Agrobacterium-mediated transformation (Hinchee et al., Bio / Technol. (1988) 6: 915 to 922), direct gene transfer (Paszkowski, J. et al, EMBO J. (1984) 3: 2,717 to 2,722) and ballistic particle acceleration using devices available from Agracetus, Inc., Madison, Wisconsin and BioRad, Hercules, California (see, for example, Sanford et al. US Patent No. 4,945,050 and European Patent Application). EP 0 332 581).

[079] It is also possible to use the method of Petition 870190082172, dated 08 / 22 / 2019, p. 52 / 248 46 / 120 transformation of protoplast to maize (European Patent Application EP 0 292 435, Patent No. 5,350,689).

[080] It is particularly preferred to use binary-type vectors of the Ti and Ri plasmids of Agrobacterium spp. Vectors derived from Ti transform a wide variety of higher plants, including monocotyledonous and dicotyledonous plants such as soybean, cotton, rapeseed, tobacco and rice (Pacciotti et al, Bio / technol. (1985) 3: 241; Byrne MC et al. Plant cell Tissue and Organ Culture (1987) 8: 3 to 15; Sukhapinda, K. et al, Plant Mol. Biol. (1987) 8: 209 to 217; Hiei, Y. et al, The Plant J. (1994) 6: 271 to 282). The use of T-DNA to transform plant cells has received extensive study and is widely described (e.g., EP-A 120 516). For introduction into plants, the chimeric genes of the invention can be inserted into binary vectors, as described in the examples.

[081] Other transformation methods are available to subject matter experts, such as direct uptake of foreign DNA constructs (see EP-A 295 959), electroporation techniques (Fromm, ME et al, Nature (1986), 319: 791 to 793) or high-speed ballistic bombardment with metal particles coated with nucleic acid constructs (e.g., the paper in (US 4,945,050). Once transformed, the cells can Petition 870190082172, dated 08 / 22 / 2019, p. 53 / 248 47 / 120 to be regenerated by experts in the field. Methods for transforming foreign genes in commercially important crops, such as rapeseed (De Block, M. et al, Plant Physiol. (1989) 91: 694 to 701), sunflower (Everett, NP et al, Bio / Technology (1987) 5: 1,201 to 1,204), soybean (EP-A 301 749), rice (Hiei, Y. et al, The Plant J. (1994) 6: 271 to 282) and corn (Fromm et al, 1990, Bio / Technology 8: 833 to 839).

[082] Experts in the field will note that the choice of method may depend on the type of plant, i.e., monocotyledon or dicotyledon.

[083] In another embodiment, the vector, as described in this document, can be directly transformed into the plastid genome. Plastid transformation technology is extensively described, for example, in documents no. US 5,451,513, no. US 5,545,817, no. US 5,545,818 and WO 95 / 16783. The basic technique for chloroplast transformation involves introducing regions of cloned plastid DNA flanking a selectable marker along with the gene of interest in a suitable target tissue, for example, using biolistics or protoplast transformation (e.g., calcium chloride or PEG-mediated transformation).

[084] Agrobacterium tumefadens cells containing a vector according to the present invention, wherein Petition 870190082172, dated 08 / 22 / 2019, page 54 / 248 48 / 120 The vector comprises a Ti plasmid, useful in methods for producing transformed plants. Plant cells are infected with Agrobacterium tumefadens, as described above, to produce a transformed plant cell, and then a plant is regenerated from the transformed plant cell. Numerous Agrobacterium vector systems useful in carrying out the present invention are known. These typically carry at least one T-DNA frontier sequence and include vectors such as pBIN19 (Bevan, Nucl. Acids Res. (1984) 12: 8,711 to 8,720).

[085] Methods using a form of direct gene transfer or Agrobacterium-mediated transfer are generally, but not necessarily, performed with a selectable marker, which can provide resistance to an antibiotic (e.g., kanamycin, hygromycin, or methotrexate) or a herbicide (e.g., phosphinothricin). The choice of selectable marker for plant transformation is not, however, critical to the invention.

[086] General methods of cultivating plant tissues are provided, for example, by Maki, KY et al, Plant Physiol. (1993) 15: 473 to 497; and by Phillips, RI et al. In: Sprague GF, Dudley JW, editions. Corn and corn improvement. 3rd edition. Madison (1988) 345 to 387.

[087] After the transformation, the plant cells Petition 870190082172, dated 08 / 22 / 2019, page 55 / 248 49 / 120 transgenic cells are placed in a suitable selective medium for the selection of transgenic cells, which are then transformed into calluses. Seedlings are developed from calluses, and plantlets generated from the seedling are grown in rooting medium. The particular marker used will allow the selection of transformed cells in comparison with cells devoid of the introduced DNA.

[088] To confirm the presence of transgenes in transgenic cells and plants, a variety of assays can be performed. Such assays include, for example, well-known molecular biological assays, such as Southern and Northern blotting methods, in situ hybridization and nucleic acid-based amplification such as PGR or RT-PCR assays, and biochemical assays, such as detection of the presence of a protein product, for example, by immunological means (ELISAs and Western blots) or by enzymatic function. The presence of enzymatically active santalene synthase can be established by chemical analysis of the plant's volatile products (santalene).

[089] A santalene synthase according to the invention can be used for the industrial production of santalene, wherein such santalene can be used in itself as a flavour or flavouring, for example, in a food product, or Petition 870190082172, dated 08 / 22 / 2019, page 56 / 248 50 / 120 as a fragrance, for example, in a household product, or as an intermediate for the production of another isoprenoid, for example, santalol.

[090] A method for producing santalene according to the invention comprises preparing santalene in the presence of santalene synthase. In principle, such a method can be based on any technique for employing an enzyme in the preparation of a compound of interest.

[091] The method may be a method in which FPP or any of its precursors (such as farnesol, IPP, isopentenyl phosphate, 3-methylbut-3-en-1-ol and even mevalonate) is fed as a substrate to cells comprising santalene synthase. Alternatively, the method may also be a method in which use is made of a living organism comprising an enzyme system capable of forming FPP from a suitable carbon source, thus establishing a complete fermentative pathway for santalene. It should be noted that the term fermentative is used in this document in a broad sense for processes in which use is made of a culture of an organism to synthesize a compound from a suitable feedstock (e.g., a carbohydrate, an amino acid source, a fatty acid source). Thus, fermentative processes, as intended in this document, are not limited to anaerobic conditions, and extend to Petition 870190082172, dated 08 / 22 / 2019, page 57 / 248 51 / 120 processes under aerobic conditions. Suitable raw materials are generally known for specific species of (microorganisms).

[092] Furthermore, the use of santalene synthase isolated from the cell in which it was produced can be made, for example, in a reaction system in which the substrate (FPP) and santalene synthase are brought into contact under suitable conditions (pH, solvent, temperature), wherein such conditions may be based on the prior art mentioned in this document and the present disclosure, optionally in combination with some routine test. The santalene synthase can, for example, be solubilized in an aqueous medium in which the FPP is still present, or the santalene synthase can be immobilized on a support material in a manner known in the art and then brought into contact with a liquid comprising the FPP. Since the enzyme has a high activity and / or selectivity with respect to the catalysis of FPP in santalene, the present invention is also advantageous for such an in vitro method, not only under acidic conditions, but also in the case of approximately neutral or alkaline pH.The appropriate conditions may be based on known methodology for known santalene synthases, for example, those mentioned in the literature cited in this document, the information disclosed in this document, knowledge. Petition 870190082172, dated 08 / 22 / 2019, page 58 / 248 52 / 120 general common and optionally some routine experimentation.

[093] In a particularly advantageous method of the invention, santalene is fermentatively prepared, that is, by cultivating cells expressing santalene synthase in a culture medium. The actual reaction catalyzed by santalene synthase can occur intracellularly or – if santalene synthase is excreted into the culture medium – extracellularly in the culture medium.

[094] The cells used in a method for preparing santalene according to the invention may, in particular, be host cells according to the invention. If desired, these host cells may be genetically modified to supply FPP to santalene synthase in increased amounts. This, for example, can be accomplished by intensifying the carbon flux relative to FPP, which, in itself, can be accomplished in different ways. In host cells with an endogenous DXP pathway (such as E. coli and R. sphaeroides), the deregulation of enzyme expression in this pathway can have a clear positive effect on isoprenoid formation. Overexpression of dxs encoding 1-deoxy-D-xylulose-5-phosphate synthase (DXP-synthases), the first enzyme in the DXP pathway and thus a major target for metabolic genetic modification, resulted in increased biosynthesis of several Petition 870190082172, dated 08 / 22 / 2019, page 59 / 248 53 / 120 isoprenoids (e.g., Matthews and Wurtzel, Appl. Microbiol. Biotechnol. (2000) 53: 396 to 400; Huang et al, Bioorg. Med. Chem. (2001) 9: 2.237 to 2.242; Harker and Lett, 1999). 448: 115 a 119; Metab Eng (2000) 2: 328 a 338; (2006) 8: 79 to 90). Furthermore, overexpression of dxr encoding for DXP isomeroreductase (also known as 1-deoxy-D-xylulose-5-phosphate reductase), the enzyme that catalyzes the second and dedicated step in the DXP pathway, can lead to increased isoprenoid production (Albrecht et al, Biotechnol. Lett, (1999) 21: 791 to 795), where such an effect can be further increased by co-expression of matrices at the same time (Kim & Keaeling, Biotechnol Bioeng (2001) 72: 408 to 415). A positive effect on isoprenoid biosynthesis was additionally obtained by overexpression of isopentenyl diphosphate isomerase (IPP isomerase, Idi), the enzyme that catalyzes the interconversion of IPP into dimethylallyl diphosphate, DMAPP (e.g., Kajiwara et al. Biochem. J. (1997) 324: 421-426); Misawa and Shimada, J. Biotech. (1998) 59: 169-181; and Yuan et al. Mctab. Eng.(2006) 8: 79 to 90) and the enzymes MEP cytidylyltransferase (also known as 4-diphosphocytidyl-2-C-methyl-D-erythritol synthase, IspD) and 2C-methyl-Derythritol 2,4-cyclodiphosphate synthase (IspF), which are transcribed as an ispDF operon in E. coli (Yuan et al. Petition 870190082172, dated 08 / 22 / 2019, p. 60 / 248 54 / 120 Metab. Eng. (2006) 8: 79 to 90).

[095] An alternative and more effective approach for genetically modified strains with an endogenous DXP pathway for high-level isoprenoid production is the introduction of a heterologous mevalonate pathway. Co-expression in E. coli of the Saccharomyces cerevisiae mevalonate pathway with an amorphous 4,11-diene synthetic synthase gene resulted in the formation of the sesquiterpene amorphadiene at titers of more than 110 mg / l when the recombinant E. coli strain was cultured in an LB+ glycerol medium (Martin et al, Nat. Biotechnol. (2003) 21: 796 a 802). This E. coli strain was subsequently enhanced by the introduction of extra copies of the fHMG1 gene encoding the C-terminal catalytic domain of the yeast enzyme 3-hydroxy-3-methylglutaryl-coenzyme A (HMG-CoA) reductase. By increasing the formation and thus the activity of this enzyme, the intracellular level of the toxic mevalonate pathway intermediate HMG-CoA was reduced, thus overcoming growth inhibition and leading to increased mevalonate production (Pitera et al. Metab. Eng. (2007) 9: 193-207). Further enhancement of flux through the heterologous mevalonate pathway was achieved by codon optimization of the first three genes of this pathway in combination with the replacement of the wild-type lac promoter with the twice-dose lacUV5 promoter. Petition 870190082172, dated 08 / 22 / 2019, page 61 / 248 55 / 120 stronger (Anthony et al, Met. Eng. (2009) 11: 13 to 19). Amorphadiene production could be further increased by replacing the yeast genes for HMG-CoA synthase and HMG-CoA reductase with equivalent genes from the gram-positive bacterium Staphylococcus aureus. In combination with an optimized fermentation protocol, cultivation of this innovative genetically modified E. coli strain produced an amorphadiene titer of 27.4 g / l (Tsuruta et al. PloS ONE (2009) 4(2): e4489. doi: 10.1371 / journal.pone.0004489). Similarly, a genetically modified E. coli strain with the mevalonate pathway of Streptococcus pneumoniae in combination with the decaprenyl diphosphate synthase gene from Agrobacterium tumefaciens (ddsA) produced coenzyme Qio (CoQio) at more than 2,400 μg / g of cell dry weight (Zahiri et al. Met. Eng. (2006) 8: 406 to 416).Increased CoQio production was also achieved by genetically modifying a strain of Rhodobacter sphaeroides with the mevalonate pathway of Paracoccus zeaxanthinifaciens in its native form (WO 2005 / 005650) and a mutated form (WO 2006 / 018211).

[096] Furthermore, host cells with an endogenous SEM trajectory (such as S. cerevisiae) have been the subject of multiple genetic modification studies to obtain isoprenoid hyperproducing strains. The introduction into S. cerevisiae of the E. coli-derived trajectory. Petition 870190082172, dated 08 / 22 / 2019, page 62 / 248 56 / 120 heterologous DXP combined with the gene encoding Citrus santalene synthase resulted in a strain that accumulates approximately 10 times more santalene compared to the strain expressing only santalene synthase (WO 2007 / 093962). Most improvements in the industrially important yeasts Candida ulilis and S. cerevisiae, however, have focused on genetic modification of the homologous SEM pathway. In particular, overexpression of the HMG-CoA reductase enzyme, believed to be the main regulatory enzyme in the DXP pathway, in its full-length or truncated version, appeared to be an effective method for increasing isoprenoid production. This stimulating effect of overexpression of N-terminal truncated HMG-CoA reductase, for example, was observed in the case of lycopene production in C. utilis (Shimada et al. Appl. Env. Microbiol. (1998) 64: 2,676 to 2,680) and epicedrol production in S. cerevisiae (Jackson et al. Org. Lett. (2003) 5: 1629 a 1.632). In the latter case, the production of this sesquiterpene can be further intensified by the introduction of upc2-1, an allele that elicits an increase in metabolic flux in sterol biosynthesis. Another method to increase flux through the MEV pathway is the use of a mevalonate kinase variant that is less sensitive to feedback inhibition by FPP and other isoprenoid precursors. Petition 870190082172, dated 08 / 22 / 2019, page 63 / 248 57 / 120 Document WO 2006 / 063752, for example, shows that Paracoccus zeaxanthinifaciens R114, a bacterium with an endogenous SEM trajectory, after the introduction of the S. cerevisiae mevalonate kinase mutant N66K / I152M and the ddsA gene from P. zeaxanthinifaciens ATCC 21588 produces significantly more coenzyme Qio than the corresponding strain of P. zeaxanthinifaciens expressing the wild-type S. cerevisiae mevalonate kinase. Similar positive results in CoQio production with P. zeaxanthinifaciens R114 were also obtained with the feedback-resistant variant K93E of the P. zeaxanthinifaciens mevalonate kinase (WO 2004 / 111214).

[097] A second approach to increased FPP amounts is based on reducing or eliminating enzymatic side activities in FPP. In yeast, the ERG9 gene encodes the enzyme farnesyl diphosphate, farnesyltransferase (squalene synthase), which catalyzes the condensation of two chemical moieties of farnesyl diphosphate to form squalene. Since this is the first disruption after FPP in sterol biosynthesis and thus regulates the flow of isoprene units in the sterol pathway, ERG9 is a frequent target in yeast metabolic genetic modification for increased sesquiterpene and carotenoid production. ERG9 disruption in combination with overexpression of reductase Petition 870190082172, dated 08 / 22 / 2019, page 64 / 248 58 / 120 tHMG-CoA in the yeast C. utilis led to increased lycopene production (Shimada et al. Appl. Env. Microbiol. (1998) 64: 2676-2680). A similar combination of tHMG-CoA reductase overexpression and ERG9 downregulation using a methionine repressible promoter increased the sesquiterpene amorphadiene production in yeast by approximately 10 times compared to the yeast strain expressing only the amorphadiene synthase gene (Ro et al. Nature (2006) 440: 940-943; Lenihan et al. Biotechnol. Prog. (2008) 24: 1026-1032). Since ergosterol is vital for yeast development and yeast cells cannot assimilate externally fed ergosterol during aerobic development, downregulation / knockout of ERG9 is frequently combined with mutations that equip the yeast strain with effective aerobic uptake of ergosterol from the culture medium. Examples include the sue allele (Takahishi et al. Biotechnol.Bioeng. (2007) 97: 170 to 181) and the upc2-1 allele (Jackson et al. Org. Lett. (2003) 5: 1629 to 1632). Takahashi et al. (Biotechnol. Bioeng. (2007) 97: 170 to 181) also investigated the effect of limiting endogenous phosphatase activity by knockout of the dppl phosphatase gene in yeast. Although this knockout clearly limited FPP dephosphorylation, reflected by much less farnesol accumulation, it did not enhance the production of... Petition 870190082172, dated 08 / 22 / 2019, page 65 / 248 59 / 120 sesquiterpene in addition to that of the combined erg9 / sue mutations under the applied developmental conditions.

[098] The reaction conditions for fermentatively preparing santalene can be chosen depending on the known conditions for the host cell species used (e.g., Rhodobacter capsulatus, Rhodobacter sphaeroides, Paracoccus zeaxanthinifaciens, Escherichia coli, Aspergillus nidulans, Aspergillus niger, Aspergillus oryzae, Saccharomyces cerevisiae, Penicillium chrysogenuni, Phaffia rhodozyma and Pichia pastoris), the information disclosed herein, common general knowledge and optionally some routine experimentation. In principle, the pH of the reaction medium (culture medium) used in a method according to the invention can be chosen within wide limits, provided that the santalene synthase (in the host cell) is active and exhibits a desired specificity under the pH conditions.If the method involves using cells to express santalene synthase, the pH is selected so that the cells are able to perform their intended function or functions. The pH, in particular, can be chosen within the range of four pH units below neutral pH and two pH units above neutral pH, that is, between pH 3 and pH 9 in the case of an essentially aqueous system at 25 °C. Good results, for example, have been achieved in a medium. Petition 870190082172, dated 08 / 22 / 2019, page 66 / 248 60 / 120 aqueous reaction having a pH in the range of 6.8 to 7.5.

[099] A system is considered aqueous if water is the only solvent or the predominant solvent (> 50% by weight, in particular > 90% by weight, based on total liquids), where, for example, a minimal amount of alcohol or another solvent (< 50% by weight, in particular < 10% by weight, based on total liquids) can be dissolved (for example, as a carbon source, in the case of a total fermentation approach) in such a concentration that microorganisms that are present remain active.

[100] In particular, if a yeast and / or fungus is used, acidic conditions may be preferable, in particular, the pH may be in the range of pH 3 to pH 8, based on an essentially aqueous system at 25 °C. If desired, the pH may be adjusted using an acid and / or a base or buffered with a suitable combination of an acid and a base.

[101] Anaerobic conditions, in this document, are defined as conditions without any oxygen or in which substantially no oxygen is consumed by the cultured cells, in particular, a microorganism, and generally correspond to an oxygen consumption of less than 5 mmol / lh, preferably less than 2.5 mmol / lh, or more preferably less than 1 mmol / lh. Petition 870190082172, dated 08 / 22 / 2019, page 67 / 248 61 / 120 Aerobic conditions are those in which a sufficient level of oxygen for unrestricted development is dissolved in the medium, capable of supporting an oxygen consumption rate of at least 10 mmol / l, more preferably more than 20 mmol / l, even more preferably more than 50 mmol / l, and most preferably more than 100 mmol / l. Oxygen-limited conditions are defined as conditions in which oxygen consumption is limited by the transfer of oxygen from the gas to the liquid. The lower limit for oxygen-limited conditions is determined by the upper limit for anaerobic conditions, i.e., generally at least 1 mmol / l, and in particular at least 2.5 mmol / l or at least 5 mmol / l. The upper limit for oxygen-limited conditions is determined by the lower limit for aerobic conditions, i.e., less than 100 mmol / l, less than 50 mmol / l, less than 20 mmol / l, or less than, up to 10 mmol / l

[102] The possibility of the conditions being aerobic, anaerobic or oxygen-limited is dependent on the conditions under which the method is performed, in particular, on the quantity and composition of the inlet gas stream, the actual mass transfer / mixing properties of the equipment used, the type of microorganism used and the microorganism density. In principle, the temperature used is not critical, provided that the synthase of Petition 870190082172, dated 08 / 22 / 2019, page 68 / 248 62 / 120 santalene (in cells), show substantial activity. In general, the temperature can be at least 0 °C, in particular at least 15 °C, more particularly at least 20 °C. A maximum desired temperature depends on the santalene synthase and the cells, in the case of a method where cells are used to express santalene synthase. The temperature is 70 °C or less, preferably 50 °C or less, more preferably 40 °C or less, in particular 35 °C or less.

[103] In the case of a fermentation process, incubation conditions can be chosen within wide limits, provided that the cells show sufficient activity and / or development. This includes aerobic, oxygen-limited and anaerobic conditions.

[104] In particular, if the catalytic reaction by which santalene is formed is carried out outside a host cell, a reaction medium comprising an organic solvent may be used at a high concentration (e.g., more than 50%, or more than 90% by weight, on a total liquid basis), in which case the santalene synthase that is used retains sufficient activity and specificity in such a medium.

[105] If desired, santalene produced by a method according to the invention, or an additional compound into which santalene has been converted after its preparation (such as Petition 870190082172, dated 08 / 22 / 2019, page 69 / 248 63 / 120 santalol), is recovered from the reaction medium in which it was produced. A suitable method is liquid-liquid extraction with an extraction liquid that is immiscible with the reaction medium.

[106] In particular, extraction with a liquid organic solvent, such as a liquid hydrocarbon, is suitable (for extraction from an aqueous reaction medium). From initial results, it is evident that this method is also suitable for extracting santalene (or additional product) from a reaction medium comprising cells according to the invention used for its production, without needing to lyse the cells for santalene (or additional product) recovery. In particular, the organic solvent can be selected from liquid alkanes, liquid long-chain alcohols (alcohols having at least 12 carbon atoms), and liquid esters of long-chain fatty acids (acids having at least 12 carbon atoms). Suitable liquid alkanes in particular include C6-C16 alkanes, such as hexane, octane, decane, dodecane, isododecane, and hexadecane.Suitable long-chain aliphatic alcohols, in particular, include C12-C18 aliphatic alcohols, such as oleyl alcohol and palmitoleyl alcohol. Suitable long-chain fatty acid esters, in particular, include C1-C4 alcohol esters of C12-C18 fatty acids, such as isopropyl myristate and ethyl oleate. Petition 870190082172, dated 08 / 22 / 2019, p. 70 / 248 64 / 120

[107] In an advantageous embodiment, santalene (or an additional product) is produced in a reactor comprising a first liquid phase (the reaction phase), said first liquid phase containing cells according to the invention in which the cells produce santalene (or an additional product), and a second liquid phase (an organic phase that remains essentially in separate phases with the first phase when brought into contact), said second liquid phase being the extraction phase, for which the product formed has a superior affinity. This method is advantageous in that it allows for in situ product recovery. Furthermore, it helps to prevent or at least reduce the potential toxic effects of santalene (or an additional product) on the cells, since, due to the presence of the second phase, the concentration of santalene (or an additional product) in the reaction phase can be kept relatively low throughout the process.Finally, there is strong evidence that the extraction phase contributes to extracting santalene (or additional product) from the reaction phase.

[108] In a preferred embodiment of the invention, the extraction phase forms a layer over the reaction phase or is mixed with the reaction phase to form a dispersion of the reaction phase in the extraction phase or a dispersion of the extraction phase in the reaction phase. Thus, the phase of Petition 870190082172, dated 08 / 22 / 2019, page 71 / 248 65 / 120 extraction not only extracts the product from the reaction phase but also helps to reduce or completely avoid losses of the product formed from the reactor through the effluent gas, which can occur if santalene is produced in the (aqueous) reaction phase or extracted in the (aqueous) reaction phase. Santalene is unsatisfactorily soluble in water and therefore readily volatilizes from water. It is contemplated that santalene solvated in the organic phase (as a layer or dispersion) is at least substantially prevented from volatilizing.

[109] Liquids suitable for use as an extraction phase combine a density lower than the reaction phase with good biocompatibility (no interference with the viability of living cells), low volatility, and close to absolute immiscibility with the aqueous reaction phase.

[110] Examples of liquids suitable for this application are liquid alkanes such as decane, dodecane, isododecane, tetradecane and hexadecane or long-chain aliphatic alcohols such as oleyl alcohol and palmitoleyl alcohol or long-chain fatty acid esters such as isopropyl myristate and ethyl oleate (see, for example, Asadollahi et al (Biotechnol. Bioeng. (2008) 99: 666 to 677), Newman et al. 95: 684 to 691) and WO 2009 / 042070). Petition 870190082172, dated 08 / 22 / 2019, p. 72 / 248 66 / 120

[111] The santalene produced according to the invention can be used in this way, for example, for use as a flavor or fragrance, or as an insect repellent, or it can be used as a starting material for another compound, in particular, another flavor or fragrance. In particular, santalene can be converted into santalol. The conversion of santalene to santalol can be carried out intracellularly or extracellularly. If this preparation is carried out within a cell, santalol is generally isolated from the host cell after its production. The invention further relates to a method for preparing santalol, preferably β-santalol, comprising converting FPP to santalene, preferably β-santalene, in the presence of a santalene synthase according to the invention, which further comprises converting santalene to santalol, preferably β-santalol.Preferably, santalene is prepared in a host cell, a plant or plant culture or a mushroom or mushroom culture, which expresses said santalene synthase according to the invention. In a preferred embodiment, a method for preparing santalol, preferably βsantalol, according to the invention is provided, further comprising isolating santalol.

[112] In general, the appropriate methods for preparing santalol from santalene can be divided into: i) methods Petition 870190082172, dated 08 / 22 / 2019, p. 73 / 248 67 / 120 purely chemical methods as described in Willis et al. (1985) in example 11, ii) biocatalytic methods (e.g., those using P450 monooxygenases) as exemplified by Daviet et al. (2015) which can also be carried out as a bioconversion (i.e., methods applying whole living cells) and iv. total fermentation and iii) autocatalytic oxidation of santalene as exemplified by Ngo & Brown (2000). In a specific embodiment, the conversion comprises a regiospecific hydroxylation of santalene to form santalol. It is contemplated that one or more genes encoding an enzyme or plurality of enzymes to catalyze the conversion of santalene to santalol can be incorporated into a host cell according to the invention. These enzymes can, for example, be selected from enzymes of Chlorella or Botiyosphaeria, or Premnaspirodene oxidase from Hyoscyamus muticus, or the P450cam or P450BM-3 mutants mentioned above in this document.As indicated above, the invention relates to an antibody that has binding affinity to a santalene synthase according to the invention. The term antibody includes reference to the antigen-binding form of antibodies (e.g., Fab, F(ab)2). The term antibody frequently refers to a polypeptide substantially encoded by an immunoglobulin gene or immunoglobulin genes or fragments thereof, which... Petition 870190082172, dated 08 / 22 / 2019, p. 74 / 248 68 / 120 binds to and specifically recognizes an analyte (antigen). However, although various antibody fragments can be defined in terms of the digestion of an intact antibody, a specialist will observe that such fragments can be synthesized de novo chemically or using recombinant DNA methodology. Thus, the term antibody, as used in this document, also includes antibody fragments such as single-chain Fv, chimeric antibodies (i.e., comprising constant and variable regions from different species), humanized antibodies (i.e., comprising a complementarity-determining region (CDR) from a non-human source), and heteroconjugated antibodies (e.g., bispecific antibodies).

[113] Antibodies or fragments thereof may be produced by any method known in the art for antibody synthesis, in particular by chemical synthesis or preferably by recombinant expression techniques.

[114] Polyclonal antibodies to santalene synthase can be produced by several well-known procedures in the art. For example, a heterologous santalene synthase can be administered to various host animals including, but not limited to, rabbits, mice, rats, etc. to induce the production of sera containing polyclonal antibodies specific to santalene synthase. Petition 870190082172, dated 08 / 22 / 2019, p. 75 / 248 69 / 120 santalene. Various adjuvants can be used to enhance the immune response, depending on the host species, and include, but are not limited to, Freund's mineral gels (complete and incomplete), such as aluminum hydroxide, surface-active substances such as lysolecithin, pluronic polyols, polyanions, peptides, oil emulsions, keyhole limpet hemocyanins, dinitrophenol, and potentially useful human adjuvants such as BCG (Bacillus Calmette-Guérin) and Coynebacterium parvum. Such adjuvants are also well known in the art; monoclonal antibodies can be prepared using a wide variety of techniques known in the art, including the use of hybridoma, recombinant, and phage display technologies, or a combination thereof. For example, monoclonal antibodies can be produced using hybridoma techniques, including those known in the art and taught, for example, in Harlow et al., Antibodies: A Laboratory Manual, (Cold Spring Harbor Laboratory Press, 2nd edition 1988); Hammerling, et al, in: Monoclonal Antibodies and T-Cell Hybridomes 563 to 681 (Elsevier, NY, 1981). The term monoclonal antibody as used in this document is not limited to antibodies produced through hybridoma technology. The term monoclonal antibody refers to an antibody that is derived from a single clone, including any eukaryotic, prokaryotic, or phage clone, and Petition 870190082172, dated 08 / 22 / 2019, p. 76 / 248 70 / 120 does not refer to the method by which it is produced.

[115] The methods for producing and screening for specific antibodies using hybridoma technology are routine and well known in the art. Briefly, mice can be immunized with santalene synthase and once an immune response is detected, for example, antibodies specific to santalene synthase are detected in mouse serum, the mouse spleen is harvested and splenocytes isolated. The splenocytes are then fused by well-known techniques to any suitable myeloma cells, for example, SP20 cell line cells available from ATCC. Hybridomas are selected and cloned by limited dilution. The hybridoma clones are then assayed by methods known in the art for antibody-secreting cells capable of binding to a polypeptide of the invention. Ascitic fluid, which usually contains high levels of antibodies, can be generated by immunizing mice with positive hybridoma clones.

[116] In certain embodiments, a method for generating monoclonal antibodies comprises culturing a hybridoma cell that secretes an antibody of the invention wherein, preferably, the hybridoma is generated by fusing splenocytes isolated from a mouse immunized with santalene synthase with myeloma cells and then, Petition 870190082172, dated 08 / 22 / 2019, p. 77 / 248 71 / 120 subjecting the hybridomas resulting from the fusion to hybridoma clones that secrete an antibody capable of binding to santalene synthase. An antibody according to the invention, for example, can be used in a method to isolate a santalene synthase produced according to the invention, for example, using the antibody immobilized on a chromatographic support material.

[117] Furthermore, the present disclosure is directed to a method for preparing santalene or santalol, wherein the method comprises converting a polyprenyl diphosphate substrate into santalene or santalol in the presence of an enzyme, wherein the enzyme comprises a first segment comprising a peptide tag and a second segment comprising a santalene synthase according to the invention. An enzyme comprising said first and said second segments may, in the present document, be referred to as a tagged enzyme.

[118] For the preparation of santalene, in particular, a method, an amino acid sequence, a nucleic acid sequence or a host cell may be used as described in this document. Santalol may, for example, be prepared by oxygenation / oxidation of santalene in a manner known to itself.

[119] The peptide tag is preferentially Petition 870190082172, dated 08 / 22 / 2019, page 78 / 24872 / 120 selected from the group of nitrogen utilization proteins (NusA; SEQ ID NO: 26), thioredoxins (Trx; SEQ ID NO: 27), maltose binding protein (MBP; SEQ ID NO: 28) a so-called SET-tag, SEQ ID NO: 29) and functional homologs thereof. As used in this document, a functional homolog of a peptide tag is a peptide tag that has at least approximately the same effect on the solubility of the tagged enzyme compared with the untagged enzyme. Typically, the homolog differs in that one or more amino acids have been inserted, substituted, deleted from, or extended into the peptide of which it is a homolog. The homolog, in particular, may comprise one or more substitutions of one hydrophilic amino acid for another hydrophilic amino acid or of one hydrophobic amino acid for another.The homologue, in particular, may have a sequence identity of at least 40%, more particularly at least 50%, preferably at least 55%, most preferably at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% with the sequence of a NusA, Trx, MBP or SET.

[120] Maltose-binding protein from Escherichia coli, or a functional homolog thereof, is particularly suitable. Petition 870190082172, dated 08 / 22 / 2019, p. 79 / 248 73 / 120

[121] The use of an enzyme labeled according to the invention is particularly advantageous in that it can contribute to increased production, especially increased cellular production of a terpenoid or terpene, such as α-santhalene and β-santhalene.

[122] For improved solubility of the labeled enzyme (compared to the unlabeled enzyme), the first segment of the enzyme is preferentially linked at its C-terminus to the N-terminus of the second segment. Alternatively, the first segment of the labeled enzyme is linked at its N-terminus to the C-terminus of the second segment.

[123] Furthermore, the present disclosure is directed to a nucleic acid comprising a nucleotide sequence encoding a polypeptide, wherein the polypeptide comprises a first segment comprising a peptide tag, preferably an MBP tag, a NusA, a Trx, a SET) or a functional homolog of any of these and a second segment comprising a santalene synthase or an amorphadiene synthase. The second segment, for example, may comprise an amino acid sequence as shown in SEQ ID NO: 3.

[124] Furthermore, the present disclosure is directed to a host cell comprising said nucleic acid encoding said santalene synthase. Petition 870190082172, dated 08 / 22 / 2019, page 80 / 248 74 / 120 labeled. The specific nucleic acids according to the invention that encode a labeled enzyme are shown in SEQ ID NO: 4, SEQ ID NO: 6, SEQ ID NO: 30, SEQ ID NO: 31. The host cell, in particular, may comprise a gene comprising any of these sequences or a functional analogue thereof.

[125] Furthermore, the present disclosure is directed to an enzyme comprising a first segment comprising a peptide tag and a second segment comprising a polypeptide having enzymatic activity to convert a polyprenyl diphosphate into a terpene, in particular, a santalene synthase, wherein the peptide tag is preferably selected from the MBP, NusA, Trx or SET group). The specific enzymes comprising a labeled enzyme according to the invention are shown in SEQ ID NO: 5, SEQ ID NO: 7, SEQ ID NO: 32, SEQ ID NO: 33.

[126] The invention will now be illustrated by the following examples. Figure Captions

[127] Figure 1: pm-LPppa- plasmid map CiCaSSy-mpmii alt.

[128] Figure 2 Plasmid map pm-SPppa-MBP- OiCaSSy-mpmii alt.

[129] Figure 3 Plasmid map pm-PcrtE-TRX- Petition 870190082172, dated 08 / 22 / 2019, page 81 / 248 75 / 120 CiCaSSy-mpmii alt.

[130] Figure 4 pm-PcrtE-TRX- plasmid map SaSSy-mpmii alt.

[131] Figure 5 GC chromatogram of terpene species produced by CiCaSSy in R. sphaeroides. The compounds identified by GC-MS are: α-santhalene (Retention time: 5.3 min), trans-α-bergamotene (Rt: 5.45 min), epi-p-santhalene (Rt: 5.75 min) and β-santhalene (Rt: 5.95 min).

[132] Figure 6: Production of total terpenes (A), α-santhalene (B), β-santhalene (C) and trans-α-bergamotene (D) during fed-batch fermentation of Khodobacter sphaeroides Rs265-9c strains carrying plasmids with the gene encoding santalene synthase from SaSSy or CiCaSSy. The product proportions for individual terpenes (B, C and D) are plotted as the amount (area) relative to the total area of ​​the components as indicated in the GC chromatogram of Figure 5.

[133] Figure 7: Alignment of CiCaSSY with relevant proteins. The CiCaSSY protein sequence (TS23-3) (SEQ ID NO: 3) was aligned with the protein sequences of its closest variants TS23-1 (SEQ ID NO: 10) and TS23-2 (SEQ ID NO: 11) found in C. camphora. The 21 of the 553 residues that were different between the TS23-1 and CiCaSSY proteins are highlighted. Petition 870190082172, dated 08 / 22 / 2019, page 82 / 248 76 / 120

[134] Figure 8: GC-MS analysis of terpene production in E. coli (example 5). Vector control sample: E. coli transformed with pACYCDUET.

[135] Figure 9: GC-MS analysis of terpene production in E. coli (example 5). sample of clone TS23-1: E. coli transformed with pACYCDuet_TS23-l

[136] Figure 10: GC-MS analysis of terpene production in E. coli (example 5). sample of clone TS23-3: E. coli transformed with pACYCDuet_TS23-3 (CicaSSy)

[137] Figure 11: GC-MS analysis of terpene production in E. coli (example 5). Sample of clone SaSSy: E. coli transformed with pACYCDuet_SaSSy) Examples: Example 1: GC-MS analysis of Cinnamomum Camphora

[138] A Cinnamomum camphora plant approximately 30 cm tall was purchased from Planfor (Pepinieres PLANFOR RD 651 40090 UCHACQ - FRANCE).

[139] Cinnamomum camphora is known to occur in several chemotypes. In particular, the cineole type appears to contain santalene (Stubbs et al., 2004, Pelissier et al., 1995), while other chemotypes (camphor, linalool) have not been reported as containing santalene (e.g., Frizzo 2000; Pino 1998).

[140] The plant was dissected into leaf material, Petition 870190082172, dated 08 / 22 / 2019, page 83 / 248 77 / 120 stem and root. 0.5 g of plant material was weighed into a pre-cooled glass tube, and 2 ml of dichloromethane were added. The suspension was vortexed for 1 min, sonicated for 5 min in an ultrasonic bath, and centrifuged for 5 min at 1500 g at room temperature. The supernatant was collected and filtered over a 1 g sodium sulfate column. Approximately 2 μL were analyzed by GC / MS using gas chromatography as described in detail by Cankar et al. (2015). Santalenes were identified by comparing retention times and mass spectra with those of sandalwood oil (Sigma-Aldrich). Results:

[141] The roots, leaves, and stem of C. camphora appeared to contain compounds corresponding to α-santhalene (Rt 13.17 min), α-bergamotene (Rt 13.34 min), epi-βsanthalene (Rt 13.54 min), and β-santhalene (Rt 13.69 min). The concentration of santalenes was higher in the roots. Other compounds found in the roots of the Cinnamomum plant were identified as guaiol, guaiadiene, intermedeol, eremoligenol, germacrene D, isolepidozene, safrole, limonene, pinene, camphene, myrcene, sabinene, 1,8-cineole, and camphor. Therefore, this tissue was additionally taken for RNA extraction. Example 2: RNA Extraction and Analysis Petition 870190082172, dated 08 / 22 / 2019, p. 84 / 248 78 / 120

[142] RNA from C. camphora root material was isolated as follows: approximately 15 ml of extraction buffer (2% hexadecyltrimethylammonium bromide, 2% polyvinylpyrrolidinone, K 30, 100 mM Tris-HCl (pH 8.0), mM EDTA, 2.0 M NaCl, 0.5 g / l spermidine and 2% β-mercaptoethanol) was heated to 65 °C, then 3 g of ground tissue were added and mixed. The mixture was extracted twice with an equal volume of chloroform:isoamyl alcohol (1:24), and a quarter volume of A 10,000 m LiCl solution was added to the supernatant and mixed. RNA was precipitated overnight at 4 °C and harvested by centrifugation at 10,000 g for 20 min. The pellet was dissolved in 500 ml of SSTE [1.0 M NaCl, 0.5% SDS, 10 mM Tris-HCl (pH 8.0), 1 mM EDTA (pH 8.0)] and extracted once with an equal volume of chloroform:isoamyl alcohol. Two volumes of ethanol were added to the supernatant, incubated for at least 2 ha at 20 °C, and centrifuged. 13,000 μg supernatant removed. The pellet was air-dried and resuspended in water. Total RNA (60 μg) was sent to Vertis Biotechnology AG (Freising, Germany). PolyA+ RNA was isolated, synthesized with a random primer using a randomized N6 adapter primer and M-MLV H reverse transcriptase. The cDNA was sheared and fractionated, and fragments of 500 bp were used for further analysis. The cDNAs Petition 870190082172, dated 08 / 22 / 2019, page 85 / 248 79 / 120 contigs carry, with adapter sequences A and B attached to their 5' and 3' ends as specified by Illumina. The material was subsequently analyzed on an Illumina MiSeq sequencing device. In total, 27,919,287 sequences were read by MiSeq, with a total sequence length of 10,592,407,803 base pairs. Trimmomatic-0.32 was used to trim the Illumina sequencing adapter sequences, Seqprep was used to overlay paired-end sequences, and bowtie2 (version 2.2.1) was used to remove phiX contamination (phiX DNA is used as a surge control, usually present at <1%). Paired-end and single-end reads were used in a Trinity assembly (trinityrnaseq-2.0.2). A total of 16,0871 contigs were assembled by Trinity.

[143] To identify sesquiterpene synthases, C. camphora contigs were used to create a cDNA sequence database. In this database, the TBLASTN program was deployed to identify cDNA sequences encoding proteins that show identity with sesquiterpene synthase protein sequences, including santalene synthases from Santalum album (GenBank accession E3W202), Clausena lansium (ADR71055) and Solarium habrochaites (ACJ38409), valencene synthase from Callitropsis nootkatensis. Petition 870190082172, dated 08 / 22 / 2019, page 86 / 248 80 / 120 (CDM55287) and trans-α-bergamotene synthase from Phyla dulcis (AFR23371). In total, 95 contigs in the C. camphora cDNA database were identified that have significant homology to sesquiterpene synthases. The contigs were grouped into 28 groups according to their sequence overlap. These 28 contigs were further characterized by analyzing them using the BLASTX program to align them with protein sequences present in the UniProt database (downloaded on August 28, 2015), and 14 of them were identified as putative sesquiterpene synthase sequences and another 14 as putative monoterpene synthases, according to their homology to terpene synthase sequences present in UniProt.

[144] Contigs were examined for open reading frames encoding full-length terpene synthase proteins, based on alignments provided by BLASTX analysis. The following criterion for identifying a full-length protein was used: both sesquiterpene synthases and monoterpene synthases carry an RRxxxxxxxxW (RRX8W) motif near their N-terminal start. An ATG codon in the frame must map 20 to 70 codons upstream of the region encoding the RRX8W motif, or its orthologous position, to be identified as a start codon. Petition 870190082172, dated 08 / 22 / 2019, page 87 / 248 81 / 120 Example 3: Cloning of Santalene Synthase Cinnamomum Camphora (CiCaSSy)

[145] Full-length open reading frames were amplified from C. camphora cDNA. Forward and inverse primers as shown in Table 1 were designed and used to amplify the full-length open reading frames such that the reading frame was fused to the C-terminal of its His-6 tag in the pCDF-DUET-1 plasmid (Novagen Corporation). A total of 37 different terpene synthase ORFs were cloned. Using the TS23fw and TS23re primers (Table 1), three different, very closely related cDNAs were obtained, which encoded proteins with SEQ ID NO: 10 (TS23-1), SEQ ID NO: 11 (TS23-2), and SEQ ID NO: 3 (TS23-3): Table 1 Sequence Name Clones TS23 Direct atatggatcctATGGACTCCATGGAGGTACGCCGCTCTG (SEQ ID NO: 8) TS23-1, TS23-2, TS23-3 TS23 Reverse atatgcggccgcTCATCCCAAGTTGATGGATTCCTTCAATGGCACTG (SEQ ID NO: 9)

[146] The cloned variants were analyzed by sequencing the TS insert. Different variants were introduced into chemically competent E. coli BL21-RIL (Stratagene) by heat shock transformation and selected on LB-agar with 1% glucose, 50 µg / ml spectinomycin, and 50 µl / ml of Petition 870190082172, dated 08 / 22 / 2019, page 88 / 248 82 / 120 chloramphenicol. The transformants were transferred to liquid medium (5 ml of LB) with 1% glucose, 50 ug / ml spectinomycin, and 50 ug / ml chloramphenicol and developed overnight at 37 °C and 250 rpm.

[147] 200 μL of these cultures were transferred to 20 ml of LB medium with the appropriate antibiotic in a 100 ml Erlenmeyer flask, and incubated at 37 °C, 250 rpm until the A600 was 0.4 to 0.6. Subsequently, 1 mM IPTG was added and the cultures were incubated overnight at 18 °C and 250 rpm. The next day, the cells were harvested by centrifugation (10 min 8000xg), the medium was removed and the cells were resuspended in 1 ml of resuspension buffer (50 mM Tris-HCl pH = 7.5, 1.4 mM β-mercaptoethanol; 4 °C). The cells were perturbed by shaking twice for 10 seconds with 0.2 g of zirconium sand in a Fastprep machine at speed 6.5. Insoluble particles were subsequently removed by centrifugation (10 min, 13,000 x g, 4 °C). The soluble protein was immediately used for enzyme assays. Example 4: In Vitro Enzyme Assay

[148] For the enzyme assays, in a glass tube, a mixture was made with 800 μL of MOPSO buffer (15 mM MOPSO (3-[N-morpholino]-2-hydroxypropanesulfonic acid) pH = 7.0, 12.5% ​​glycerol, 1 mM MgC12, 0.1% Tween 20, 1 mM ascorbic acid, 1 mM Petition 870190082172, dated 08 / 22 / 2019, page 89 / 248 83 / 120 dithiothreitol). 100 µl of purified enzyme solution and 5 µl of farnesyl diphosphate or geranyl diphosphate (10 mM, Sigma FPP evaporated and dissolved in 50% ethanol) and 20 µl of 250 mM Na-orthovanadate. This mixture was incubated at 30 °C with gentle stirring for 2 hours. Subsequently, the water phase was extracted with 2 mL of ethyl acetate. The ethyl acetate phase was collected, centrifuged at 1200xg, dried on a sodium sulfate column, and analyzed by GC-MS.

[149] GC-MS analysis was performed on an Agilent Technologies system comprising a 7980A GC system, a 597C inert MSD detector (70 eV), a 7683 autosampler and injector, and a 30 m long x 0.25 mm internal diameter Phenomenex Zebron ZB-5ms column with a 0.25 µm stationary phase, with a Guardian pre-column (5 m). In this system, 1 µl of the sample was injected. The injection chamber was at 250 °C, the injection was split-free, and the ZB5 column was maintained at 45 °C for 2 minutes after which a gradient of 10 °C per minute was initiated, up to 300 °C. Peaks were detected in total ion count chromatograms. The compounds were identified by their retention index and by their mass spectrum in combination with a comparison of the mass spectrum to the libraries (NIST8 and in-house).

[150] It was found that the Clone TS23-3 (SEQ ID NO: Petition 870190082172, dated 08 / 22 / 2019, page 90 / 248 84 / 120 3) produces santalenes in this in vitro assay and thus encodes a santalene synthase, and was named CiCaSSY. Closely related clones TS23-1 (SEQ ID NO: 10) and TS23-2 (SEQ ID NO: 11) did not produce any santalenes or other sesquiterpenes in the in vitro assay. Example 5: Santalene Syntase Expression in E. coli

[151] For the production of sesquiterpenes in E. For coli, terpene synthase must be supplied with the FPP substrate. Cicassy was therefore co-Rhodobacter with a plasmid containing all the genes necessary for FPP synthesis (pBb A5c-MevT-MBIS-NPtll). This plasmid is a variant of the pBbA5c-MevT(CO)-MBIS(CO, IspA) plasmid (Peralta-Yalta et al, 2011), in which the chloramphenicol resistance marker was replaced by a kanamycin resistance marker (NptII). From this plasmid, a base pair 728 fragment ranging from the Apal site to the chloramphenicol acyltransferase (CAT) initiation site was amplified using Phusion polymerase and the primers P7 GCTGTTAGCGGGCCCATTAAG (SEQ ID NO: 12) and P2 GATATTCTCATTTTAGCCATTTTAGCTTCCTTAGCTCCTG (SEQ ID NO: 13), and the neomycin phosphotransferase II (NptII) gene from pBINPlus (van Engelen 1995) using the primers P5 C AGGAGCTAAGGAAGCT AAAAT GGCT AAAATGAGAAT AT C (SEQ ID NO: 14) and P6 C CAAGC G AGCT C GAT AT C AAACT AAAAC AATT C ATC C Petition 870190082172, dated 08 / 22 / 2019, page 91 / 248 85 / 120 AG (SEQ ID NO: 15). The fragments were isolated from gel and used as a template for a fusion PCR, using primers P6 and P7, and amplified a 1524 bp fusion fragment. This fragment, and pBbA5c-MevT(CO)-MBIS(CO, IspA) were both digested with Apal and Sacl restriction enzymes, and the pBbA5c-MevT(CO)-MBIS(CO) vector fragment and the digested fusion PCR fragment were ligated and transformed into E. coli DH5alpha by electroporation, and recombinant colonies were selected on LB+kanamycin. The presence of genetic elements, including the MEV trajectory operon and the NptII gene, was confirmed by isolating plasmid DNA from miniprep and analyzing this DNA by digestion with Apal and Sacl, producing bands of approximately 12,000 bp and 1,500 bp. The resulting plasmid was named pBbA5Sc-MevT-MBIS-NptII.

[152] In addition, the 1,670 bp fragment of BamHI NotI from the pCDF-DUET-1 vector, in which CiCaSSY had been cloned, was transferred to pACYC-DUET-1 (Novagen Corporation), for a fair comparison with SaSSY, which had also been introduced into pACYC-DUET-1 in this way.

[153] The first pBbA5c-MevT-MBIS-NPtII plasmid was heat-shock transformed into commercially available competent BL21DE3 cells (New England Biolabs cat C2527). Transformants were selected on LB plates containing 50 ug / ml kanamycin and 1% glucose. Petition 870190082172, dated 08 / 22 / 2019, page 92 / 248 86 / 120

[154] A pBbA5c-MevT-MBIS-NPtII transformant was developed and competent cells were produced using the CaCl2 method. Briefly, 10 ml of culture of this transformant in LB + 1% glucose + 50 ug / ml of Kanamycin values ​​were developed up to A600 = 0.5.

[155] Subsequently, the cells were centrifuged at 8000xg for 5 min, resuspended in 1 ml of 100 mM ice-cold CaCl2, centrifuged again for 5 minutes at 8000xg, the supernatant was discarded, and the cells were resuspended in 1 ml of 100 mM ice-cold CaCl2, and 50 µl of these cells were used for transformation. The cells were transformed by heat shock with 50 ng of pACYCDuet, pACYCDuet_TS23l, pACYCDuet_CicaSSy, and pACYCDuet_Sassy plasmids. The transformants were selected on LB plates containing 50 µg / ml kanamycin, 50 µg / ml chloramphenicol, and 1% glucose.

[156] A tube containing 5 ml of LB medium with 50 µg / ml kanamycin, 50 µg / ml chloramphenicol, and 1% glucose was inoculated with a colony containing both plasmids and grown overnight at 37 °C. The overnight culture was used to inoculate 20 ml of LB plus antibiotic (but without glucose) at an OD of 0.1. The culture was grown at OD 0.45 to 0.55 and then induced with 20 µl of 1M IPTG. The culture was overlaid with 2 ml of dodecane to prevent evaporation. Petition 870190082172, dated 08 / 22 / 2019, page 93 / 248 87 / 120 sesquiterpenes from the bottle and developed overnight at 28°C and 250 rpm.

[157] For GC-MS analysis, dodecane was separated from the culture by centrifugation and diluted 200 times with ethyl acetate. 2 μL were analyzed by GC / MS using gas chromatography as described in detail by Cankar et al. (2015).

[158] Ts23-1 does not produce a detectable amount of any terpene in this system (Figure 9). The main product of SaSSY in this system was found to be trans α-bergamotene (Figure 11); the main product of CiCaSSY was found to be α-santhalene (Figure 10). Example 6: RS102 middle

[159] 20 g / l of yeast extract (Gistex, DSM) and 0.5 g / l of NaCl are dissolved in distilled water, the pH is brought to 7.4 with NaOH. Distilled water is added to a volume of 930 ml, and the medium is sterilized with steam.

[160] One ml of sterile 0.5 g / ml MgSCU-TELO, 2 ml of sterile filtered microelements (80 g / l (NH4)2Fe(SO4)2-6H_O; 6 g / l ZnSO,i-7H2O; 2 g / l MnSOiILO; (0.2 g / l NiSO,-6H2O, optionally); 2 g / l Vitamin C), and 2 ml of autoclaved CaFe solution (75 g / l CaCl2H2O; 5 g / l FeCl3-6H2O; 3.75 ml HCl (37%)) and 66 ml of glucose solution are added to the sterile medium. Petition 870190082172, dated 08 / 22 / 2019, page 94 / 248 88 / 120 Example 7: Bacteria and Culture Conditions

[161] The Rhodobacter sphaeroides Rs265-9c strain was obtained from Rhodobacter sphaewides strain ATCC 35053 [acquired from the American Type Culture Collection (ATCC Manassas, VA, USA - www.atcc.org); number 35053; Rhodobacter sphaeroides (van Niel) Imhoff et al., isolated from a sewage settlement lake in Indiana and deposited as Rhodopseudomonas sphaeroides van Niel] after two mutagenesis cycles and was used as the base host for the construction of recombinant strains that have enhanced santalene production. All R. sphaeroides strains were grown at 30 °C in RS102 medium, unless otherwise mentioned.

[162] E. coli strains were cultured at 37 °C in LB medium (Becton Dickinson, Sparks, MD, USA). For plasmid maintenance in recombinant E. coli and R. sphaeroides strains, ampicillin (100 mg / l), chloramphenicol (30 mg / l) and / or neomycin (25 to 50 mg / l, depending on the plasmid) were added to the culture medium. Liquid cultures were routinely grown aerobically in a rotary shaker. When solid media were required, agar (1.5% final concentration) was added. Example 8: Cloning of Santalene Synthase (CiCaSSy) Petition 870190082172, dated 08 / 22 / 2019, page 95 / 248 89 / 120 and plasmid construction pm-LPppa-CiCaSSy-mpmii alt

[163] For the expression of santalene synthase of In the case of Cinnamomum camphora in R. sphaeroides, the full-length ORF was custom synthesized and optimized for codon usage by Genscript USA Inc. (Piscataway, New Jersey, USA). Additionally, the sequence for the LPppa promoter was added at 5' of the gene (SEQ ID NO:2). The construct was delivered cloned onto the pUC57 plasmid. The complete construct was excised from the plasmid using the restriction enzymes EeoRI and BamHI (at 5' and 3', respectively). The fragment containing the promoter and gene (1758 bp) was ligated into the previously digested pm-mpmii alt vector using the same two restriction enzymes. The ligation mixture was transformed into E. coli S17-1 cells. Transfer of pm-LPppa-CiCaSSy-mpmii alt (Figure 1) from S17-1 to R, sphaeroides Rs265-9c by conjugation was performed using standard procedures (US Patent No. 9260709B2).The nucleotide sequence of the LPppa-CiCaSSy construct is provided in SEQ ID NO:2; the protein sequence is represented in SEQ ID NO:3. Example 9: Plasmid construction pm-SPppa-MBP-CiCaSSy-mpmii alt

[164] For the expression of the CiCaSSy gene in combination with the MBP tag under the regulation of Petition 870190082172, dated 08 / 22 / 2019, page 96 / 248 Using the 90 / 120 SPppa promoter, the SPppa-MBP construct was synthesized by Genscript USA Inc. (Piscataway, New Jersey, USA). The gene encoding MBP was codon-optimized for expression in R. sphaeroides. The SPppa-MBP construct was then fused into the CiCaSSy sequence and cloned into the previously delayed p-mmpmii alt vector with EcoRI and BamHI. The SPppa-MBP construct was subsequently amplified using primers 5'-CTGTCCATGATCTTGTCGTCGTC-3' (SEQ ID NO: 16) and 5'-ACTGGCCTCAGAATTCAAATTTATTTGCTTTGTGAGCGGATAAC-3' (SEQ ID NO: 17), and CiCaSSy was amplified with primers 5'-CAAGATCATGGACAGCATGGAAGTC-3' (SEQ ID NO: 18) and 5'TTTATGATTTGGATCCTCAGCCCA-3' (SEQ ID NO: 19). The digested amplicons and vectors were then assembled using Clontech's InFusion® enzyme mixture. The reaction mixture was transformed into E. coli S17-1 cells.

[165] Transfer of pm-SPppa-MBP-CiCaSSy- mpmii alt (Figure 2) from S17-1 into R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (US Patent No. 9260709B2). The nucleotide sequence of the SPppa-MBP-CiCaSSy construct is provided in SEQ ID NO:4; the protein sequence is plotted in SEQ ID NO: 5. Example 10: Plasmid construction pm-PcrtE-TKX-CiCaSSy-mpmii alt Petition 870190082172, dated 08 / 22 / 2019, p. 97 / 248 91 / 120

[166] For expression of the CiCaSSy gene in combination with the TRX tag under PcrtE promoter regulation, the PcrtE-TRX construct was synthesized by Genscript USA Inc. (Piscataway, New Jersey, USA). The E. coli TRX-encoding gene was codon-optimized for expression in R. sphaeroides. The PcrtE-TRX construct was then fused following CiCaSSy and cloned into the previously delayed pm-mpmii alt vector with EcoRI and BamHI. The PcrtE-TRX construct was subsequently expanded using the primers 5'CTGTCCATAATCTTGTCGTCGTCAT-3' (SEQ ID NO: 20) and 5'ACTGGCTCCAGAATTCCGCTGOTGAACG-3' (SEQ ID NO: 21), and CiCaSSy was expanded using the primers 5'CAAGATTATGGACAGCATGGAAGTCC-3' (SEQ ID NO: 22) and 5'TTTATGATTTGGATCCTCAGCCCAGGTT-3' (SEQ ID NO: 23). Digested amplicons and vectors were then assembled using the enzyme mixture.

[167] InFusion® from Clontech. The reaction mixture was transformed into E. coli S17-1 cells.

[168] Transfer of pm-PcrtE-TRX-CiCaSSympmii alt (Figure 3) from S17-1 to R.sphaeroides Rs265-9c by conjugation was performed using standard procedures (US Patent No. 9260709B2).

[169] The nucleotide sequence of the PcrtE-TRX-CiCaSSy construct is given in SEQ ID NO: 6; the sequence Petition 870190082172, dated 08 / 22 / 2019, p. 98 / 248 92 / 120 of proteins is represented in SEQ ID NO: 7. Example 11: Construction of the pm-PcrtE-TRX-SaSSy-mpmii alt plasmid.

[170] For expression of santalene synthase of Santalum album (SaSSy) in R. sphaeroides in combination with the TRX tag under the regulation of the PcrtE promoter, the full-length ORF along with the E. coli TRX was custom synthesized and optimized in terms of codon usage for R. sphaeroides by Genscript USA Inc. (Piscataway, New Jersey, USA). Additionally, the sequence for the PcrtE promoter was added at 5' of the gene (SEQ ID NO: 24). The construct was delivered cloned onto the pUC57 plasmid. The complete construct was excised from the plasmid using the restriction enzymes EcoRI and BamHI (at 5' and 3', respectively). The fragment containing the promoter and genes (2335 bp) was ligated into the pm-mpmii alt vector previously digested with the same two restriction enzymes. The ligation mixture was transformed into E. coli SI7-1 cells.

[171] The transfer of pm-PcrtE-TRX-SaSSy-mpmii alt (Figure 4) from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (US Patent No. 9260709B2).

[172] The nucleotide sequence of the construct PcrtE-TRX-SaSSy is provided in SEQ ID NO: 24; the sequence Petition 870190082172, dated 08 / 22 / 2019, page 99 / 248 93 / 120 of proteins is represented in SEQ ID NO: 25. Example 12: Shaking Flasks in Developmental Conditions

[173] Seed cultures were carried out in 100 ml shake flasks without baffles with 20 ml of RS102 medium with 100 mg / l neomycin and a glycerol stock cycle. The seed culture flasks were cultured for 72 hours at 30 °C in a shake incubator with a 50 mm orbit at 110 rpm.

[174] At the end of the 72 hours, the OD600 of the culture was evaluated to calculate the exact volume of culture to be transferred to the larger flasks.

[175] Shake flask experiments were performed in 300 ml shake flasks with 2 bottom baffles. Twenty ml of RS102 medium and neomycin at a final concentration of 100 mg / l were added to the flask along with 2 ml of sterile n-dodecane. The inoculum volume was adjusted to obtain a final ODG00 value of 0.05 in 20 ml of medium.

[176] The flasks were kept for 72 hours at 30 °C in a shaker incubator with a 50 mm orbit at 110 rpm. Shaker flask experiments were performed in replicates. Example 13: Sample Preparation for Content Analysis Petition 870190082172, dated 08 / 22 / 2019, pages 100 / 248 94 / 120 Isoprenoid in Organic Phase

[177] Cultures were collected 72 hours after inoculation in pre-weighed 50 ml PP tubes which were then centrifuged at 4500xg for 20 minutes. The n-dodecane layer was transferred to a microcentrifuge tube for subsequent GC analysis.

[178] Ten microliters of ethyl laurate were weighed into a 10 ml glass ampoule to which 800 μL of the isolated dodecane solution were added and weighed. Subsequently, 8 ml of acetone were added to the ampoule to dilute the dodecane concentration for a more accurate GC analysis. Approximately 1.5 ml of the terpene-containing dodecane in acetone solution were transferred to a chromatography ampoule. Example 14: Gas Chromatography

[179] Gas chromatography was performed on a Shimadzu GC2010 Plus equipped with a Restek RTX-SSil MS capillary column (30 m x 0.25 mm, 0.5 µm). The injector and FID detector temperatures were set to 280°C and 300°C, respectively. The gas flow rate through the column was set to 40 ml / min. The initial oven temperature was 160°C, increased to 180°C at a rate of 2°C / min, further increased to 300°C at a rate of 50°C / min, and maintained at this temperature for 3 min. The volume Petition 870190082172, dated 08 / 22 / 2019, page 101 / 248 The 95 / 120 sample injected was 1 µl with a split ratio of 1:50, and the nitrogen constitution flow rate was 30 ml / min. Example 15: Analysis of terpenes produced by CiCaSSy in R. sphaeroides

[180] Figure 5 shows the chromatogram obtained by analyzing the organic phase isolated from all R. sphaeroides cultures expressing the CiCaSSy gene (example strains 9, 10 and 11). Four main compounds were identified: α-santhalene (A), trans-α-bergamotene (B), epi-ε-santhalene (C) and β-santhalene (D). The most abundant terpene was α-santhalene, followed by trans-α-bergamotene and β-santhalene. Since no purified santalene is available to be used as standards, the terpene titration was calculated based on the GC response factor obtained with the valencene terpene. The total terpene production (cumulative area under the curve for all 4 terpenes) obtained in the strains from examples 9, 10, and 11 are 5.3 ± 0.12 g / kg of dodecane, 5.8 ± 0.29 g / kg of dodecane, and 3.4 ± 0.09 g / kg of dodecane, respectively. The ratio for the four terpene species was conserved in all strains. Example 16 Seed Medium for Fed-Batch Cultivation Petition 870190082172, dated 08 / 22 / 2019, page 102 / 248 96 / 120

[181] The following components were dissolved in 1 l of water: 20.8 g of yeast extract, 10.3 g of MgSO₄·7 H₂O, 86 mg of ZnS₃C₅·7H₂O, 30 mg of MnSC₂X₅ H₂O, 1.1 g of CaCb -2H20, 0.96 g of FeS047H20, 1.44 g of KH2PO4 and 1.44 g of K2HPO4. The pH was adjusted to 7.4 with 10 M NaOH. After sterilization (121 °C, 20 min), 50 ml of 50% (w / w) glucose and 1 ml of 0.1 mg / ml neomycin sulfate is added per liter of the medium. Example 17 Medium for Fed-Batch Culture

[182] The following components were dissolved in 1 l of water: 25 g of yeast extract, 1.7 g of MgSO4-7H2O, 0.10 g of ZnSO4-7H2O, 35 mg of MnSO4-7H2O, 1.3 g of CaCl2 -2H20, 0.17 g of FeCl2 -6H20. 1.7 g of K2HPO4, 1.7 KH2PO4, 1.1 g of (NH4)2Fe(SO4)2 KH20, 2.8 g (NH4)2SO4, 1.1 g (NH4)H2P04, 1.9 g MgCl2 ·6Η20 and 1 ml of antifoam.

[183] ​​After sterilization (121 °C, 20 min), the pH is adjusted to 7.0 with 25% ammonium hydroxide solution. Per liter of sterile medium, 60 ml of 50% (w / w) glucose, 1 ml of 0.1 mg / ml neomycin sulfate, 4 mg niacin, 8 mg thiamine.HCl, 4 mg nicotinamide, 0.2 mg biotin and 150 ml n-dodecane were added. Example 18 Fed-Batch Fermentation under Culture Conditions Petition 870190082172, dated 08 / 22 / 2019, page 103 / 248 97 / 120

[184] Seed cultures for fed-batch cultures were prepared by inoculating 500 ml of sterile seed medium (example 16) by adding 1 ml of glycerol stock of the appropriate strain of Rhodobacter sphaemides, and incubating for 48 hours at 30 °C.

[185] In a 1 l fermenter vessel, 350 ml of fed-batch medium were sterilized and supplemented with sterilized glucose (filter), Neomycin, vitamins, and n-dodecane as indicated (example 17). By adding 50 ml of seed culture, the medium was inoculated and incubated for approximately 24 hours at 30 °C, agitation of 600 rpm, airflow of 0.3 w / m, and a pH of 7 (adjusted by automated addition of 12.5% ​​ammonium hydroxide solution). After 24 hours, agitation was increased to 1200 rpm, and 450 to 500 g of 50% (w / w) glucose was fed to the fermenter over 100 hours. Example 19 Fed-Batch Fermentation Sample Preparation GC

[186] Broth samples of approximately 20 ml were collected during a fed-batch culture. Ten microliters of ethyl laurate were weighed into a 10 ml glass ampoule to which 0.8 ml of broth sample was added and weighed. Subsequently, 8 ml of acetone was added to the ampoule and the broth mixture was... Petition 870190082172, dated 08 / 22 / 2019, page 104 / 248 98 / 120 acetone was incubated at room temperature for 25 minutes while being stirred at 400 rpm. Approximately 1.5 ml of the acetone broth mixture containing terpenes was transferred to a chromatography ampoule and used for analysis according to Example 14. Example 20 Comparison of terpene production by Rhodobacter spaeroides strains bearing CiCassy and SaSSy.

[187] Rhodobacter spaeroides strains bearing CiCaSSy (pm-SPppa-MBP-CiCaSSy-mpmii alt, example 10) or SaSSy (pm-PcrtE-TRX-CiCaSSy-mpmii alt, example 11) were grown in fed-batch mode according to example 18. Samples were taken from the fermentation broth at various time points and analyzed for terpene production according to examples 19 and 14. The total terpene concentration produced (cumulative areas of peaks A to D in Figure 5) was almost equal for both strains throughout the cultivation (Figure 6A), and a final terpene concentration of approximately 5.5 g / kg of broth was obtained. Over 120 hours of fed-batch cultivation, the terpenes produced by the CiCaSSy strain consisted of approximately 49% α-santhalene (Figure 6B), 21% β-santhalene (Figure 6C), and 25% transα-bergamotene (Figure 6D), while the SaSSy strain produced approximately 28% α-santhalene (Figure 6B), 14% β-bergamotene (Figure 6D), and 25% transα-bergamotene (Figure 6D). Petition 870190082172, dated 08 / 22 / 2019, page 105 / 248 99 / 120 santalene (Figure 6C) and 52% trans-α-bergamotene (Figure 6D). The percentages of α-santhalene and β-santhalene produced by the CiCaSSy strain were 1.8 times and 1.5 times higher, respectively, than that of the SaSSy strain. In contrast, the fraction of trans-α-bergamotene produced by the CiCaSSy strain was 2 times lower than that of the SaSSy strain. REFERENCES

[188] Amick, JD, Julien, BN, 2015. Modified Santalene polypeptide synthetase, encoding of nucleic acid molecules and uses thereof. WO / 2015 / 153501

[189] Baldovini, N., Delasalle, C, Joulain, D., 2011. Phytochemistry of the heartwood from fragrant Santalum species: a review. Flavor Fragr. J. 26, 7 to 26.

[190] Cankar, Jongedijk, Klompmaker, Majdic, Munun, Bouwmeester, Bosch & Beekwilder (2015) (+)-Valencene production in Nicotiana benthamiana is increased by negative regulation of competitive pathways. Biotechnol. J. 10, 180 a 189

[191] Chapuis, C., 2012. Intermediates for the preparation of Beta-Santalol. WO / 2012 / 110375

[192] Daviet, L., ROCCI, L. and Schalk, M. (2015). Method for producing fragrant alcohols. (WO2015040197)

[193] Diaz-Chavez, M.L., Moniodis, J., Madilao, LL, Jancsik, S., Keeling, CI,

[194] Barbour, EL, Ghisalberti, EL, Plummer, Petition 870190082172, 08 / 22 / 2019, pág. 106 / 248 100 / 120 JA, Jones, C.G., Bohlmann, J., 2013. Biosynthesis of Sandalwood Oil: Santalum album CYP76F Cytochromes P4S0

[195] Produces Santalols and Bergamotol. FLoS ONE 8.

[196] Frizzo, Caren D. et al. Essential oils of camphor tree (cinnamomum camphora nees & eberm) cultivated in Southern Brazil. Braz. arch. biol. technol. [online]. 2000, volume 43, n.3 [cited 26 / 02 / 2016], pages 313 a 316.

[197] Jones CG, Moniodis J, Zulak KG, Scaffidi A, Plummer JA Ghisalberti EL, Barbour EL, Bohlmann J. (2011) Sandalwood fragrance biosynthesis involves sesquiterpene synthases of both the terpene synthase (TPS)-a and TPS-b subfamilies, including santalene synthases. J Biol Chem. 286(20): 17445-54. Ngo, K.-S., and Brown, G.D. (2000). Autoxidation of a-santalene. Journal of Chemical Research 2000, 68 a 70.

[198] Pelissier, Y., Chantal, M., Prunac, S., Bessiere, J. 1995. Volatile components of leafs, stem and bark of Cinnamomum camphora Nees et Ebermaier. J. Essent. Oil Res. 7, 313 a 315.

[199] Peralta-Yahya PP, Ouellet M, Chan R, Mukhopadhyay A, Keasling JD, Lee TS. (2011) Identification and microbial production of a terpene-based advanced biofuel. Nat Commun. 2:483. Petição 870190082172, de 22 / 08 / 2019, pág. 107 / 248 101 / 120

[200] Pino JA & Fuentes V. 1998, Leaf Oil of Cinnamomum camphora (L.) J. Presl. de Cuba. Journal of Essential Oil Research 10, 531 a 532

[201] Schalk, M., 2016. Method for producing a- santalene. US9297004.

[202] Schalk, M., 2014. Method for producing beta -santalene. EP2376643.

[203] Schalk, M., 2006. Novel sesquiterpene synthases and methods of their use. WO / 2006 / 134523.

[204] Stubbs, B J., Specht, A. e Brushett, D. 2004. The Essential Oil of Cinnamomum camphora (L.) Nees and Eberm.— Variation in Oil Composition Throughout the Tree in Two Chemotypes from Eastern Australia. Journal of Essential Oil Research 16, 9 a 14. Teixeira da Silva, J.A., Klier, M.M., Soner, D., Page, T., Zhang, X., Nataraj, M., Ma, G., 2016. Sandalwood: basic biology, tissue culture, and genetic transformation. Planta 243, 847 a 887.

[205] van Engelen FA Molthoff JW, Conner AJ, Nap JP, Pereira A, Stiekema WJ. (1995) pBINPLUS: An improved plant transformation vector based on pBIN19. Transgenic Res. 4(4):288-90.

[206] Willis, BJ, Eilerman, RG, Christenson, PA and Yurecko Jr., JM 1985.

[207] Functionalization of terminal trisubstituted alkenes and derivatives thereof. Patent No. US 4510319 Petition 870190082172, dated 08 / 22 / 2019, pp. 108 / 248 102 / 120

[208] Zulak, K., Jones, C, Moniodis, J., Bohlmann, J., 2016. Terpene synthases from Santalum. US9260728. Sequences Seq Id No: 1 CiCaSSy - Cinnamomum camphora Nucleotide Sequence ATG G ACTCC ATG G AG G T ACGCCGCTCTGC AATCTATC ACTCG ACCTTTTGG G AT AUG AT A G C ATTCG CG CC CTG CTC GC AAG AA G AG ACTGC A CTG CTG C AG CTG CATTG AGTC CTG AC C AT CACAAAA GACTC A AGG AAA G A ATTC AG CG CGG GCTAC A AG A C ATCACA CAGCCACA CC ATCTG CTTG G AHGATCG ACG CTGTCC AACGC CTC GGTG TG G CCTACCAGTTTG AG G AAG AA ATCAGTG AC G C ACTG CAT G G GCTTC ACTCA G AG AAC ACGG AGC ATGC AATT A AG G AC A G TCTG C AC CACAC ATCTCT CTATTTT AG ATTG CTTAG GCA A C ATG G GTGTA ACC TTTCATCAG AC AT ATT C A A C A A ATTT A AG A AG G A AG GA G C AG GTTT C A A GG CAAG CCT ATGTG AGG ATG C AATGGG ACTTTTG AG CTTGTATG AGG CTG TACGTCTT AGCGTCA A AG GTG A AGC CATCTTGG AG GAA G CTC AGG TCTTCTCG ATCG CG AATTTG A AG ATTCTG ATG G A,A AG GG TGG AG AGG A AGCTG G CAG AT AG A AT AG A AC ATG CCTTG G AG ATCCCCTTG TATTG GAG G GCG CCG AG ACTGG AAGCT AG ATG GTAC ATAG ATG TAT AT GAAAAGGAAGATGGGAGGATTGATGACTTGCTTGATTTTGCAAAGCTAGATTTCAACAGGGTGCAAATGT TGTACCAAACCG A ACTGA AG GA ATT ATCAATG TG G TG G GA ATTGCTG GGG π ACC AG CG AAG ATG GG GTT CTTCCG AG AC A GA CTATTGG AG A ACC AT CT CTTTTC A ATTG CA GTG G TTGTCG AG CCTC AAT ACT CCC AGTG TAGAGTA GC AATT ACAA A AG C CAT AG T CCTT ATG ACAGC AATGG AT GACTTTT ATG ATGTG CATS G TTTGCC AG ATG AG CTAAA AGTCTTC AC GG AC ACCGTT A ATCGGTG GG ATTTAGAG GG A ATTG ATC A ACT ACC AG AG TATATG AA G CTGTA CT ACTT GG CGTTATATA ATACAACCA ATG AG ACC GC ATACATC ATCCTCA AGG AG AA, GGGA TTCAATG CT ACACATTATCTG A AG AA ACT GT GGGC A ATG CA AAGTAACG CG T ACTTTCG GGAAGCTC A ATGGTTC A ACAGTG GTTACAT ACCT AA ÁTTTG ATG AGT ATTTAG ACAATG CTTTA GTCTCAGTTG GG GCGC CCTTTG T ATTG G GT CTCT C ATÂCCCC ATG AT ACAAC AACAAAT ATC AAAG G AG GAA ATTG ATTTAATCCCCG AAG ATCT A AAT CTCCKCGTTGG GC ATC G AT CAT ATTTCG ACTATATG ATG ATTTG GC CA CTTC A AAGG CTG AGC A AC AAC GTGG GG ACGTGCC AA A ATCC ATC C AAT GTT ATATGCATG A AACTG GTAG TTC GG AGG A AGT TGCAGCAAACCATATCAGGGACCTCATCAGTGATGCTTGGAAGGAAGTGAATGCAGAGTGTTTGAAACCT ACTTCTCTGTC AA AGCATTACGTGG GAGTAG CTCCAAATTCG G CT AGG TCTG GA GTGCTGATGT ACC ATC A TGACTTTGATGGCTTTGCAAGTCCCCATGGCAGGACTAATGCTCATATCACGTCAATATTTTTTGAACCAGT G CC ATT GAA GGA ATCC ATC AA CTTGG G ATG A Seq Id No: 2 Petition: 870190082172, on August 22, 2019, page. 109 / 248 103 / 120 LPppa-CiCaSSy - Cinnamomum Camphora Sequência of Nucleotídeos (Otimizada with Code) A.-L4 7Y'A TA .4.4.4.4.4 TTTÀ T'H'GCITTG TGAGCGGA TA4C.4 A 7T4 77)4 / / 4 TTCACCGG CGAGCCA G( 11GG. ;tccggccgt< ogcgati :taccacaí ic ac< sttcti.iggacatcg acagcatccg GGCGCTCCTCXKI.K?GGCGGGACT(XJACGGCGGCCGCGGCCCTCTCGCCCGACCA CCATAaGCGCCíOAAÍKIAGGCATCCAGCGCCCCCCAGGATCACCCAGCCAGCCAG CGACCATGIY^nXT^JGCTGATCGACGCCGTGCACtCGCCTílGGCiTrGGC.CCAC.CA GT4TGAGGAAGAGATC1VGGACGCGCTGCACGGCCTCCAT1^X1GAGAACACCGA GCA(X;CCATCAAGGACTCGCTGCACCATACGTCGCTGCCTTΑGCCTTΊ GGCTGCAACC'I'G I CGTCGGACATC'I 1CAACAAG'JTCAAGAAGGAAGGC ggcggcttcaagcctcgctctgcgaggacgccatgggcctgctctcgctgtat CIAGGCCGTíICGCCTCCTIITGAAííItGCííAGíjCCATCCAGGGAGGAGGAGCC TTCTCGATCtlCCAACCTLlUaATCCTCATGGAGCGCGTGGAGCGCAAGCTCGCC GACC-GCATCGAGCATGCCCTGGAGATCCCGCTCTVrTGGCGCGCCCCGCGIOTG G AGGCCCGCTGGT ACATCGACGTGTATG AG AAG G AAGACCGGCCACGCATCG CTGCTCGAl..TirGCGAAGCTGGAC'l TCA^CCGCGTGCAtLVI'GC / rCl ATCAGACCG AGC4GAAGGAGC1OTCG.VIG1OGTGGGAGCTGC7'G(^CCTGCCCGCCAAGATGG GClTCTTCCGCGAiTGCCTGCTCGAGAACCACCTmVlCGATCGCCGTGG'lGGT GGAG CCCCAiri'ACTOGCAGTG CCG14 ΪΠΐί JCC ATC ACCAAGi iCCGA lGGG' AGCGGG! ATG G ACGACTTCTATGACGTCl CATG C CCTGCCGC ACGAG CTCAAGGT gttcaccgacagggtgaaccgctgggacctggaggcatggaccagctccccga G TAG ATGAAGC T GTACTATCTG G CG C TCTACAAG AC C ACG A \CG AG ACG GAT OCT! AniATCClOAAGGAGAAliGGCTTCAACGCCACGCVI'TACCTGAAixAAGC'I'CTGG GCCATlJCACTlOGAACGiXJTATTCCtlClkACTlHXZCAGTGGTTCAAC'rCGGtlCTAC A'rCCCGAAGl'TCGACGAGTAn'CTGCCCAGCGCGCGCGCGCGC ΤΊ 'CGTGCTGG GC CTCI 'CGTATC CC AT< í ATC C AGO AG C AG A' I'CTCG AAGG AA< SAG A TOGACCTGATCCCCGAC GACCTCXkCCTGCTCCGCTG GG CCTCGATCATCTTCCG amíTA(XkACGACCTGGC4'AC<:TCGAAGGCCGAGCAGCAííCGCGGCGAi:GTGCC CAAt jTC G ATCCACtTG C TAT ATG CATGAG AC GG GOT CG TCGG AG G.YAG TG GCGG C CjAACCATAI'CCGCGACC'I'GA'J'CTCGGACGCGI'GGAAGGAAG'J'GA.-V'GCCGAGIO.

[209] Sequence in italics is the LPppa promoter. Seq Id No: 3 Petition 870190082172, 08 / 22 / 2019, pág. 110 / 248 104 / 120 CiCaSSy - Cinnamomum Camphora Sequência de aminoácidos MDSMEVRRSATYTLSTFVVDTDSTRAI.LARRDCTAAAAl.SPDTIIlKRl.KERIQR.RLQDI TQPHHLlÁiblDAYQKJ jGVAYQFEEEJ SDAI .H GLHSENTEHAIKJJSLH HTSLYFlil 4, RQHGCNTSSDIFNIG^GGlGGGFKASLCEDAMGLLSLYEAVRLSVKGEAILEEAQV FSIANbRIIAIPYiVERKLADRI EH ABE] PbYWR.APR]..EtARWY[l)VYEKEDG RIDDLE DIL\KLDFNRVQMLYQTELKELSMWWELLGL1\U <MGF1'RDRLLEN11LFS1AVVVE P(ÍYS(íCRVAITKAI\ / LMTAMDDFYDVllGrJ:'J')E] J<VFT] H’VNRWD] JiGIDtjLPEYM ELY YL AL ΥΝΊΤΝΕΤ A Y11LKE KGF ΝΛΤ11 YLKKL W ΑΛ 1Q SN A YFRE AQ WFNSG Y11 ’K FDEYLDNALVSVG AI’FVIJILSYI’MIQQQJSKEHI IJbIPWLNL] .RWAS11FR] ADD J. ATSKA EQQ RG1.) VPKSJ i^CYM H ETGSSE E VAANH1K DI. IS DA WKE VNAECLK1 ’TSI, SKI IYVG VAPNS ARSGVLMYT ΠITJFDGFASFHGRTN AITTTSTFFEPXTÚ ACESINLG Seq Id No: 4 SPppa-MBP-CiCaSSy - Nucleotide Sequence .4AAT7T i ITl'GCTrrGTG. 1GCGGA 734ACL 177'A ΤΤΛ GA HX:A CCGGCGA GCCAGC ARGUMENT. t PTTCA CTCTAG.· 17Ki CAGG4 f ?f7GAC4 7ΐ 14 7TACG ACG AGG ACA AGATCTTC CAGG AC AAG GTCr]nAT(? CG TTC At: (m)G <VACG (A XFIX3 (Y) (^TAC AACGGC A AG CT< j ΔΤίΙΰΟίη / ΑΤα^ΠΑΤ^σ^αΟΤίΚ^ΙΚΧ^ΩΤίΊΤΐΧΧΤΓίΑΤί^ΑΤΑΑΐ / ΑΑίΚίΑυΩ^Κ: TCmiAACCCGCQCA^ AGGCCAAGCGCAAGTCGt^CGCTCATGrrCAACCrGCAGGAGCCGl^mOACCT GGCCCmATCGCGGCCGACGG.CG.GCTA(X;CGTTCAAG.TATGAGAACGGO\AGT ATGACATCAAGGACGTGGGCGTGGACAACGCGGGCCAAGGCCGTGCCTT A(l\ACAAGCACArrGAACf^ (TGAt lYCGGACT ACTCtLVF CGCGGAGGCCGCGTrCAACAAGG(;CGAt3ACCGCCATC}ACGAfrCAACGGCCCGTC Petition 870190082172, of 22 / 08 / 2019, p. 111 / 248 105 / 120 ggcgtggtcgaacatcgacacctçXjAaggtgaactatggcgtgaccgtgctccc CACGTTCAAGGGCaCGrXOTCGAAGCCCTTCGTGGGCGTGCTGTCGGCGGGCAT CAACGCCGTCGCCGAACAAGGAGCTCGCGAAGGAGTCCCTGGAGACTCT gí3tí:accüaogai;ggíií3tg(;aí}gccgtgaaí.l\ag(;acaagcç(:!(^tgggcg(:çgt GGl'CCTG.MGTCCtTATCtAGG / W^AGCTG.GTGAWAC'CCGCCiCATCGCGGCCAC .CATfíGAGAACGCCGCAGAAGGGCGAGATCATG,CCGAACATCCCCCAt}ATGTCGGC CTTCTGGTATGCGÍHOCGCACCGGTGATCAAGGCGGCCTCGGGCCGCCAGAC.ÇGTGGACCtAGGCCCTCAAGGACGCCCACLXCCGGCGACGACGACGACAAGATCàT GGACAGCATGGAAGTCCGGCGGTCGGCGATCTÀCCAGCACGTTCTGGGACAT C(}\í lAGC ATCCGG* XOCTCCTGGCGCGGa JGGACTGCACf íGCGGCCC1 TÇTCGCCCííACCACCATAAGGGCCTt^AAGGAGCGCATCCAGGÍ^CGCCrCCAGG AC' A TC ACCCAG CCGCACCATCTC CTCGGCCTCATCG ACGCCGTGCAGCG CCTGG GCGTGGCCIIACCAGTTCGAGGAAGAGATCTCGGACGCGCT G ACT CG CTG CACC AT AC G' ICG 0' I'CT VJ TT CCGCCrGCTCCGCCAGCA'I'G* ICTGCAAtlOTCGTCGGACA'lVHA'AACAAGTTC AAGAAGG A AGGCGGCGGCTTC A AGG C-CTCGCTCTGCG AG G AGCGGCT CTCTCGCIXSTATGAGGCCGTG CGCCTi TCGGTGAAt KXKGAGGiXATCCTGGAG G.AAGCCCAG GT< TTIOTCGATCGCCAACCTG AAGATCCTCATGGAGCGCGTG G AG CGCA WCTCGCCGACCG CATCGAG CATG CCCTt J GAG ATTCCCTCGCGC11 GCCCCGCCyrC'l'GGAGGCCCiXG'GGTACATCTiACGTGTA'l'GAC^AAGGAAGACGCiC CGCAirGACGACCTGClXX}ACJ'T(;GCGAAGCJ'GGACTTCAACCGCGTGCAGA'JY.i CTCTAr[,(?AGACC(}AGCT,GAAGGAGC,[,(^TCGATGr['GGTGGGAGCr(}CTCiGGCCTG (XiCG(XLAAi?iAl'GG(St?lTO'Ti;CtX^GACC1GCCTGC']'(XAAC,AACCA.CC'[,CTTCTCGA 'I'aJCCGTGGTCARrGClAGCiJCCAGTACrOlCLAGTGCCGCGrCtGCCATCACCAAilG (XIATCGTGCTGATGACGGCGATGGACGACTTCTATGACGTGCATGGCCTGCCGG AC( 1 AG CTCAA( iGTGTTC ACC<1\CAC( KTJ'GA AC( OCTGGGACCIX! G AG GG CATCG ACC AG CTCCCC G AGT AC ATG AVK'TG TACTATÍ ' TG (JCG CTCTAC AACACC AC' G.\A CGAi^ACCiGCClAl'ATCATCC'rGAAGGAGAACJGGCTTC.AXCGCCACGCAl'TACCT GA.XGAjXGCl'CTGGGCCATGCAGTCGA.XCGCGTArrTCOTCGAWGCCCAGTGGT'l' (14A('1,CGGGCTACATCCCGAACTTTCc,TACGAGl,ATCTGGA(.'AACG(?CCTCGTG,rCG GTGG G CG CCCCGTTCGTG CTGGGCCTCTCGTATCCC ATGAl'CCAC CAG CAG ATCT (X3 A AG GAAG AG AT CG ACCTG ATC CCCG AGG AC CTCA ACCTGCTCC GCTGG Gi :CT C< lATCATCTPCCGa m ;tacg acgacctggccacctcg aaggccg AG( \agcagc. Petition: 870190082172, on August 22, 2019, page. 112 / 248 106 / 120 G CGGCG ACG TGCCC AAG TCGATCCAG TGCT AT ATG GAT G AGAC-GG GCTCGTCGG AGGAAGTGG CGG GCA ACC A'J A1OCGCG AC CTGATCT CGG ACG CGTGI L\ AGG A AG TGjUCGCCGAGTGCCTGA.kGCCGACCTCGCTCTiXLUGCACTACGTGGGCGTGG (XK'CCAACTCG GCCCG CTCGGGCG TG (

[210] The italicized sequence is the SPppa promoter; the underlined sequence is the MBP with an optimized codon. Seq Id No: 5 MBP-CiCaSSy - Protein Sequence* V MK2EE GKl Λ1W1 NG ü KG Y N11L A1A G 1\KEE K1)TG T VE 1 Ill »KLE E KíTu V. C VlG IJG PPI JF WAI1J ORFGG YAQSG L] ..YEITRrinaAGorKKYRGG WILY VR YNG KJ JAYPIAV EA [ ,S IJ YNK11 í J TNPPKTWE E Ϊ PA] JOKE 1 J <AKG K SAI Al I Ml Λ) EP YFTW PI J A ADG G Y Al·’ K YENG KYDJ K DVCWNAG AK AG 1 TF1ΑΌIJ KN Kl IΜ N ΑΩΤ1.1 YS1AEA A ENK GETAA1TIN<lPWAWSNIDTSK\^YG\M.PTFK^PSI<PFVr^^AG1NIAASPXKE íjAKEFEEN^T.ETDE6ÍT.EAVNKDKPÍ.GAVAI.KS\tEEETÀ7KDPRÍAATMENAQKGEI MPNIPÜMSAFWYAVTtTAVlNAASGRQTVIlEAIJÍIlAQTGDDIlllKlMDSM EVRRSAI YIISTFWDIDSIRALLARRDCTAAAALSPDIIHKIíLKERIQRRLQDITQPIIHLLGLID AVQR [ Ái VAYQFEEE ISCAI Ή GLH SENTE H AI KI ISLHHTSl A’ F K 1.1 ,R Q H G C NI .SS1 )I FNKFKí<EGGGFKASLCEDAMGΓA□SΓ A7EAVRl.SVKGEAII .EEAQVFSIΑΝΊ f EMER \·Έ.ΕΚ[Λ1·> ΒΙΕΗΑΙΙΙ<11ΙΛΛνΚΑΡΚΕΕ·ΑΙΝ\·Υ]|.Ν·ΑΕΚΕΙΙ1ΩΙ?Ι1Ι)ΕΕ1)Ι·,ΑΚΙ,ΟΝΚ.ν^ ΜΙ AQTEI.K El SM WWE1 J.GLPA K MG I'Fl i I )R LLENJILFS1AVVVEPQ YSQCRVA1T K ΑνΤδίΤΑΜΠΠΓ^Τί\ΤιαΤ.·ΓΟΕΚ\ΤΟΟΤνΚΜΙ)Ε€Ινί^ΕΡΕΥΜΚΙΑΥΤ.ΑΕΥΝΤΤ N ET A Y11LKE KGFNATHYLKKL WAMt JSNAY FKEAQ WFNSGYIPKEJ1EY l JONAL VS VG.APFYTGLSYPMIQQQISKEIDLIPEDLNLLRWASIIFRLYDDLATSKAEQQRGD. V PKS K ÍCY Μ H ETGSS EE V.AtN Η1RI) IJ SI )A WK Η VN AECLK1 'TS1

[211] The underlined sequence is the MBP. Seq Id No: 6 PcrtE-TRX-CiCaSSy - Nucleotide Sequence Petition 870190082172, of 22 / 08 / 2019, p. 113 / 248 107 / 120 ​7'6(14 / WGCGGGGGíXÀ I GGCt 1ACGGCCAWGCGCTG'G. rn;rac;7TGi:GR:G.4CCT(77CTTC(;GT6C'C i tgcccgtga cgcgatg tggc AGG(XiCATGGGGC.GTTGCGG. t TCCGGTCGC. 17'6.1 CTG. 4 CGCÀA CGAA GGCACA T ATGTCGGACAAGATCATCCACCTGACCGACGACAGCTTCGACACCGACGTGCTG AAfí(jCC;GACGfKIG.CCAT5XTC.GTC(LATCT.CTG.Gl»CCG.AAT<Ífí.TG.CGGCC.C.CTfj.L· .1V}ATG ATCG(XH'X'GAT (Ύ ΊΤ?(.ίACGAGATC GC CGACGΑΛΤATCAG GG ÇAAGÇ1G ACCGTCGCCAALíCTÍÍAACATCíACCAG.AACCiCiGGCACGGCGCCG.VAATACGGC AAGGTCGGCGCGCTGTCGAAGGGCCACXTl’GAAGGAGT'JXX^TCGATGCGAACCTC GCC.G.GTG.GT(MTGACQACGACAAGATTA1GGACAGCATGGAAGTCCGGCGGTCG G CG ATCTACCACAG CACGTTCTGGGACATCGACAGCATCCGGG CG CTCCTGGCG CGG CGG G ACTG C ACGG CG G CC GCGGCCCTCTCí 4CCCG AC C A C C ATA AGCG (' f ΊΌ ;ÍA(4G AG CGC ATCCAG CGCCGC CTCCAGGACATC ACCCAGCCCCAC C ATCTGCTC GGCrrCArCiJACGCCGTGCAGCGCCl'GGGCGrGGCUTACCAGTTCGAGGA^VGAG ATIITCGGACGCGCTGCAWGCCTCCATTCGGAGAAI'ACCGAGCACGCCAI'CAAG GACT<XK1TGQ\CCATACGTCGC1X.,TAT7’4CCGCCTGCTCCGI:CAGCATL1GCTG(:A ACCTGTCGTCGGACÀTCTTCA.ACAAGrl1TCAAGAA(4(iALÀGGCGGCGGClG'CAA(.;G CGTCGCTCKtCGAGGACGCCATGGGCCTGCTCTCGCTGTATGAGGCCGTGCGCC T( lOGGTGAAGGGCG AGGCCATí ,'CTGG AGG AAGCCCAGÍ JTGl’I’t )TCG ATCG CCA ACCTGAAGATCCTCATGGAGCGCGTGGAGCGCAAGCTCGCCGACCGCATCGAGC ATGCCí rGGAGATCCCül XCl’ATTGGCGCG C CCCGCGTim'iGAGilCCCGCTGflT ACATCGA(1(7rGTATGAG.AAGGXAGA.CGGCCGCATCGACGACCTCK1TCGACTTCG (,GA.\GCTGGACTrrCAAC!CGCG,r,GCAGArrGCTCTÀTCAGA(.,CGAGC,í,GA;\GGAGC K7rCGÁTGTCGniGGAGCTCC1X}GGCCTGCCCGCCAAGATGGGCTTCr['TCCGCG ACCGCCTGC1XJGAGAA(?CL\CCCTCTTCJ7 / GA4'CGCC(ri,C1<,yrGGTOGAGCCCCAGT AC' 'CGGGAGGCCG Γ GGCGC ACC A AGG CG ATCGTGCTG A' J 'GA CG G CG ATGG AaU(^TCTATGACGT(.}Cí\TGGC-(7rGC(.:GGACGAGCTCA2\GGTGTrrCACC-GACA ( TGTACTATCTGGCCiC'T'CTACAACACCACGAACGAGACGGi-CT.-ntATCATCCTGAA (KiAGAjVlGilCTTC.AACilCCACGl'ATTACCTGA.AGAiYGC'rCTllGClCCATGCAGTC GAACGC GT ATTTCCG CG AG G CCCAC AG CCC ACTG ACTG ACTG ACTG CCG A AGTT C. Petition 870190082172, of 22 / 08 / 2019, p. 114 / 248 108 / 120 GACGAGTATCTGGACAACGCCCTCGTGTCGGTGGGGCGCCCCGTTCGTGt.'TGGGC C / l'CTCGTATCCCATGAJOCAGCAGCAGATCTCGAAGGAAGATCGACC'J'GATCU CCGAGGACCTC / AV'CTGCTUCGCT(1GGCCTCGATCATCTTACCCCTGGACCGGATCGACTGGACCGATΧ AAGGCCGAG (IAG( 'AGCGCGGCGACGTl JCCC AAGTCGATCí ?AGTG í ?ΤΛΤΛτα c ATG AGACGGG CTCGT CGG AG GAAGTGG CG GCC AXCCATAT CCG CGACCTGATCTCGGACGCG1OGAAGGAAGGAGGAGGACCGAGCGAGGAGGGAGGAGGAGGAGGAGGAGGAGGGG ('TCGCTCTCGAAGCACTACGTGGGCGTGGCCCCCAACTCGGCCCGCTCGGGCGT GCTCATÍÍTATCCACCATÍΪACTrCt5ACGGCTTCGCGTí'GCCCCATGGCCG* ACGAAC GCCCACATÍACGAGCATCTTCTTCGAGCCG

[212] Sequence in italics is the PcrtE promoter; the underlined sequence is the TRX with optimized codon. Seq Id No: 7 TRX-CiCaSSy - Protein Sequence MSnKTIIlLTDDSFDTDn.KADGAILVDF^LAEWCGPCKANAPIT^EIADEYQGKLTV DDPKIMDSMEVTRSAlYIISTFWDIDSIRALLxARRDCTAAALSPDHIIKRLK.ERIQR R]í .QDITQPHHIJG1J1JAVQRI jG VAYQFEEEISDALHGI jHSENTEHAI KDSLHHTSJ, YFRI JJ{QI IGCNLSSDÍFTIFEGGGFKASI -CEDAMGLI.S1ATAVRLSVKGEAILE EAQVFSI ANl.KI J ΛΙ ERV E liki ADR IEIJ ALE lí 'LA WR AI 'RI ,E ARVVYIDVY E KE DG RI ϋηΕΕΟΕΑΚΕυΚΝΚν(ίΜ1.Υί3ΤΕΕΚΕ[.5νΐνν ννΕΙ.Ε<ΪΙ.ΡΑΚΛΗ11'Ί·,ΚΙ)Κυ.ΕΝΗΕΕ8ΙΑ \ΛΆΈΡ4Υ8(ΜνΑΙΤΚΑΊ\·ΊΛΓΓΑΜΒηΡΥΠνΝΤαΕΓΏΕΜ<\ΤΤητνΝΒΛΛΌΤΕΕΕΜΕΜΕΜΜ. ΛΎ LA 1ΛΤΝ Ί TNE ΤΛΥ11LKE KGF NAT I imílíl AV Λ MQSN ΛΎI' K I'AQ WFN S GYIPKFDEYLDNALVS^OAPFXTGLSYPMIQQQISI^EIDLIPEDLY RQRVIRL Y I) VPKSIQCYΜ H ETGSSEEV AA N Η I RD IJ SD AW KE V NA EC L lí PTSLSIGnT7G\t\I)Nfij\IíSGYTMYIIIIDFDGFÃSr)II(3riTN.\IIITSIFFEP\TLKESIN LG

[213] The underlined sequence is TRX. Seq Id No: 8 TS23 FW - Nucleotide Sequence Petition 870190082172, of 22 / 08 / 2019, p. 115 / 248 109 / 120 thing^waterATGGACTCCATGGAGGTACGíXGCTCTG Seq Id No: 9 TS23 RE - Sequence of Nucleotídeos ar.al.gcgg <ygcTCATCCC.LMmGArlOGAnX’:ClT(L\,¥rGGCACTG Seq Id No: 10 TS23-1 - Protein Sequence ΜI )S Μ KV fíl í SA NYHS'J'E W DI I )S1 HA IJ ,Λ R ft I )CTVA A AI ,SH IlH HKRT JCER1Q It It I jQ ​​I) ITQPIIULLG LIDA VQRLGVAYQFEEEISDALHGLI ISE NTEHMKDtíLIIHTSLYritL VEST S NLKI EMEItt 'ERKLAD It ID 11ALEIΓ L YW1ΪΛ1 Ίΐ Έ2 YR W Y1D V YE KEDG RI DDL LJ )FAK1. DEN R VQM J A'QTE 14\EJ SM WW EIJ Ό1J Έ KMG EE RD KJ JdESJ I Li'S IGV W EPQYSQCRVAITKAIΛΊ JG AMÍJIJFYDVI IGLPEELKVrrDTVNRWDLEGl DQLPEY MK1A Υ1^1ΛΝΓΙΊ,ΝΕΤΑΥυΕΙ\ΕΚϋΙ'ΜΑΊΊΙ,ϊΈΚΚΙΛνΑΜ(ί$ΝΗΥΡΚΕΑί^ν\ΤΝ8αΥ1Ι' KFDE\TDNJAI,VSVGVPLLJ .GfKYPMK^^I IISKAEI DI JPEDLNTLWASl TERLYND LATSKAEQCjRGDVTKSlLjCA'MTTETG.SSEEi'AANinriDI JSDAWKELNAECLKFTSL SKI IYVGVAPNSARSGVLMYIIJ IDl'DGFASPIISRTNAI UTSIFJ-'EPVP LKESINLG Seq Id No: 11 TS23-2 - Protein Sequence

[214] MDSMEVltltSANYHSTlA\DlDSlRjYLL_UtRDCT\iVY\LSlIDllHMtLKERlQRRl / ÍD ITQPH11LLG1J J) AVQRI JIVA YQI ΈΕΕIS DA I ,HG IJ I SEN TEI 1 AVHI KlHI YFR ), ITSL]] LJiQIlGCNLSTDIENKFlíFÍEGGGFÍASLClCDAMGl.l^lATAAIlLGXqíSEAILEEAQ λΤ5Τ8ΝΕΚΤΤΛ[ΕΤ{\ΈΗΚΕίλΠΠΙϋΤΪΑΕΕΤ ΡΪΛΥνΕΛΡΚΥΈΛΚ\\πΠη\ΊΈΚΕ·θυΤΐΙΟΠΕ LJ )FAK1.1) ΕΝ K VQM AND QTE1 KE J ,SM W WEI J.G1 .PEKMG EE RD KJ .LES11LFSIG VAT ΕΡί^^ίΚΒλοίΙΤΙ^ΧΕνΕΓΤΑλΙΒΒΓΥϋπίΟίΡΕΕΕΚνΤΤΤνΝΊΑΥϋΕ MKLYYJ ΛΙΛΝΊΊ Ν ETAY 111 KE KGFNATHYL KK LW AM QSNSY ER EAt í WFNSUYIP 1G-DE YLD N AL VS VG VIJLLL GLS YPMIQQI11SIÚAE1DLI PEDLI LW1 LW1 LW YN LW LATSKAEQ(jRGDVPKSlQC!YMHETGSSEEVAANI IIRDL1 SDAWKEVNAItGI KPTS LSK]JYVGVAPNSARSGVIΔ1ΊΉ1IDFDGFASPTISRTNAlIÍTSTPI EFVPlKESTNLG Petition 870190082172, of 22 / 08 / 2019, p. 116 / 248 110 / 120 Seq Id No: 12 P7 - Nucleotide Sequence GCTGTTAGCGGGCCCATTAAG SEQ ID NO: 13 P2 - Nucleotide Sequence GATATTCTCATTTTAGCCATTTAGCTTCCTTAGCTCCTG Seq Id No: 14 P5 - Nucleotide Sequence CAGGAGCTAAGGAAGCTAAAATGGCTAAAATGAGAATATC Seq Id No: 15 P6 - Nucleotide Sequence CCAAGCGAGCTCGATATCAAACTAAAACAATTCATCCAG Seq Id No: 16 Nucleotide Sequence CTGTCCATGATCTTGTCGTCGTC Seq Id No: 17 Nucleotide Sequence ACTGGCCTCAGA\TTCA'A\TTTATTTGCTTTGTGAGCGGATA\C Seq Id No: 18 Nucleotide Sequence CAAGATCATGGACAGCATGGAAGTC Seq Id No: 19 Nucleotide Sequence Petition 870190082172, dated 08 / 22 / 2019, p. 117 / 248 111 / 120 TTTATGATTT GGATCCTCAGCC C AGGTT Seq Id No: 20 Nucleotide Sequence CTGTCCATAATCTTGTCGTCGTCAT Seq Id No: 21 Nucleotide Sequence ACTGGCCTCAGAATTCCGÜTGCTGAACG Seq Id No: 22 Nucleotide Sequence CAAGATTATGGACAGCATGGAAGTCC Seq Id No: 23 Nucleotide Sequence TTTATGATTTGGATCCTCAGCCC AGGTT Seq Id No: 24 PcrtE-TRX-SaSSv Nucleoside Sequence Petition 870190082172, dated 08 / 22 / 2019, pp. 118 / 248 112 / 120 CGC7'GC7'GAAC(r'CGA TGGCGi. CCTG T1'G TGGITGCGTCGACCTGTf TIXIGGGCCA TGCCCGTG. i CGCG.4 TGTGGC aggcgcatggggcgttgccgatccggtcgcatg. í ctg icgcaacgaaggcacat ATV^l1 <?(lGACAAGA,T,CAr11CCACCTGACCGACGACA(lCTTCGACACCGACG,i,CTiHTG AAGGCOGACGGCflCCATCCTCGTGGATTTCTGGGCCG.AATGGTGCGGCCCGTGC AAGA,rGA,rCí.K.:{}(3GGA1'CGr€GACCÍAGA1,CGCC{jACÜAArrATCAGGÍjGAAGU'],G Α^ίΐΤίΧίίΧΙΑΑσΟΤΐί.ΑΑΟΑΤαΐΑί / ΟΑσ .ΛΑΟσ^ΟΌΰίΙΑΟίίίίσΡΟί^τΑΑΑΤΑΟί^κ: ATCCGCGGCATCCCGACGCTGCTGCTCTTCAAG.AACGG()GAGGTGGCGGCCACC .AV.;GTGGGCGCGCTGrl1CCÍAAGGGCCAG <H1G.A-\GGAG;J'rrGCTCCÍArlGCG.AÍ(JCTC· GCCOGTGGTGATGACGACGACAAGATTATGGACAGCAGCliCCGCGACCGCCATG ACCGCCCirCl'TCATiXlACCCC^AClTGACCACGTGAACCTCAAGAiXXkACACCGACG aiAGCGAGWYCt^GrCijCATt^KiCWXCrACWVGCCGTCGATClXjG.VXC'lAWACT Petition 870190082172, of 22 / 08 / 2019, p. 119 / 248 113 / 120 TCCTGGAGAGC CT CGCC ACC CACCAT AÀC AT C GTG G AG GAG CG CC ACCTCA At} C TGGCGGA· jAAGCTGAAG <iGCCAGGTCAAG'I TCATG'I L CGGCGCCCCTATGi 1AGC CCCTCGCGAAGCTCGAGCTGGTCGACGTGGTCCAGCGGCTCGGCCTG.UCCACC ' IX .ITTCG AG ACC G A ç ATCAAGG A AGCC CTCT TC T CG ATC T AC A AGG ACGG CA GCA ACC GGTG GTG G TTC G G CCAC C TG CATG C G ACGTC G CTCCG C TTC’CG G CTG GTC C GCCAGTGCGGtTOTGTIOATCCCCCAGGACGTGTTCXULACCTTi'CAGAACAiVGAC GG G Í.XJ AGTTCG ACATG A AG CTCTGCGACAACGTCAAGGf > CCTGCT CTCGCTGTA CG At iGCCAGt ITATt 'TCG GCTGGAAG GGCG AG AACATÍ X(TGGACG At 1GCCAAGGC GTTCACXAAtXiAAGTGCCTCAAGTCGGCCTGGGAGAACATCAGCGAGAAGTGGCT t}G CGA AG CG CGT AGCGTCGGGGTC(ΛGCCG' C CGCT C CATTGGCGCGTCC C CCG GATíXLAGíXXtCGGTGGTTCATCGAGGCCTÀTGAGCAGGAAGCCAACATGAACCC GACCCTGCTCAAG CTC G CCA ACKd'CG AC'TTCAAC ATG GTGCAGTCG ATCCACC AG AAGGACkATCGGCGAGCrGGCGCGGTGGTGGGTCACCACGGGCOTGGACAAGCTC GC Crrrct iCG OGG AAC AAC CrΓΰ C Ί Ό C AGTC GT AC XTGTG GAG CTG C ​​GC C ATCG CG; ΊΧΧ1ΠΛ0(?(?·ΰΑΛ{?τΤΊ,1\Α4(.·τυΤΙι(ϊ(ΧΧ / Ίτί·'ΟΑ(ιΑί?0Λ'1,υ<1Τ{ΪΟΑ(.τΛΊ'(3ι1(Ίτ((Ί'ε(Ίτ(1Ίτ'(Ίτ'(Ί'ε(Ίτ(1Ί'')(ΐ ATGGCAGCATCGACGAG CTGGAC CTCTATACCTCGAGCl TTGGAG (X '.GTGGTt X1TGCGTCGAGATCXCACA AGCTCCCG AACACCCra.AAGCTCATCTTCATGTCGATGTCCAACAAGACG.AACtLXGGTCGClCC TÍX?GCGTCCW1CAT11AGCGGÍ1GCTACA;\CTCX1AT(X'CCACGTTCATCAA(3GC('!T GGGTGGAt iCAGTGCA AGTCGTA'I'CAGA AGG AAGCCCGCTGGTTCCACCX1TGGCC A'lWCXlCCGCCCCl'GGAGttAGTAC'IOGCTGAACGGCCTCG'I'GAGCA'rCGGClTtC CGCTC7CTCCT(iAirAC(JGGCTATGTt;GCCATCGCGiiAGAACGAGGCCCTC(ICT({G ACAAGGTCCACCCGCTCCtXXlACC'rGCTGCAI'TACTCGAGCCTCCTG'l'CGCGCCT GArl'CA;\CGACATCGGCA(X'AGCCCCGACGAGATGGC«CGGGGCCTACAACCrrCAA}GGCGTCATCGAGGAGAACT( JGAAGATCt Yl'C AACCAi ÍTt JCTGCTT CG AC CAG TC GCAGTT CCAGt 2 AGC CG TTCAT CACCTT (A1ACCT C-kACT CGGTG CG C GGCAGCCATΊ TCTTCTAÍJGAGTTCCGGGACCGGGACTGGGGGGGGGG ACGAAtiGT(.UTACATGAAGAGC(lTGCTGATCGACCCCATCCCCCTGGGCGAGGAG TGA.

[215] The italicized sequence is the PcrtE promoter; the underlined sequence is TRX with an optimized codon. Seq Id No: 25 Petition 870190082172, dated 08 / 22 / 2019, pp. 120 / 248 114 / 120 TRX-SaSSy - Protein Sequence MSDKTnil / J'DDSPDTlJVLKAJXhMLVDFAYAEWCGPCKMIAPILDEIADEYQGKLTV AKLNT DQNI 'GTAPKYGTRGIPTIJ ..LFKNGEVA'XTKVG ALSKGQLKEITDA-NLAGG I> DDDKI M DSSTATAMTAPFl DPTDH VNLKTIJTDAS ENRRMt 5 NYKPS1WNYDF1 .QSL ATI JI INI VEERl I J. KI ΛΕΚΙ ,KGQ VKFM FG Al'MEPLAKLELVDWQKLGLNl ILFETEI KEALFS1YK DGSNG WWFG1! 1.11 ATS!Jd'RI ,1J1QCGLFI I >QDVF KTFQNKTGEFI) MK LCDNVKGLLSLYEASVLUWKGENILDEAKAEn'KC'LKSAWHNJ.SICKWLAKRVKH AI ,AI .F LH W ft X PR.IEA RW Fl RA Y EQ ΚΑΝ Μ N FT IJ Ji 1,Α K LD FN VIVQSIH QK RI GET A RWWATTOLDKkW.XRNNLLQSYlVIWSCYU.^SDPICFKL.XRETI^IGSATTX-AODGY DVYGS1 DEIJ.H ATSSVERWSCVEf BKLPNTI Ji] J FMSMFNKTNEVG] □RVQHERi i Y MSI 1 TT IKAWVE Q CKS YQ ΚΕΛΚ WF1LGG H TRI 'LE E YSL NGL VS IG FPLLLITG Y VM A EVAREHIKGVIEENWKIJ AQCCFDQSQFQEPFITFNl ,NSVItGSl J FFYEFGDGFGVT DS WT K VD M KS XT. 11) 1111TGEE

[216] A sequence linked to TRX. Seq Id No: 26 NusA - Sequência de Proteínas MNIAT;1E.\XAELV8KEIU\EP1TEEK1EE;iEE^\TATLIIID<KYE^E1DVN0IUTIITK80OE OTEKK\vGL'\I)E\T(^'TKEGTEEAAH.YEI)E«I.NIOBYAEPITIILEEKVNTKnKITT£ / GAK(i VIX'QKXTiE.YElLUlVVDQniEIIE'GEllTGXA'KKVNliDNlSLDLGNNjYEAMLREDM LFRENFEFGnRX RGVLYSX RPEARQAiilJ XI'ItSKPEMlJELJ RIEYFEIGEEXTEIK A-^ARDPCTSrUKTAVKrNDIGilDPVGACX'GMRGARVQAVSTEUJGERIDIXTAVDDN PAQFXT NAMAPADXIXSTXTDEDKT TTMDIA VEAG NT AQAIGH NGQNATtLASQLSG XX' ELNXlm'XTHlIXMKHQAEAHAAJDTFWYLDlDEDlhVlVTXTEGFSTLEElAYVTM KELLEIEGLDEPTATALRERAKNALATIAQAQEESLGDNKIADDLNLLEGVDRDL AFKI .AARG VCT [□EDI □AEQGIDIJ LA DI E( Ϊ1IFDEKAGAJ MAA RNIC WFG DE'A Seq Id No: 27 TRX - Sequência de Proteínas MSDKTIHLTDDSFDTDXTJÍADGATLVDFWAEXVCGPGKMIAPILDEIADEYQGKITV AKENIIIXΙΝΡΟΤΛΡΚΧυΙβΤΙΡΤΕΕΕΓΝΟΕν^ΓΝυΑΕΚΚβΙΕΙΕΙΕυΛΝΝυΑαδΙΙ) DDDKI Petition 870190082172, de 22 / 08 / 2019, pág. 121 / 248 115 / 120 Seq Id No: 28 MBP - Sequence of Proteins ΜΚΤΕΕΟΚΊ A'i W1N< jDKGYNGLAEYG KKFK K DTG Í KVTVEHPDKLE EK FPQVAATt ΐ IJGPGlllWAl I DKF.QGYAQSGLLAEITPI.IKAFQI)KLYPFTVVDA VILLAYVLAYV ,SIJYNKDIJTNPPKTWl·:KI PA' / )ΙχΈί,Κ.ΛΚ(!KSYl AIFNLQEPY FTWPIJ AAf)G GYAFKYENGKYDIKDAOWNACm^GLTFLWLIKNKIIM^YDTDYSL^AAFMK NGTFLWLIKNKIIM^YDTDYSL^AAFMK NGMT1 VGJPT VGJPT VG. FKGQPSKI 'FVGVLSAGl NAASPXK E LAKE FLE N YLLT DE GLE AVTJ KDKPLG A V1ÍLKS Y EE ​​E ΕΛΊίΡΡΚI AATME(NAQ KG EI Μ PN11>QMSAΙ '^ΎΑΑίίίίίQA I)1 <Aί JÍI)AQTGIJDDUK[ Seq Id No: 29 Set - Protein Sequence EE ASVTSTKETl ZIFA( ^E AA KT] ίΛΑΝΚΑΚΚΕΑΕ I -AAATAEQ Seq Id No: 30 NusA-OiCaSSy - Nucleotide Sequence ATGA.YCAi\GGAGATCCTC.GCGGTGGTGGAGGCGGTGTCG;LV?GAGA\GGCGCTG (x:ccgcgagaagatcttqiàggccctggagtcgg(.xxtggccaccgcgaccaag AAGAMrrACGAGCAGGAGATCGACGTtXXXXGCAGAGCGGCGCGCGC GACTTÇGACACGTTCCGCCGCTfÍGCTCGTGGTGGAlFGAtÍGTGACCÍlAíiCL CAOG AYGGAGATCAC.CCT.GGAÍyÍj-CGÍÍ.GCC(Íf)TATGAGCÍACGAGT.CG.CT.GAACCrTGGGG ga(:iaix.;3x;c;aí}g.íCí'ag;\tc(.;agiogG4t.;a(x;ttuga(:cgcaiiíiacci^ <kiag ACG f t C G AA G G A GGTG ATCGT’G CA G. L4GG Ί GCGCGAf Ί G CGG AGC G CGC CATGGO1j 1GTGGAGUAGTTCCG C-GAGCACG AQGG C.'OAGA,l1CArT1CACCGÍJCtyrGGl1G.4AG AA CCrrGUGCGAG(3ACArl1GCT(X:CGCGGGAGAACTTCCGCCCGGGCC;ACCGCGrrdCG aiGCGTGCTGTATTCt FrGGGGCCCGAGGCGt:GTGG(:GC(X;\GCTGTTCGTGAC Petition 870190082172, of 22 / 08 / 2019, p. 122 / 248 116 / 120 CGGCGAGGAAGTGATCGAGATCAAGGCGGCCGCCCGCGACCCGGGCTCGCGCCG (XíAAGATCOCCGTGAAGACCAACGACxAVjÍXjCATCGACCCCGTCGGCGCCTGÜG tgggcatgcgtggcgcccgcgcggccgtgtcgaccgacgcggcggcgcgg aiÍLVrCGAtjATCGTOíUrf^GGt^CGAÍ^AACÍAIGGfXlílAGTTCílTGATÍlAACCÍCC ATG GC CÇC GG ÇGGA CG TGGCCTCG ATCGT GG TGG ACGAG G ACAYAG CAT ACCATG GACATCGCCGTGGAGGCGGGCxAACCTGGCCQAGGCCATCGGCCGCAACGGCCAG AACGTGCGCCTGGCCTCGCAGCTCTCGGGCTGGGAt^CTíÍAACGTGATGACGGTG GACG ACCTGCAGGCCAAG ί AITCAGGC :CGAGGGCarrGCC AGC-XCCATCGAT? ACGAAGTACCTCGACAlGGACGAGGACrtTCGCGACVGTGCTOTGGAGGAAGGC TTCTCGACGCIVíGAGGAGCIVCCCTATGTGCCGATGAAGGAGCTGCTCGAGATC GAGGGCCTÍi}AC<^AGCCGA<^GG:rGGAG<^GCri^CG<-GAAA!)<-ACGCGAGGCGA cctggccaccatcgcccaggcccaggaagagtccctgggcgacaaagccggc (JGACGACCTGCTCAACCTGGAGGGCG'rGGxAUCGtJGACCTGGCCTTCAAGCTCGC (y'lCCCGCGGCGTGTGCxACGCrCGAGGACCTGGCCGAGCAGGAGCGAGGACCGGCCGGCCG ATCGxAGGGCt JTC At J CGACG At tAAGGCCGGCGCC (.''PG ATC ATGGC CG CCCGCAAÇ ATCTGCTCGTTCGGCGA CGAGG CG ATGGAf'AGCATGG A ACiTJX '.GGCG rfTCGGC(^ATCTACCACAGCACGTTt:TGCCACATCGACAGt:ArrCCGGGCGCTCCT GGCGCGGCGGGACTGCACGGCGGCCGCGGCCCTCTCGCCCGACCACCATA.WCG CCTGA;\GGAG<y]CATÍXW.lCílC(VrCCTCCA<.lGACArIOA.CCCAGCCCCACClATCT οχ·;Ί'(.·,Γτί.·;(χ?Ί'(λγ[’ϋοτΑ(ι.(·?τ(Χ’ίΐΊ'(;ΓΑ<^·(:κ,ΓΊ,(]σ(·;απΊχ·χϊί)οτ.Α€€Α(ΐττσ(}.Α(ΐ(ΐ.\ AGAGATGrCGGACGCGCTGCACGGCCTCCAlUGGGAGAACACCGAGCACGCCAl· CAAGGACTCGCrrGCACCATACGTC(B^rCTArJ,frTCCGCCTGCTCCGCCAGt?AT(}GC TtlCAACi / TGTCGTCiJGAtlATi'T’TCAACAAG'lTCAAG.AAGikAAGGCGGCGi ICITC A.XGGCCTCGCTCTGCGACTCTACGCCATGtSGUCTGC'l’CTCGC'rGTAn’GAGGCCG'rG (XICCTGTCGGTGAAGGG CGAG GCCATCCTGGAGG AAGCCC AGGTGTTCTCGATC GCtASAtxrrGAAGATCClCATGilAGCGCGTGiiAGCGCAAtii^TCGCCGACi’GCATC GAG CAT G CC CTGGAG ATC CCGC TCT ATTG G CGC GC C C CG CG TCTGGAi j GCCCGC TGGTACATCGACGTGTATGAGAAGGÀAGACGÍ^CGCATCGACGACCTGCTCGAC 1''I,CGCGAAGC'rGGACTrrCAACCGCGTGCAGATGCrrCTATCAGACCGAGCTGAAG aArtCTCTCGATC^GTGGGAGC4X;CTG(.;GCCTCCC(XlCCAAGATi;GGC'rTCn'C CG (XIACCG CCTGCTCC: AG AACC! ACCTCTTCTCG ATCC CCGTGGTGGTGG AG CCC CAGTACTCGCAGTGCCGCGTGGCCATCACCAÁGGCGATCGTGCTGATGACGG ATG GGCCATCAT(ITGGCTGGCTGATGGCTGATGGCTGGGGGGGAT) JCCGÍLWGAGCTCAAGGTÍ HTCACC. Petition 870190082172, of 22 / 08 / 2019, p. 123 / 248 117 / 120 GACACGGTGA.\CCGCTGGGACCTGGAGGGCATCGACCAGCTCíX:CGAGTACATG AAGCTOTACTATCTOGCGCJITTACAACACibACGAACGAGACGGCCTATATCATCC TGjA\GGAGAAGGGCTTCA.\C,GCCAC(j1CATTACCTGA2\GA2\GGACTGGCCATGGGAGGGAGGGAGGA2 GTATTTCCGCG AGG CCCAGTl IGTTC A Λ CTÍ XJ GG CT A ( ' ATCCC GA AGTTCG ACGAGT ATCTG GAG AACG C GCTCG TG TCGG TG GGCG C CCCG TT CGTG C lOGGC-CTCTCGTATtXCATGATCCAGATCGATCGAGGATCGAGGA GArCCCCGAGGACCrCAACÍ^GCTCCGCTGGGCCTCGArCATCTTCCGCCTGTAC GACGACCGTGGCCAtXTCGAAGGcXOAGCAGCAGCGGCGACGGTGCCCAAGTCG ACC'11GATdrCGGACííCG TGG A\GG AA GTGAACG CCGAGTG CCTG A AG CC< JACCTCGCTt^TíXJAAGCACTACGTííGGrCGTGGCCCCCAACTCGGCCCGCTCG GGrxriOCTCATGT ATCACCATGACTTCGACTCGGTCCGTCGCCTC (.XlAAClICCCAC^TAACGAGCA'IOTTCTrCGAGCCGGTCCCCCTCAAGtkAGAGCAT CAAÍVTGGGCTGA

[217] The underlined sequence is the optimized codon NusA Seq Id No: 31 SET-CiCaSSy - Nucleotide Sequence Petition 870190082172, dated 08 / 22 / 2019, pp. 124 / 248 118 / 120 ATGGAGGAGGCCAGCGTGACCAGCACCGAGGAGACCCTGACCCCGGCCCAGGAG GCCXjCCCGCACCCGCCGCCAACAAGGCCCGCAAGGAGGCCGAGCTGGCCGCC GÇCACaiCÇGAGCAGGCCGCeAlGÍjACAGGATGGAAGTCCGGCGGTCGGA CAGTC ACGTC ACCGGA GG AC AT CG AC AG C Ar[ CCGG G CGCTC CTGGC G CG GCGG GACTi X1ACGGCGGCCGGCCCTCTCGCCCGACCACCATAAGGGCCTGAAGGAG (X3 C ATCC AG CG CCG C CT CC AG G AC ATC A CC CAG CCCC ACCG TTCCACTCG TíXIAíOCCGTGCAGCGLJCTGGGCGTOGCíTACCAG riOGAGGAAGAGATC'rCGG ACGCGCTGCACGGCCTCCATTCGGAACACCGAGCACGCCATCAAGGACTGCGC 1'G(:ACCAT'AC(.riX,GCM,Cr'.\TI^'CCGCCrGíirCCGCCA<7CA'rGí.;C,rGCAACC,rG'l,C GTCGCkXCATCTTC^kCAÍGTTC^iGXXGGXUjGraGCGGCTTCzUGGCCTCGCTGCTG (^GCGAGGACGCCATGGGCCTGCTCTCGCTGTATtlAGGi'CGTGCGCCTC'rCGGT GAAGGGCGAGGCCATCCTGGAGGAAGCCCACKH'GTTCTCGATCGCCAACCTGAA (LA'lOCTCCA'lOOAGCGCGlOGAGCCGfLXAGCTCGGACCGACCGACCATGCCCTGAA GGAGATCCCG(nVi\AlTOGCGCGCCCQjCGTCTGGAGGCCCGCTGGTACATCGA (ΧΙΌ nWTGAGAAGG AAGACG GCCGCATCGACGACCTGCTCG ACTTCG CGAAGCT GG ACTTC A ACC G CGT G CAG ATG CT CTATC AG ACCGA G CTGAAGG AG CT CTC ' .1 AT GKKiTGGGAGCTGC'l'GGliCCTGCCCGCCAAGArGGGClTCTTCCGCGACCGCCT gctcgaga^xccaccctctctc GTGCCGCGTGGCí ' ATC ACCAAGGCG ATCGTG CTG ATGACGGCGATí 1 (lACGAGrr CTATG ACGTGCATG G CCTGCCGG ACGAG CT (?AAG GTGTT CAC CGACAC G GT GA\ CCGC1'GGGA(X:TGGAGGGCATCGACCAGCTCCCCGAGTACArrtiAAGCTGTACTA TCTGGCGCTCTACAACACCACGAACGAGACGGCCTATATGAICCTG AAGGAGAA GGG in :rn,CAACGC(jACGCarn,At;Crrt JAAÍ; GTAn'TCC(l(X]AGGC(XAGTO(}rrTCAACTCGG(}Cr]'ACATCC(?GAAGTTC<.iACGA GI'ArCTGGACAACGCCiTrCGIirrCGGl'GGGCGCCCiXTrTCGTGCTGGGiX / TCrC (ÍTATCCCATCAGGATGACCA<ΤΤΤΤΤΤ ÍGAAÍ ÍAGATCG ACCTGATCCCCGA GG ACCTCAACCTC CTCCGCTGGGCCIXOATC.ATCTTCCGCCTGTACG A CG A CCTG G(.TACX1TCGAAGGCCGAGCAGCAGCGCGGCOACGTGCCCA.AGTCGATCCAGTGC TATATGCATGAGACGGGCTCGTa^GAGGAÀGTGGCGGaAÁACeATATCCGCGAC (. CTCTCGAAGCACTACGTGGGCGTGGCClJCCAAí.TOSGCCCGCTCilGGCGTGCTC ATT 1 TATCACCATGAtvrTCGACGGCTTCGCGTCGCCCCAT(^GCCG CACGAACG CCC ACATCACGAGCATCTTCTTCGAGCCGGTCCCCCTC.A.\GGAGAGCAT(LAACCT(!GG CTG A Petition 870190082172, dated 08 / 22 / 2019, pp. 125 / 248 119 / 120

[218] The underlined sequence is the optimized codon SET tag Seq Id No: 32 NusA-CiCaSSy - Protein Sequence h1NKETLAV\^AVSNECALPREKTFEAT,ESALATKKKYEQETDXTlVQTDRK8GDF YIXOKXTtE.AER^iXAOQFREHEGEIITGXATtKVNRDNISLDLGNN.AEAXTLREDM LPKENFRPGDRVRGVI Λ SYR]* 1EARGAQLFVTKSKPEMIJ ELEK IE yPEIGEEVI E1K A^XRDPGSR.ÜÍIAVdÍTNTlCRIDPVGAC.AOMmVÍVQAVSTELGGERIDIVLWDN PAQFVr NA \f ΛΡΑ DVASI VVDEhKIITM T JIAVEAG NI -AQAIG R MGQNVRI ASG I ,SGW ELNWAKWDΑHWD. AAWTFTKYLD1DE1) FAT VLVEEG FSTLE E1,A YV PM KELLEIEGI JjEPTVEAl JiERAKNALATlAGAOEESl XiDNKltADDLLMhEGVDRDL AHvLAAKGVCTLE 1)1 AEQGl1 )I )LA I >1EG1 ZHJEKA MEGA CAMW1 E^RSAlYHSTFVXOIDSIRALLARRDÇTAAAALSPDIIIIiníLKERIQRRLQDITQPII IILLGLTDAVQRIOVAYQFEEEISn.ÀLUGTJlSENTEHAIKDSLnrTTSTATRLT.RQTIG CNLSSI) JJNK FKKEGGGFKASLCEJMMGJ ,1 .S LYE OF K LSV KGEAl LEEAÍ i VFS IAN ΕΙ·αΕΜΕΓί\ΈΕΙίΕ.1ΣΑ:ΕΠ^ΗΙΓΕΥ\\ΤΐνΑ1Ε3ΑνΛ10\ΑΈΚΕ0ϋΚΙϋ0Ε1ΌΐΐΡΐΐΕ. I) FNRVQMIAQTEI .KE J .SM WWE 1.11JI JAK MGF FKIJ RLLENH ] .FS1AV\ΛΈΡ^ YS^ CRYÜYITKXIVLMTAMDUFYDVHGLPDELKVFTDTYNIAVDLEGIDQLPEYAIKLYL Al Λ'Ν'ΓΤΝΕΤΑΥί ILKEKGFNAT1IYLKKIAVAMQSNAYFREAQWFNSGYl PKFDEYl, DNAI A'SVG APFVLGLSXTMIQQQ í SKEEIDIJ PEDI .LRWAS11 b RI A EKDDMSQDSIQGY 1”[ GSSEEYAYNl 11 RI )1 JSIJAWKEVNAEC1KPTSLSK1IYV GVAPNSARSGVTMYHl IDFDGFASP1IGRTNAH1TSI l· FEPVP1JÍESLNLG

[219] The underlined sequence is NusA Seq Id No: 33 SET-CiCaSSy - Protein Sequence Petition 870190082172, of 22 / 08 / 2019, p. 126 / 248 120 / 120 ME EAS VTSTEETl .TP \OEA4 RTR Λ.Λ N KARKEAELA YXTAEQM Í XS Μ E λΉ HSA ϊ YHS ΊΊΛνυΐϋ8ΙΗΑ1.ΙΑΙ <ηη<:τααααι^1ί)υπκ1ΐεκεει^]ΐ1ΐεΐ^)γ1^1ήιιι.1 υ.ιεα\^ rixjvayqfeeeisdamkslhsentoilukdslhiitseyfrljjíqiigcnessdifnk fkke g gfk aslced am gij .slye ave lsuig e ai le aq \t si a ni jill λγερλέκ κεαϋριειιλεε1ριαανΐίλριΐεεαιί\ν¥ΐυνυεκ]<1χ;εΐυΐοεευΐ'1ακει)1'’νενίΐμιλ· qtelkelshlwytllillp.akaittffrdrlleniilfslavaa^r'qysqcrvaitkyil’ lmtamddfy dvhgi .pl )e1 .kvftdtvnrwdlegidqt .peymkj λϊ1.α1 .ynttnet ayi lekekgenati i yekkewamqsnayfre aqwl· nsgyip1í1’deylrnalvsmia pfvlgls ypm1 qqqis keeid li ped ln1j ji was11hi lydl11 xatskaeqq rg dm ’ k siqc ym11etgsseevaa n í 11rdij si) wke vn ecek ptslsk! yy’gvapns arsg yijviyiiitdfdg faspi ic rtnatittstffepvplkestnt λί

[220] The underlined sequence is SET. Petition 870190082172, dated 08 / 22 / 2019, pp. 127 / 248

Claims

1 / 3 claims 1. Expression vector characterized in that it comprises a nucleic acid of SEQ ID NO: 1 or SEQ ID NO: 2, and degenerate sequences thereof, encoding the amino acid sequence of SEQ ID NO: 3 with santalene synthase function.

2. Microbial host cell, characterized in that it comprises an expression vector comprising a heterologous nucleic acid sequence as defined in claim 1.

3. Host cell, according to claim 2, characterized in that the host cell is a bacterial cell selected from the group of Gram-negative bacteria, in particular, from the group of Rhodobacter, Paracoccus and Escherichia.

4. Host cell, according to claim 3, characterized in that the host cell is a fungal cell selected from the group of Aspergillus, Blakeslea, Penicillium, Pfaffia, (Xanthophyllomyces), Pichia, Saccharomyces and Yarrowia.

5. Transgenic mushroom or culture comprising transgenic mushroom cells, characterized in that said mushroom or culture comprises host cells as defined in claim 2, wherein the host cell is selected from Schizophyllum, Agaricus and Pleurotis.

6. A method for preparing santalene, characterized in that it comprises converting a farnesyl diphosphate into santalene in the presence of a santalene synthase as shown in SEQ ID NO: 3 or a functional homolog thereof, said homolog being a santalene synthase comprising an amino acid sequence having a sequence identity of at least 60% (or at least 70%, or at least 80%, or at least 90%, or at least 95%) with SEQ ID NO: 3 and having a specificity with respect to catalyzing santalene formation expressed as the molar ratio of santalene to bergamotene of at least 1.5:

1.

7. Method for preparing santalene, according to claim 6, characterized in that the santalene is prepared in a host cell as defined in claims 2, 3 or 4, or a mushroom or mushroom culture as defined in claim 5, which expresses said santalene synthase.

8. Method according to claim 6 or 7, characterized in that it further comprises isolating the santalene.

9. A method according to any one of claims 6 to 8, characterized in that the molar ratio of santalene to bergamotene is at least 1.5:1 and the α-santalene to α-bergamotene ratio is greater than 1 and / or the β-santalene to α-bergamotene ratio is greater than 0.5:

1.

10. Method, according to any one of claims 6 to 9, characterized in that the ratio of santalenes (α- and β-santhalene) to α-bergamotene is greater than 2:

1.

11. Method for preparing santalol characterized in that it comprises converting FPP into santalene in the presence of a santalene synthase as shown in SEQ ID NO: 3 or a functional homolog thereof, said homolog being a santalene synthase comprising an amino acid sequence having a sequence identity of at least 60% (or at least 70%, or at least 80%, or at least 90%, or at least 95%) with SEQ ID NO: 3 and having a specificity with respect to catalyzing santalene formation expressed as the molar ratio of santalene to bergamotene of at least 1.5:1, further comprising converting santalene into santalol.

12. Method for preparing santalol, preferably β-santalol, according to claim 11, characterized in that santalene is prepared in a host cell as defined in claims 2, 3 or 4, or a mushroom or mushroom culture as defined in claim 5, which expresses said santalene synthase.

13. Method according to claim 11 or 12, characterized in that it further comprises isolating santalol. Petition 870260065022, dated 02 / 07 / 2026, p. 14 / 19

Images