Means and methods for alpha-farnesene production

Abstract

The present invention relates to the field of the field of recombinant manufacture of farnesene and related products. In particular, it relates to a method for producing alpha-farnesene. The method comprises at least the following steps: providing at least one polypeptide exhibiting alpha-farnesene synthase activity, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; an amino acid sequence encoded by a nucleotide sequence having at least 70%, sequence identity to the nucleic acid sequence of any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; an amino acid sequence as set forth in SEQ ID NO:1; an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting alpha-farnesene synthase activity, or exhibiting alpha-farnesene synthase activity; and converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide. The invention further relates to a composition comprising alpha-farnesene in an amount of at least 40 % (w / w) and melonal. Moreover, the present invention contemplates a recombinant nucleic acid molecule encoding an alpha-farnesene synthase and a vector and host cell comprising the same.

Description

[0001] Means and methods for alpha-farnesene production

[0002] The present invention relates to the field of recombinant manufacture of sesquiterpenes and related products. In particular, it relates to a method for producing alpha-farnesene. The method comprises the following steps: providing at least one polypeptide exhibiting alpha-farnesene synthase activity, wherein said polypeptide comprises an amino acid sequence selected from the group consisting of amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; an amino acid sequence encoded by a nucleotide sequence having at least 70%, sequence identity to the nucleic acid sequence of any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; an amino acid sequence as set forth in SEQ ID NO:1 ; an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1 ; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting alpha-farnesene synthase activity, or exhibiting alpha- farnesene synthase activity; and converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide. The invention further relates to a composition comprising alpha-farnesene in an amount of at least 40% (w / v) and melonal. Moreover, the present invention contemplates a recombinant nucleic acid molecules encoding an alpha-farnesene synthase and a vector and host cell comprising the same.

[0003] Alpha-farnesene is a natural organic compound belonging to the sesquiterpene family that is abundantly found in plants, particularly in fruits and vegetables. To-date alpha-farnesene is applied or considered for application in a broad range of different sectors within the chemical industry. For example, its pleasantly, fruity aroma, make it an interesting ingredient for flavour and fragrance manufacturing. Moreover, the hydrogenated product of alpha-farnesene exhibits a high combustion heat, which makes it an intriguing bio-based alternative for aviation fuel [1], In addition, alpha-farnesene has recently gained attention as the starting point for the chemical synthesis of vitamins A and E [2-4], These vitamins are widely applied in personal care, as well as feed and food additives. The global, annual vitamin A and E market alone exceeds 5 billion USD.

[0004] T o cater such broad range of different industries including the bulk chemical production of aviation fuel and vitamins, a cost competitive access route to alpha-farnesene is essential. In addition, the alpha-farnesene must come with a favourable side-product composition to allow for its use in off- odour sensitive flavour and fragrance applications. As extraction from natural sources is not cost- competitive, the most promising route to alpha-farnesene is currently via biotechnological terpenoid synthesis. Here, farnesenes are either synthesized from a natural carbon source (e.g. glucose) via the MVA- and I or MEP pathway [5, 6], Or alternatively via non-natural carbon sources such as isopentenols, via the isopentenol utilization pathway [7-9], Independent of the starting material biotechnological sesquiterpenoid production proceeds via the universal terpene precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP). These two are condensed in 2:1 molar ratio to farnesyl diphosphate (FPP), which in a final step is converted to alpha-farnesene by a terpene synthase (alpha-farnesene synthase, Fig. 1). Terpene synthases are generally regarded as one of the key bottlenecks in biotechnological terpene production and thus critical for the final production costs [5, 10], Furthermore, terpene synthases are notoriously promiscuous and often yield a broad spectrum of products from the same substrate (here FPP) [11 , 12], To ensure applicability of terpene synthases in industrial biotech processes a favourable side component portfolio is desired, especially when the product is destined for flavour and fragrance applications.

[0005] (E / E)-Alpha farnesene is a terpene of particular interest for flavour and fragrance applications as it serves as a building block in the synthesis of several vitamins, and serves as a flavour and fragrance product. It can be produced by biotechnological means in high enantio-purity. To cater both markets, biotechnological production of alpha-farnesene needs to be at low cost. A key enzyme in the biosynthetic pathways to the product is alpha-farnesene synthase. Like other terpene synthases, the class of alpha-farnesene synthases is considered to be inefficient and thereby negatively influence the productivity and hence production costs. Alpha-farnesene is also the precursor of sinensal, a valuable fragrance and flavour ingredient.

[0006] Hence, there is a need for more efficient terpene synthases, including more efficient alpha- farnesene synthases and production processes utilizing the same leading to high levels of a specific terpene product, in particular alpha-farnesene, and favourable side components.

[0007] Furthermore, there is a constant need for terpene compositions, in particular alpha-farnesene compositions with novel or improved olfactory properties.

[0008] The technical problem underlying the present invention may be regarded as the provision of means and methods for complying with the aforementioned needs. The technical problem is solved by the embodiments characterized in the claims and herein below.

[0009] It is in particular solved by the provision of novel alpha-farnesene synthases which enable higher productivities and titers in the biotechnological production of alpha-farnesene.

[0010] The present invention relates to a method for producing alpha-farnesene, the method comprising at least the following steps:

[0011] A) providing a polypeptide exhibiting alpha-farnesene activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) an amino acid sequence encoded by a nucleotide sequence having at least 70% sequence identity to the nucleic acid sequence of any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; c) an amino acid sequence as set forth in SEQ ID NO:1 ; d) an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1 ; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting farnesene synthase activity, or exhibiting farnesene synthase activity; and

[0012] B) converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide.

[0013] Advantageously, it has been found in accordance with the present invention that the polypeptides exhibiting alpha-farnesene synthase activity identified in the studies underlying the present invention are particularly useful for converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide. Thereby, the invention provides for a method for producing alpha-farnesene which is surprisingly reliable and efficient and therefore reduces productions costs. Moreover, it results in a desired product composition that is particularly suitable in flavor and fragrance applications and for further downstream processing. Still further, the desired product composition advantageously results in improved organoleptic characteristics, in particular improved olfactory properties and / or gustatory properties.

[0014] It is to be understood that as used in the specification and in the claims, “a” or “an” can mean one or more, depending upon the context in which it is used. Thus, for example, reference to “a host cell” can mean that at least one host cell can be utilized.

[0015] As used in the following, the terms “have”, “comprise” or “include” or any arbitrary grammatical variations thereof are used in a non-exclusive way. Thus, these terms may both refer to a situation in which, besides the feature introduced by these terms, no further features are present in the entity described in this context and to a situation in which one or more further features are present. As an example, the expressions “A has B”, “A comprises B” and “A includes B” may both refer to a situation in which, besides B, no other element is present in A (i.e. a situation in which A solely and exclusively consists of B) and to a situation in which, besides B, one or more further elements are present in entity A, such as element C, elements C and D or even further elements. The term “comprising” also encompasses embodiments where only the items referred to are present, i.e. it has a limiting meaning in the sense of “consisting of’, or not.

[0016] Further, as used in the following, the terms "particularly", "more particularly", “typically”, and “more typically” or similar terms are used in conjunction with additional or alternative features, without restricting alternative possibilities. Thus, features introduced by these terms are additional or alternative features and are not intended to restrict the scope of the claims in any way. The invention may, as the skilled person will recognize, be performed by using alternative features. Similarly, features introduced by "in an embodiment of the invention" or similar expressions are intended to be additional or alternative features, without any restriction regarding alternative embodiments of the invention, without any restrictions regarding the scope of the invention and without any restriction regarding the possibility of combining the features introduced in such way with other additional or alternative or non-additional or alternative features of the invention. Not mandatory, but preferred features are also characterized by the term “preferably”, “more preferably” or “most preferably”. Further, it will be understood that the term “at least” means that the item or parameter to which the term refers is limited in one direction but open ended in one or more other directions.

[0017] The term “about” as used herein means that with respect to any number recited after said term an interval accuracy exists within in which a technical effect can be achieved. Accordingly, about as referred to herein, preferably, refers to the precise numerical value or a range around said precise numerical value of ±20 %, preferably ±15 %, more preferably ±10 %, or even more preferably ±5 %. "%" in the context of the concentration of a solution or composition as referred to herein means percentage (mol / mol) if not indicated otherwise.

[0018] It is to be understood that this invention is not limited to the particular methodology, protocols, reagents etc. described herein as these may vary. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to limit the scope of the present invention that will be limited only by the appended claims. Unless defined otherwise, all technical and scientific terms used herein have the same meanings as commonly understood by one of ordinary skill in the art.

[0019] According to the method of the present invention, a polypeptide exhibiting alpha-farnesene synthase activity is provided.

[0020] The terms "protein" or "polypeptide" are used interchangeably and refer to molecules consisting of a chain of amino acids, without reference to a specific mode of action, size, 3-dimensional structure or origin. Typically, the term “polypeptide" refers to a molecule consisting of a contiguous sequence of at least 20, amino acids that are covalently linked to each other by peptide bonds. Molecules consisting of less than 20 amino acids covalently linked by peptide bonds are usually considered to be "peptides". Preferably, the polypeptide comprises of from 50 to 1000, more preferably of from 75 to 1000, still more preferably of from 100 to 500, most preferably of from 110 to 400 amino acids.

[0021] The term “alpha-farnesene activity” as used to herein refers to an activity of a polypeptide, in particular of an enzyme, that is capable of converting farnesyl pyrophosphate to alpha-farnesene, more particularly to as ( ,6£)-3,7,11-Trimethyl-1 ,3,6,10-dodecatetraen (CAS: 502-61-4). A polypeptide exhibiting alpha-farnesene synthase activity as provided in the method according to the invention is hence typically understood as a polypeptide capable of converting farnesyl pyrophosphate to alpha-farnesene. More typically, said polypeptide is capable of catalyzing the following reaction (2£,6£)-farnesyl diphosphate (3£,6£)-a-farnesene + diphosphate.

[0022] More typically, said polypeptide exhibiting alpha-farnesene synthase activity is an alpha- farnesene synthase, even more typically the polypeptide is specified as a member of enzyme class EC 4.2.3.46.

[0023] In line with the present invention, said the polypeptide exhibiting alpha-farnesene synthase activity comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleic acid sequence of any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; c) an amino acid sequence as set forth in SEQ ID NO:1 ; d) an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 ; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting alpha-farnesene synthase activity, or exhibiting alpha-farnesene synthase activity.

[0024] Preferably, the polypeptide exhibiting alpha-farnesene synthase activity is from or based on a polypeptide from the genus of Scoparia, for example, but not limited to, from Scoparia dulcis (Sd). Said polypeptide exhibiting alpha-farnesene synthase activity may have an amino acid sequence as set forth in SEQ ID NO:1. More preferably, the polypeptide exhibiting alpha-farnesene synthase activity is from or based on the SdAFS gene specified as SEQ ID NO:2 elsewhere herein. The SdAFS gene may be codon-optimized for expression in a specific host cell; in particular, said gene may have a nucleic acid sequence of any of SEQ ID NO:3, or SEQ ID NO:4.

[0025] It is understood that the method according to the invention comprises a step of providing said polypeptide exhibiting alpha-farnesene activity.

[0026] The “sequence identity” referred to herein above defines a relationship between amino acid sequences or nucleic acid sequences and can be determined by comparing those sequences. Usually, sequence identities are determined by comparing two sequences over the whole length of the sequences but may also be compared only for a part of the sequences aligning with each other. Preferably, the sequence identities are compared over the whole length of the sequences, herein. Sequence identity refers to the degree of relatedness between polypeptide sequences or nucleic acid sequences. It will be expressed in the percentage of identical amino acids or nucleotides in two sequences compared to each other. Accordingly, upon aligning two sequences, the number of matching amino acids or nucleotides between those sequences is, in general, determined and put into relation to the total number of amino acids or nucleotides in the aligned sequence or sequence part. For instance, variant sequences may be defined by their sequence identity when compared to a parent sequence, i.e. an amino acid sequence as shown in any one of SEQ ID NO:1 , or a nucleic acid sequence as shown in SEQ ID NO:2. To determine the percent-identity between two sequences in a first step a pairwise sequence alignment is generated between those two sequences, wherein the two sequences are aligned over their complete, entire or full length (i.e., a pairwise global alignment). The alignment is generated with a program or software described herein. The preferred alignment for the purpose of this invention is that alignment, from which the highest sequence identity can be determined. Variant sequences may typically include codon-optimized nucleic acid sequences that are in particular adapted for the expression in the host cells used.

[0027] Ways of codon optimization or codon pair optimization, are known in the art and may be based on a method as described in WO 2008 / 000632 or as offered by commercial DNA synthesizing companies like DNA2.0, Geneart, and GenScript. Examples of codon optimized sequences in line with the present invention are given in SEQ ID NO:3, and SEQ ID NO:4.

[0028] Sequence alignments can be generated with a number of software tools, such as Needleman and Wunsch algorithm - Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology 48 (3): 443-453. This algorithm is, for example, implemented into the “NEEDLE” program, which performs a global alignment of two sequences. The NEEDLE program, is contained within, for example, the European Molecular Biology Open Software Suite (EMBOSS). EMBOSS - a collection of various programs: The European Molecular Biology Open Software Suite (EMBOSS), Trends in Genetics 16 (6), 276 (2000). BLOSUM (BLOcks Substitution Matrix) - typically generated on the basis of alignments of conserved regions, e.g., of protein domains (Henikoff S, Henikoff JG: Amino acid substitution matrices from protein blocks. Proceedings of the National Academy of Sciences of the USA. 1992 Nov 15; 89(22): 10915-9). One out of the many BLOSUMs is “BLOSUM62”, which is often the “default” setting for many programs, when aligning protein sequences. BLAST (Basic Local Alignment Search Tool) - consists of several individual programs (BlastP, BlastN) which are mainly used to search for similar sequence in large sequence databases. BLAST programs also create local alignments. Typically used is the “BLAST” interface provided by NCBI (National Centre for Biotechnology Information), which is the improved version (“BLAST2”). The “original” BLAST: Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410; BLAST2: Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402.

[0029] Sequence identity as used herein is, preferably, the value as determined by the EMBOSS Pairwise Alignment Algorithm "Needle". In particular, the NEEDLE program from the EMBOSS package can be used (version 2.8.0 or higher, EMBOSS: The European Molecular Biology Open Software Suite - Rice, P., et al. Trends in Genetics (2000) 16: 276-277; http: / / emboss.bioinformatics.nl) using the NOBRIEF option ('Brief identity and similarity' to NO) which calculates the "longest-identity". The identity between the two aligned sequences is calculated in such a case as follows: Number of corresponding positions in the alignment showing an identical amino acid in both sequences divided by the total length of the alignment after subtraction of the total number of gaps in the alignment. For alignment of amino acid sequences the default parameters are: Matrix = Blosum62; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5. For alignment of nucleic acid sequences the default parameters are: Matrix = DNAfull; Open Gap Penalty = 10.0; Gap Extension Penalty = 0.5. Variant amino acid or nucleic acid sequences as referred to herein may be naturally occurring variations such as allelic variants or orthologous, paralogous or homologous variants. Alternatively, such sequences may be artificially generated, e.g., in an attempt to improve a property of the enzyme or nucleic acid (e.g., improved expression of the enzyme or increased enzymatic activity of the enzyme) by a biological technique known to the skilled person in the art, such as, e.g., molecular evolution or rational design, or by using a mutagenesis technique known in the art and described elsewhere herein (random mutagenesis, site-directed mutagenesis, directed evolution, gene recombination, etc.). Typically, variants of the polypeptides with alpha- farnesene synthase activity according to the invention are polypeptides with one or several amino acid substitutions compared to the amino acid sequence of any of SEQ ID NO:1 , preferably, artificial amino acid sequences.

[0030] Variant nucleic acid sequences encoding an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, preferably as shown in SEQ ID NO: 2, or an amino acid sequence encoded by a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 , preferably as shown in SEQ ID NO: 2, may differ from the nucleic acid sequences shown in SEQ ID NO: 2, SEQ ID NO:3, or SEQ ID NO:4 , preferably as shown in SEQ ID NO: 2, for reasons set forth elsewhere herein due to at least one nucleotide substitution, addition and / or deletion. It will be understood that polynucleotides comprising such variant nucleic acid sequences as referred to herein, preferably, are capable of hybridizing to each other under stringent hybridization conditions. Stringent hybridization conditions as referred to herein are, preferably, 6 x sodium chloride / sodium citrate (SSC) at approximately 45°C, followed by one or more wash steps in 0.2 x SSC, 0.1 % SDS at 50 to 65°C. The skilled worker knows that these hybridization conditions differ depending on the type of nucleic acid and, for example when organic solvents are present, with regard to the temperature and concentration of the buffer. For example, under “standard hybridization conditions” the temperature differs depending on the type of nucleic acid between 42°C and 58°C in aqueous buffer with a concentration of 0.1 to 5 x SSC (pH 7.2). If organic solvent is present in the abovementioned buffer, for example 50% formamide, the temperature under standard conditions is approximately 42°C. The hybridization conditions for DNA: DNA hybrids are, preferably, 0.1 x SSC and 20°C to 45°C, preferably between 30°C and 45°C. The hybridization conditions for DNA:RNA hybrids are, preferably, 0.1 x SSC and 30°C to 55°C, preferably between 45°C and 55°C. The abovementioned hybridization temperatures are determined for example for a nucleic acid with approximately 100 bp (= base pairs) in length and a G + C content of 50% in the absence of formamide. The skilled worker knows how to determine the hybridization conditions required by referring to textbooks such as the textbook mentioned above, or the following textbooks: Sambrook et aL, "Molecular Cloning”, Cold Spring Harbor Laboratory, 1989; Hames and Higgins (Ed.) 1985, ’’Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford; Brown (Ed.) 1991 , "Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford. Thus, variant nucleic acid sequences can be derived from polynucleotides which are capable of hybridizing under stringent hybridization conditions to nucleic acid sequences encoding an amino acid sequence encoded by a nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4 or an amino acid sequence encoded by a nucleic acid sequence which is at least 60%, at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% identical to the nucleic acid sequence as shown in SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4.

[0031] A fragment as referred to above may be a polypeptide consisting of any amino acid sequence of the above-mentioned sequences and sequence variants that is of sufficient length of exhibiting a alpha-farnesene synthase activity specified above. It is, thus, preferably envisaged that a fragment having the aforementioned biological activity of the polypeptide comprises the amino acid sequence of the catalytically active region of an alpha-farnesene synthase. Typically, a fragment consists of at least 20, at least 30, at least 40, at least 50, at least 100, at least 150 or at least 200 contiguous amino acids in length from the above-mentioned sequences or sequence variants.

[0032] The aforementioned polypeptide exhibiting alpha-farnesene synthase activity may also be comprised in a fusion polypeptide. Such a fusion polypeptide comprises in addition to the amino acid sequence of the polypeptide exhibiting alpha-farnesene synthase activity one or more additional amino acid sequences. Said additional amino acid sequences may be polypeptides having other enzymatic activities, such as farnesyl pyrophosphate synthases or cytochrome P450 monooxygenases or polypeptides or peptides having marker or label functions for, e.g., monitoring proper expression or for purification purposes, such as tags (e.g., MYC tag, FLAG tag, His tag, MISTIC tag, P17 tag, SUMO tag, CaBP tag, MBP tag, Trx tag, GST tag, Z-basic tag, Fh8 tag, Skp tag, NusA tag, mysB tag, Tsf tag, RpoA tag, Ecotin tag, etc.) or fluorescent proteins (e.g. RFP, GFP, BFP, YFP or CFP) or enzymes from the upstream pathways (FPP synthase, IPP isomerase, isopentenylphosphate kinase, .isopentenol kinase, HMG-CoA reductase, etc.).

[0033] The method according to the invention comprises a step of converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide. Typically, the conversion takes by said alpha-farnesene synthase activity place at a temperature in the range of 5 °C to 60 °C in an aqueous environment, more typically at a temperature in the range of 20 °C to 40 °C in an aqueous environment.

[0034] The aforementioned conversion step, i.e. step of converting farnesyl pyrophosphate to alpha- farnesene may be carried out in vitro, i.e. in a suitable reaction vial containing all components required for the conversion as described above. The skilled person is well aware of how to adjust the reaction conditions such that the reaction will be carried out efficiently. For example, suitable buffers may be used to provide the components in an environment having a suitable pH and suitable salt concentrations. A suitable temperature in such a setting can be applied as well without further ado. Typically, the step of converting farnesyl pyrophosphate into alpha-farnesene is carried out in a host cell. In other words, the conversion of farnesyl pyrophosphate into alpha-farnesene is carried out by the polypeptide exhibiting alpha-farnesene synthase activity of the in a host cell.

[0035] A further embodiment of the invention is a recombinant microorganism comprising said recombinant construct or said recombinant vector. The recombinant microorganism may typically be a prokaryotic cell. Suitable prokaryotic cells include Gram-positive, Gram negative and Gramvariable bacterial cells, preferably Gram-negative.

[0036] Thus, prokaryotic microorganisms that can be used in the present invention include, but are not limited to, Gluconobacter oxydans, Gluconobacter asaii, Achromobacter delmarvae, Achromobacter viscosus, Achromobacter lacticum, Agrobacterium tumefaciens, Agrobacterium radiobacter, Alcaligenes faecalis, Arthrobacter citreus, Arthrobacter tumescens, Arthrobacter paraffineus, Arthrobacter hydrocarboglutamicus, Arthrobacter oxydans, Aureobacterium saperdae, Azotobacter indicus, Brevi bacterium ammoniagenes, Brevibacterium divaricatum, Brevibacterium lactofermentum, Brevibacterium flavum, Brevibacterium globosum, Brevibacterium fuscum, Brevibacterium ketoglutamicum, Brevibacterium helcolum, Brevibacterium pusilium, Brevibacterium testaceum, Brevibacterium roseum, Brevibacterium immariophilium, Brevibacterium linens, Brevibacterium protopharmiae, Cereibacter sphaeroides, Corynebacterium acetophilum, Corynebacterium glutamicum, Corynebacterium callunae, Corynebacterium acetoacidophilum, Corynebacterium acetoglutamicum, Enterobacter aerogenes, Erwinia amylovora, Erwinia carotovora, Erwinia herbicola, Erwinia chrysanthemi, Flavobacterium peregrinum, Flavobacterium fucatum, Flavobacterium aurantinum, Flavobacterium rhenanum, Flavobacterium sewanense, Flavobacterium breve, Flavobacterium meningosepticum, Micrococcus sp. CCM825, Morganella morganii, Nocardia opaca, Nocardia rugosa, Pianococcus eucinatus, Proteus rettgeri, Propionibacterium shermanii, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Rhodococcus erythropolis, Rhodococcus rhodochrous, Rhodococcus sp. ATCC 15592, Rhodococcus sp. ATCC 19070, Sporosarcina ureae, Staphylococcus aureus, Vibrio metschnikovii, Vibrio tyrogenes, Actinomadura madurae, Actinomyces violaceochromogenes, Kitasatosporia parulosa, Streptomyces avermitilis, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces antibioticus, Streptomyces cacaoi, Streptomyces lavendulae, Streptomyces viridochromogenes, Aeromonas salmonicida, Bacillus pumilus, Bacillus circulans, Bacillus thiaminolyticus, Escherichia freundii, Microbacterium ammoniaphilum, Serratia marcescens, Salmonella typhimurium, Salmonella schottmulleri, Xanthomonas citri, Synechocystis sp., Synechococcus elongatus, Thermosynechococcus elongatus, Microcystis aeruginosa, Nostoc sp., N. commune, N.sphaericum, Nostoc punctiforme , Spirulina platensis, Lyngbya majuscula, L. lagerheimii, Phormidium tenue, Anabaena sp., Leptolyngbya sp. Eukaryotic microorganisms that can be used in the present invention include, but are not limited to Saccharomyces spec, such as Saccharomyces cerevisiae, Hansenula spec, such as Hansenula polymorpha, Schizosaccharomyces spec, such as Schizosaccharomyces pombe, Kluyveromyces spec, such as Kluyveromyces lactis and Kluyveromyces marxianus, Yarrowia spec, such as Yarrowia lipolytica, Pichia spec, such as Pichia methanolica, Pichia stipites and Pichia pastoris, Zygosaccharomyces spec, such as Zygosaccharomyces rouxii and Zygosaccharomyces bailii, Candida spec, such as Candida boidinii, Candida utilis, e.g. described in https: / / microbialcellfactories.biomedcentral.eom / articles / 10.1186 / 1475-2859-13-83, Candida glabrata and Candida sonorensis, Schwanniomyces spec, such as Schwanniomyces occidentalis, Arxula spec, such as Arxula adeninivorans, Ogataea spec such as Ogataea minuta, Klebsiella spec, such as Klebsiella pneumonia, Aspergillus spec, such as Aspergillus niger or Myceliophthora thermophila, such as Ashbya gossypii, such as Cutaneotrichosporon sp..

[0037] Preferred microorganisms of the invention comprise Rhodococcus rhodochrous, Aerococcus sp., Ashbya gossypii, Aspergillus sp., Bacillus pumilus, Bacillus subtilis, Bacteroides thetaiotaomicron, Cereibacter sphaeroides, Clostridium algidicarnis, Corynebacterium efficiens, Corynebacterium glutamicum, Cutaneotrichosporon oleaginosus, Escherichia coli, Haloferax volcanii, Lactobacillus casei, Methanocaldococcus jannaschii, Methanothermobacter thermautotrophicus, Myceliophthora thermophila, Pichia pastoris, Pseudomonas synxantha, Pseudomonas azotoformans, Pseudomonas jluorescens, Pseudomonas ovalis, Pseudomonas stutzeri, Pseudomonas acidovolans, Pseudomonas mucidolens, Pseudomonas testosteroni, Pseudomonas aeruginosa, Pseudozyma tsukubaensis, Ralstonia eutropha, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae, Shigella boydii, Sinorhizobium meliloti, Streptomyces antibioticus, Streptomyces avermitilis, Streptomyces cacaoi, Streptomyces coelicolor, Streptomyces flavelus, Streptomyces griseolus, Streptomyces lavendulae, Streptomyces lividans, Streptomyces olivaceus, Streptomyces tanashiensis, Streptomyces virginiae, Streptomyces viridochromogenes, Thermoplasma acidophilum, Vibrio natrigens or Yarrowia lipolytica.

[0038] Especially preferred microorganisms are Ashbya gossypii, Bacillus subtilis, Cereibacter sphaeroides, Corynebacterium glutamicum, Cutaneotrichosporon oleaginosus, Escherichia coli, Pichia pastoris, Pseudomonas aeruginosa, Pseudomonas putida, Rhodobacter sphaeroides, Rhodococcus opacus, Saccharomyces cerevisiae and Yarrowia lipolytica.

[0039] It is to be understood that the host cell shall be capable of producing alpha-farnesene. If necessary, the host cell may be genetically modified in order to express enzymes or proteins required for the alpha-farnesene synthesis including the aforementioned alpha-farnesene synthase. Suitable cellular expression systems are well known in the art and for example described in WO2014 / 014339.

[0040] The host cell shall be cultivated under conditions and for a time sufficient to allow expression of the aforementioned enzymes and for conversion of farnesyl pyrophosphate into at least one alpha-farnesene, see e.g. Fig. 1. Particular preferred conditions are also described in the accompanying Examples, below, or known to those skilled in the art.

[0041] The cultivation of the host cell is preferably a fermentation process as disclosed for example in WO2014 / 014339, EP 3017050, W02023 / 212400; Nature, volume 537, pages 694-697 (2016).

[0042] Yet, the conversion step of the method of the present invention may also be carried out in an organism, typically a multi-cellular organism such as the transgenic non-human organism referred to elsewhere herein. Typically, said organism is genetically modified such that the enzymes required for conversion of farnesyl pyrophosphate into at least one alpha-farnesene are expressed by said organism. The skilled person is, however, well aware of what conditions need to be applied for culturing the non-human transgenic organism and in particular for producing alpha-farnesene in said organism.

[0043] The term “transgenic non-human organism” as used herein refers to an organism which has been genetically modified in order to comprise the polynucleotide, vector or gene construct of the present invention. Said genetic modification may be the result of any kind of homologous or heterologous recombination event, mutagenesis or gene editing process. Accordingly, the transgenic non-human organism shall differ from its non-transgenic counterpart in that it comprises the non-naturally occurring (i.e. heterologous) polynucleotide, vector or gene construct in its genome. Non-human organisms envisaged as transgenic non-human organisms in accordance with the present invention are, preferably, multi-cellular organisms. Moreover, the non-human organisms are, preferably, animals or plants. Preferred animals are mammals, in particular laboratory animals such as rodents, e.g., mice, rats, rabbits or the like, or farming animals such as sheep, goat, cows, horses or the like. Preferred plants are crop plants or vegetables, in particular, selected from the group consisting of Arabidopsis spp., Nicotiana spp, Cichorum intybus, Lactuca sativa, Mentha spp, Artemisia annua, tuber forming plants, oil crops, e.g. Brassica spp. or Brassica napus, flowering plants (angiosperms) which produce fruits, and trees.

[0044] Methods for the production of transgenic non-human organisms are well known in the art; see, e.g. Lee-Yoon Low et aL, Transgenic Plants: Gene constructs, vector and transformation method. 2018. DOI.10.5772 / intechopen.79369; Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M. and Robl, J. M. (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington D.C); Sambrook, loc.cit, Ausubel, loc.cit).

[0045] Preferably, said non-human transgenic organism is a plant or a non-human animal to be sacrificed. Accordingly, it accordance with the latter, methods of treating animals are not encompassed within the methods of the present invention.

[0046] In case, the method of the invention is carried out in vivo, i.e. in a host cell or a non-human transgenic organism it will be understood that the said host cell or non-human transgenic organism shall express the polypeptide exhibiting alpha-farnesene synthase activity as specified above such that the conversion of farnesyl pyrophosphate into at least one alpha-farnesene can be carried out in said host cell or non-human transgenic organism. Preferably, said polypeptide exhibiting alpha-farnesene synthase activity is encoded by a heterologous polynucleotide, a vector or a gene construct.

[0047] The term “heterologous polypeptide” in this context means that the polynucleotide encoding the polypeptide exhibiting alpha-farnesene synthase activity is not naturally occurring in the host cell or organism into which it is introduced. Thus, a heterologous polynucleotide originates from a first species or is an artificially modified polynucleotide, while the host cell or non-human transgenic organism is from a second species that differs from said first species. A heterologous polynucleotide may be comprised in a vector or gene construct as specified herein below. Alternatively, it may be introduced into the genome of a host cell or non-human transgenic organism such that upon integration into the genome, the polypeptide exhibiting alpha-farnesene synthase activity encoded by said heterologous polynucleotide is expressed. Typically, the heterologous polynucleotide shall be integrated into the genome of the host cell or non-human transgenic organism at a locus that allows expression of the heterologous polynucleotide, e.g., in proximity to an endogenous promoter.

[0048] The term "nucleic acid" or “nucleic acid molecule” as used herein, includes reference to a deoxyribonucleotide (DNA) or ribonucleotide (RNA) polymer, i.e. a polynucleotide, in either single- or double-stranded form, and unless otherwise limited, encompasses known analogues having the essential nature of natural nucleotides in 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 full- length or a sub-sequence of a native or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the specified sequence as well as the complementary sequence thereof. Thus, DNAs or RNAs with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Moreover, DNAs or RNAs comprising unusual bases, such as inosine, or modified bases, such as tritylated bases, to name just two examples, are "polynucleotides" as the term is used herein. It will be appreciated that a great variety of modifications have been made to DNA and RNA that serve many useful purposes known to those of skill in the art. The term "polynucleotide" as it is employed herein embraces 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 otherthings, simple and complex cells. Every nucleic acid sequence herein that encodes a polypeptide or enzyme such as the alpha-farnesene synthase as defined herein also, by reference to the genetic 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 which encode identical or conservatively modified variants of the amino acid sequences due to the degeneracy of the genetic code. The term "degeneracy of the genetic code" refers to the fact that a large number of functionally identical nucleic acids encode any given protein. For instance, the codons GCA, GCC, GCG and GCU all encode the amino acid alanine. Thus, at every position where an alanine is specified by a codon, the codon can be altered to any of the corresponding codons described without altering the encoded polypeptide. Such nucleic acid variations are "silent variations" and represent one species of conservatively modified variation.

[0049] The term “vector”, preferably, encompasses phage, plasmid, cosmids, viral vectors as well as artificial chromosomes, such as bacterial or yeast artificial chromosomes (YAC). The vector encompassing the polynucleotide of the present invention, preferably, further comprises selectable markers for propagation and / or selection in a host. The vector may be incorporated into a host cell by various techniques well known in the art. If introduced into a host cell, the vector may reside in the cytoplasm or may be incorporated into the genome. In the latter case, it is to be understood that the vector may further comprise nucleic acid sequences which allow for homologous recombination or heterologous insertion. Vectors can be introduced into prokaryotic or eukaryotic cells via conventional transformation or transfection techniques. The terms “transformation” and “transfection”, conjugation and transduction, as used in the present context, are intended to comprise a multiplicity of prior-art processes for introducing foreign nucleic acid (for example DNA) into a host cell, including calcium phosphate, rubidium chloride or calcium chloride co-precipitation, DEAE-dextran-mediated transfection, lipofection, f-mating, natural competence, carbon-based clusters, chemically mediated transfer, electroporation or particle bombardment. Suitable methods for the transformation or transfection of host cells, including plant cells, can be found in Sambrook et al. (loc. cit.) and other laboratory manuals, such as Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey. Alternatively, a plasmid vector may be introduced by heat shock or electroporation techniques. Should the vector be a virus, it may be packaged in vitro using an appropriate packaging cell line prior to application to host cells.

[0050] Preferably, the vector referred to herein is suitable as a cloning vector, i.e. replicable in microbial systems. Such vectors ensure efficient cloning in bacteria and, preferably, yeasts or fungi and make possible the stable transformation of plants. Those which must be mentioned are, in particular, various binary and co-integrated vector systems which are suitable for the T DNA- mediated transformation. Such vector systems are, as a rule, characterized in that they contain at least the vir genes, which are required for the Agrobacterium-mediated transformation, and the sequences which delimit the T-DNA (T-DNA border). These vector systems, preferably, also comprise further cis-regulatory regions such as promoters and terminators and / or selection markers with which suitable transformed host cells or organisms can be identified. While cointegrated vector systems have vir genes and T DNA sequences arranged on the same vector, binary systems are based on at least two vectors, one of which bears vir genes, but no T-DNA, while a second one bears T DNA, but no vir gene. As a consequence, the last-mentioned vectors are relatively small, easy to manipulate and can be replicated both in E. coli and in Agrobacterium. These binary vectors include vectors from the pBIB-HYG, pPZP, pBecks, pGreen series. Preferably used in accordance with the invention are Bin19, pBI101 , pBinAR, pGPTV and pCAMBIA. An overview of binary vectors and their use can be found in Hellens et al, Trends in Plant Science (2000) 5, 446—451. Furthermore, by using appropriate cloning vectors, the polynucleotides can be introduced into host cells or organisms such as plants or animals and, thus, be used in the transformation of plants, such as those which are published, and cited, in: Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6 / 7, pp. 71-119 (1993); F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1 , Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38; B. Jenes et aL, Techniques for Gene Transfer, in: Transgenic Plants, vol. 1 , Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143; Potrykus 1991 , Annu. Rev. Plant Physiol. Plant Molec. Biol. 42, 205 225.

[0051] More preferably, the vector of the present invention is an expression vector. In such an expression vector, i.e. a vector which comprises the polynucleotide of the invention having the nucleic acid sequence operatively linked to an expression control sequence (also called “expression cassette”) allowing expression in prokaryotic or eukaryotic cells or isolated fractions thereof. Suitable expression vectors are known in the art such as Okayama-Berg cDNA expression vector pcDV1 (Pharmacia), pCDM8, pRc / CMV, pcDNAI , pcDNA3 (Invitrogen) or pSPORTI (GIBCO BRL). Further examples of typical fusion expression vectors are pGEX (Pharmacia Biotech Inc; Smith 1988, Gene 67:31-40), pMAL (New England Biolabs, Beverly, MA) and pRIT5 (Pharmacia, Piscataway, NJ), where glutathione S transferase (GST), maltose E-binding protein and protein A, respectively, are fused with the recombinant target protein. Examples of suitable inducible nonfusion E. co / / expression vectors are, inter alia, pTrc (Amann 1988, Gene 69:301-315) and pET 11d (Studier 1990, Methods in Enzymology 185, 60-89). The tar-get gene expression of the pTrc vector is based on the transcription from a hybrid trp-lac fusion promoter by host RNA polymerase. The target gene expression from the pET 11 d vector is based on the transcription of a T7-gn10-lac fusion promoter, which is mediated by a co-expressed viral RNA polymerase (T7 gn1 ). This viral polymerase is provided by the host strains BL21 (DE3) or HMS174 (DE3) from a resident lambda-prophage which harbours a T7 gn1 gene under the transcriptional control of the lacUV 5 promoter. The skilled worker is familiar with other vectors which are suitable in prokaryotic organisms; these vectors are, for example, in E. coli, pLG338, pACYC184, the pBR series such as pBR322, the pUC series such as pUC18 or pUC19, the M113mp series, pKC30, pRep4, pHS1 , pHS2, pPLc236, pMBL24, pLG200, pUR290, plN-111113-B1 , lambdagtl 1 or pBdCI, in Streptomyces plJ101 , plJ364, plJ702 or plJ361 , in Bacillus pUB110, pC194 or pBD214, in Corynebacterium SKn or pAJ667. Examples of vectors for expression in the yeast S. cerevisiae comprise pYep Sec1 (Baldari 1987, Embo J. 6:229-234), pMFa (Kurjan 1982, Cell 30:933-943), pJRY88 (Schultz 1987, Gene 54:113-123) and pYES2 (Invitrogen Corporation, San Diego, CA). Vectors and pro-cesses for the construction of vectors which are suitable for use in other fungi, such as the filamentous fungi, comprise those which are described in detail in: van den Hondel, C.A.M.J.J., & Punt, P.J. (1991 ) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et aL, Ed., pp. 1-28, Cambridge University Press: Cambridge, or in: More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego). Further suitable yeast vectors are, for example, pAG-1 , YEp6, YEp13 or pEMBLYe23. As an alternative, the polynucleotides of the present invention can be also expressed in insect cells using baculovirus expression vectors. Baculovirus vectors which are available for the expression of proteins in cultured insect cells (for example Sf9 cells) comprise the pAc series (Smith 1983, Mol. Cell Biol. 3:2156-2165) and the pVL series (Lucklow 1989, Virology 170:31-39).

[0052] Yet, the vector may be an integration vector. An integration vector refers to a DNA molecule, linear or circular, that can be incorporated, e.g., into a microorganism's genome, such as a bacteria’s genome, and provides for stable inheritance of a gene encoding a polypeptide of interest, such as the alpha-farnesene synthase of the invention. The integration vector generally comprises one or more segments comprising a gene sequence encoding a polypeptide of interest under the control of (i.e. , operably linked to) additional nucleic acid segments that provide for its transcription.

[0053] Such additional segments may include regulatory sequences, for example promoter and terminator sequences, and one or more segments that drive the incorporation of the gene of interest into the genome of the target cell, usually by the process of homologous recombination. Typically, the integration vector will be one which can be transferred into the target cell, but which has a replicon which is non-functional in that organism. Integration of the segment comprising the gene of interest may be selected if an appropriate marker is included within that segment. One or more nucleic acid sequences encoding appropriate signal peptides that are not naturally associated with a polypeptide to be expressed in a host cell of the invention can be incorporated into (expression) vectors. For example, a DNA sequence for a signal peptide leader can be fused in-frame to a nucleic acid of the invention so that the alpha-farnesene synthase 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 instance, enhances extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to certain organelles, like the chloroplasts, mitochondria and peroxisomes. The signal peptide can be cleaved from the polypeptide upon transportation to the intended organelle or from the cell. It is possible to provide a fusion of an additional peptide sequence at the amino or carboxyl terminal end of the polypeptide.

[0054] The term “gene construct” as used herein refers to polynucleotides comprising the polynucleotide of the invention and additional functional nucleic acid sequences. A gene construct according to the present invention is, preferably, a linear DNA molecule. Typically, a gene construct in accordance with the present invention may be a targeting construct which allows for random or site- directed integration of the targeting construct into genomic DNA. Such target constructs, preferably, comprise DNA of sufficient length for either homologous or heterologous recombination as described in detail below. In both cases, the construct must be, preferably, impeccable, with regulatory sequences, typically to control gene expression, such as a promoter, a site of transcription initiation, a site of polyadenylation, and a site of transcription termination.

[0055] Preferably, the method of the present invention comprises the step of obtaining said manufactured composition comprising at least on alpha-farnesene. “Alpha-farnesene” in line with the present invention refers to all known stereoisomers thereof such as (3£, 6£)-3, 7, 11-Trimethyl-1 ,3,6,10- dodecatetraen (CAS: 502-61-4); (<3 ^E)-3, 7, 11-T rimethyldodeca-1 ,3, 6,10-tetraen; 3E,6Z)- 3,7, 11 -T rimethyldodeca-1 ,3,6,10-tetraen; and (3Z,62)-3,7, 11 -T rimethyldodeca-1 ,3,6,10-tetraen; preferably to (3£,6£)-3,7,11-Trimethyl-1 ,3,6,10-dodecatetraen.

[0056] The term “obtaining” as used herein refers to any way of receiving at least one desired product such as alpha-farnesene and optionally side components described elsewhere herein, in a composition at any degree of purity. Accordingly, the composition may essentially comprise alpha-farnesene, typically in a purified form, in a suitable solvent or may be a mixture comprising additional components, such as side components defined elsewhere herein, besides alpha- farnesene. Suitable solvents in line with the present invention include are described for example in EP3017050, and may include: dodecane, isopropyl myristate, oleyl alcohol, white oil, sunflower oil, tributyl phosphate, etc.. “Essentially comprising alpha-farnesene” typically refers to a composition comprising at least 40 % (w / v) of alpha farnesene, more typically even higher amount of alpha-farnesene as specified elsewhere herein. “Essentially consisting of alpha-farnesene” typically refers to a composition comprising at least 80 % (w / v), more typically at least 90 % (w / v) of alpha farnesene

[0057] Thus, the method of the invention may encompass one or more purification steps. The purification techniques which need to be applied depend on how the method of the present invention has been carried out. For example, if the method has been carried out in vitro, i.e. in reaction vials using isolated components such as isolated enzymes, adducts and auxiliary components such as reaction buffers, it will be understood that minimal purification may be necessary. However, if the method is carried out in vivo, i.e. in a host cell as defined elsewhere herein, further purification and pre-treatment steps may be necessary.

[0058] More typically, the step of obtaining alpha-farnesene comprises at least one or more of the following steps: precipitation, adsorption, solid-liquid separation, filtration, centrifugation, chromatography, extraction using a hydrophobic solvents; phase separation, solvent evaporation, distillation, even more typically solvent evaporation and distillation.

[0059] Even more typically, the method according to the invention comprises a step of purifying the desired product, still more typically purifying the obtained alpha-farnesene or composition comprising alpha-farnesene. Still more typically, said purifying comprises distillation of the composition comprising alpha-farnesene. Method and means for distillation are known in the art.

[0060] The method in line with the present invention is in particular advantageous as the desired product can be obtained in high amounts and with desirable side components. The composition comprising desired side components can be characterised by improved organoleptic properties, in particular improved olfactory and / or gustatory properties.

[0061] Moreover, if the steps are carried out in vivo, e.g. in animals or plants, even further pre-treatment and / or purification steps may be required. The skilled person is well aware of suitable pretreatment and / or purification steps depending on the given circumstances under which the method may be carried out. Purification techniques to be envisaged may be extraction techniques, chromatography, such as LC, GC or HPLC, size-exclusion chromatography, affinity chromatography, distillation, centrifugation, precipitation, adsorption, solvent evaporation, filtration and the like. Pre-treatment steps to be envisaged may be harvesting, heat treatment, ultra-sonic treatment, treatment with chemicals and / or enzymes, and the like.

[0062] Particular preferred measures are described below and / or in the accompanying Examples.

[0063] Typically, the method for producing alpha-farnesene according to the invention further comprises: cultivating the host cell in a fermentation broth under conditions and for a time suitable for alpha-farnesene production by the alpha-farnesene synthase activity of the polypeptide; and obtaining a desired product from the fermentation broth, preferably obtaining alpha- farnesene or a composition comprising alpha-farnesene or a composition derived from alpha-farnesene from the fermentation broth.

[0064] The term “fermentation broth” or “culture broth” is used in a broad sense to include any and all fermentation medium during or after fermentation, i.e. during or after contacting with the host cells. Preferably, the fermentation broth is the fermentation medium comprising host cells, which are cultivated typically to express alpha-farnesene synthase and to produce the alpha-farnesene and optionally side components. Preferably, the fermentation broth is the liquid as it is obtained at the end of a fermentation process without any further treatments. However, as used herein, the aforesaid terms also include the fermentation broth as obtained in the fermentation process, as well as a product obtained therefrom by removal of cells and / or cell fragments; the latter may also be referred to as "cell-free fermentation broth". In accordance, the terms “fermentation broth” or “culture broth” also include aliquots, i.e. sub-portions, of the fermentation broth, as well as fractions of the fermentation broth, i.e. parts of the fermentation broth obtained by removal of parts of its constituents; thus the fermentation broth may also be a cell-free fraction of the fermentation broth initially obtained. Preferably, the fermentation broth is a cell-containing fermentation broth, a cell-free fermentation broth, or and aliquot and / or fraction thereof.

[0065] The term “fermentation medium” as used herein refers to a water-based solution containing one or more chemical compounds that can support the growth of the host cells. Preferably, the fermentation medium is suitable for the respective host cell organism. The person of skill in the art knows how to select a suitable fermentation medium for the respective host cells. Common fermentation media for different host cells are disclosed for example in EP3017050, Nature, vol. 537, pages 694-697, 2016, WO2023 / 105080 such as LB medium for Hco / Zhost cells.

[0066] According to the method of the present invention, the polypeptide exhibiting alpha-farnesene synthase activity is preferably expressed from a recombinant nucleic acid molecule, also referred to as a polynucleotide present in the host cell.

[0067] The term “recombinant” is known to the skilled artisan. In particular, "recombinant" (or transgenic) with regard to a cell or an organism means that the cell or organism contains a heterologous polynucleotide also referred to as “expression construct” which is introduced by man by gene technology and with regard to a polynucleotide or a nucleic acid molecule includes all those constructions brought about by man by gene technology I recombinant DNA techniques known in the art.

[0068] For the purposes of the invention, "recombinant" (or transgenic) with regard to a cell in particular means that the cell contains a polynucleotide which is introduced by man by gene technology and with regard to a polynucleotide the term “recombinant” includes all those constructions brought about by man by gene technology / recombinant DNA techniques in which either

[0069] (a) the sequence of the polynucleotide or a part thereof, or

[0070] (b) one or more genetic control sequences which are operably linked with the polynucleotide, including, but not limited thereto, a promoter, or

[0071] (c) both a) and b) are not located in their wild-type genetic environment or have been modified.

[0072] In line with the present invention, the recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence having at least 70 % of sequence identity to any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1 ; d) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; e) a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting farnesene synthase activity.

[0073] The recombinant nucleic acid molecule typically further comprises gene regulatory sequences suitable for driving expression of the nucleic acid molecule in the host cell as known in the art. These typically include promoter and terminator sequences known in the art and elected according to the host cell or host organism utilized. More typically, the regulatory sequences comprise T7, Ptac, Ptrc, Plac, PBAD, Prha, Ptet, sigma70-based promoter sequences; and T7term, rrnBterm, etc. terminator sequences, and for example as disclosed in WQ2018 / 160066.

[0074] In the method for producing alpha-farnesene according to the invention preferably said cultivating in step B) comprises cultivating the host cell in an aqueous fermentation medium at a temperature between 5 °C and 60 °C, more preferably a temperature between 20 °C and 40 °C, and a pH in the range of 3 to 10.

[0075] The cultivation conditions in particular depend on the host cell and are known to the person skilled in the art.

[0076] Typically, said obtaining of alpha-farnesene comprises at least one or more of the following steps: precipitation, adsorption, solid-liquid separation, filtration, centrifugation, chromatography, extraction using hydrophobic solvents; phase separation, solvent evaporation, distillation, more typically solvent evaporation and distillation.

[0077] The present invention moreover relates to a recombinant nucleic acid molecule comprising at least: i) a nucleotide sequence encoding an alpha-farnesene synthase, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:1 ; c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; ii) regulatory sequences suitable for driving expression of the nucleic acid molecule in a host cell.

[0078] Particularly preferred is a recombinant nucleic acid molecule comprising at least: i) a nucleotide sequence encoding an alpha-farnesene synthase, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence as set forth in SEQ ID NO:2; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:1 ; c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; ii) regulatory sequences suitable for driving expression of the nucleic acid molecule in a host cell.

[0079] Furthermore, the present invention relates to a vector comprising the nucleic acid molecule according to the invention. Still further, the invention contemplates to a host cell comprising the nucleic acid molecule according to the invention, or the vector according to the invention.

[0080] As specified elsewhere herein, the host cell is preferably a microbial cell, such as a fungal cell, an archaea cell, an algae cell, a protozoa cell or a bacterial cell; more preferably a fungal or a bacterial cell, even more preferably a bacterial cell , still more preferably a cell of a Rhodobacter species or an Escherichia species,. Optionally, the host cell is a non-plant host cell.

[0081] Moreover, the invention may encompass a non-human transgenic organism comprising the nucleic acid molecule according to the invention, or the vector according to the invention, or the host cell according to the invention. Optionally, the invention may encompass a non-human nonplant transgenic organism comprising the nucleic acid molecule according to the invention, or the vector according to the invention, or the host cell according to the invention.

[0082] Still further, the invention encompasses the use of the nucleic acid molecule according to the invention, or the vector according to the invention, or the host cell according to the invention in the production of alpha-farnesene or in the production of a composition comprising alpha- farnesene.

[0083] Specifically, the production of alpha-farnesene is a fermentation process comprising the cultivation of a recombinant host cell under conditions suitable for the expression of alpha- farnesene synthase and alpha-farnesene synthesis or alpha-farnesene production.

[0084] The term “fermentation process” is typically understood as a method for cultivating the host cells in a fermenter at a suitable scale, such as 0.1 L to 500’000 L depending on the desired scale and application. Typically, the fermenter is adapted to provide the conditions for supporting host cell cultivation; this may include means for supplying fermentation medium into the fermenter, means for removing fermentation broth from the fermenter, means for adjusting the pH and / or means for adjusting the temperature of the fermentation broth. The fermentation medium is typically an aqueous fermentation medium with at least one carbon source and specifically suitable for supporting the cultivation and growth of the host cell or host organism and for the production of the desired product. More specifically, the fermentation medium additionally comprises micronutrients. The fermentation medium may be a chemically defined medium. Further suitable fermentation media and processes are known in the art and described for example in WO 2023 / 012111 , WO2023 / 105080 and EP 3017050 B1 . The fermentation media and processes are in particular dependent on the host cells such as specified elsewhere herein.

[0085] The term “cultivating” or “cultivation” as used herein refers to keeping alive and / or propagating host cells comprised in a fermentation broth at least for a predetermined time. The term may typically encompass phases of exponential cell growth at the beginning of growth after inoculation as well as phases of stationary growth. The cultivation typically results in the production of the desired product. Specifically, a desired product according to the invention comprises alpha- farnesene and optionally desired side components.

[0086] The composition obtained by and / or obtainable by the method of the invention comprises alpha- farnesene in an amount of at least 40 % (w / v); more preferably in an amount of at least 50 % (w / v); still more preferably in an amount of at least 60 % (w / v), still more preferably in an amount at least 70 %, at least 80 %, at least 90 %, at least 92 %, or at least 95 % (w / v). Examples of alpha-farnesene purity of a composition according to the invention are shown in Fig. 3. The composition according to the invention is advantageous as high amounts of alpha-farnesene, e.g. high purities thereof, can be reached. Said composition is typically understood as a “desired product” according to the invention. The amount of alpha-farnesene can be determined by techniques as known in the art, such as gas chromatography. Specifically, gas chromatography combined with a flame ionization detector (GC-FID) and hydrogen as a carrier gas may be used. Gas chromatography may be coupled to a quadrupole MS (GC-MS) in order to identify alpha- farnesene and potential side components using helium as a carrier gas. Further details are given in the examples below. The composition according to the invention may preferably comprise side components such as melonal (2,6-dimethyl-5-heptenal) in a desirable amount.

[0087] A “desirable amount” regarding the side components is typically in the range of 0.01 to 1 g / L, more typically in the range of 0.05 to 0.5 g / L of the composition. The amount of side components, in particular melonal, can be determined by techniques as known in the art, such as GC-FID and GC-MS. Desirable amounts of side components, in particular melonal, are specified elsewhere in herein in more detail.

[0088] Hence, the present invention further contemplates a composition comprising alpha-farnesene in an amount of at least 40 % (w / v) and melonal. The amount of melonal in the composition is preferably at least 1 pg / L. More preferably, the amount of melonal is at least 5 pg / L, at least 10 pg / L, at least 20 pg / L, at least 30 pg / L, or at least 40 pg / L, or at least 50 pg / L, or at least 100 pg / L. The amount of melonal in the composition may even be higher such as around at least 1 mg / L, or at least 5 mg / L, or at least 7 mg / L. Still more preferably, the amount of melonal does not exceed 1 g / L, even more preferably the amount of melonal does not exceed 500 mg / L, still even more preferably the amount of melonal does not exceed 200 mg / L, still even more preferably the amount of melonal does not exceed 100 mg / L or does not exceed 80 mg / L, or does not exceed 75 mg / L. The composition according to the invention is advantageous as it has a pleasant odor and / or pleasant taste due to the combined presence of alpha-farnesene and melonal. A pleasant odor and / or pleasant taste may be understood as an improved sensorial perception, e.g. improved olfactory and / or gustatory perception, in an organoleptic analysis in comparison to a control composition, for example in comparison to an alpha-farnesene composition without melonal, i.e. without melonal in any detectable amount.

[0089] A pleasant smell and / or pleasant taste may in particular refer to a more fresh, more soapy, and more fruity smell and / or taste perception in comparison to a control composition.

[0090] More preferably, the composition in line with the present invention comprises alpha-farnesene in an amount of at least 40 % (w / v) and melonal, preferably in an amount of at least 1 pg / L.

[0091] The composition is preferably a solution of the desired product in a suitable solvent, or as an oil, typically as an essential oil; in particular an oil essentially comprising, or essentially consisting of, alpha-farnesene. In particular, if comprising high amounts of alpha-farnesene, such as above 60 % (w / v), the composition may be an essential oil. The composition may be obtained or is obtainable by the method according to the invention.

[0092] The composition according to the invention may be purified for example by distillation, specifically for removing side components. Moreover, it may be used in the manufacture of vitamin A or vitamin E, and / or aviation fuels, and / or flavor and fragrance compositions, preferably in the manufacture of sinensal. The invention further relates to the use of the composition according to the invention for producing vitamins, such as vitamin A or vitamin E, and / or for producing aviation fuels in particular aviation fuels based on sesquiterpene skeletons, as for example disclosed in Advanced Science, Volumel O, Issue 23, 2023, 2300889 and / or for producing flavor and fragrance compositions, preferably flavor and fragrance compositions including compositions comprising sinensal.

[0093] Advantageously, it has been found in accordance with the present invention that the polypeptides exhibiting alpha-farnesene synthase activity identified in the studies underlying the present invention are particularly useful for converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide. Thereby, the invention provides for a method for producing alpha-farnesene which is surprisingly reliable and efficient and therefore reduces productions costs. Moreover, it results in a desired product composition that is particularly suitable in flavor and fragrance applications and for further downstream processing.

[0094] The explanations and interpretations of the terms given above in this specification apply for all embodiments characterized herein. The following embodiments are particular preferred embodiments according to the present invention.

[0095] 1 . A method for producing alpha-farnesene, the method comprising at least the following steps:

[0096] A) providing at least one polypeptide exhibiting alpha-farnesene activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) an amino acid sequence encoded by a nucleotide sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the nucleic acid sequence of any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; c) an amino acid sequence as set forth in SEQ ID NO:1 ; d) an amino acid sequence having at least 70%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity to the amino acid sequence of SEQ ID NO:1 ; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting alpha-farnesene synthase activity, or exhibiting alpha-farnesene synthase activity; and

[0097] B) converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide.

[0098] 2. The method according to embodiment 1 , wherein the step of converting farnesyl pyrophosphate into alpha-farnesene is carried out in a host cell.

[0099] 3. The method according to the preceding embodiment, wherein the host cell is a microbial cell, such as a fungal cell, an archaea cell, an algae cell, a protozoa cell or a bacterial cell; more preferably a fungal or a bacterial cell, even more preferably a bacterial cell, still more preferably a cell of a Rhodobacter species or an Escherichia species.

[0100] 4. The method according to the preceding embodiment s further comprising: cultivating the host cell in a fermentation broth under conditions and for a time suitable for alpha-farnesene production by the alpha-farnesene synthase activity of the polypeptide; and obtaining a desired product from the fermentation broth, preferably obtaining alpha- farnesene or a composition comprising alpha-farnesene or a composition derived from alpha-farnesene from the fermentation broth.

[0101] 5. The method according to any one of the preceding embodiment s, wherein the polypeptide exhibiting alpha-farnesene synthase activity is expressed from a recombinant nucleic acid molecule present in the host cell.

[0102] 6. The method according to the preceding embodiment 5, wherein the recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of: a) a nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence having at least 70 % of sequence identity to any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1 ; d) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; e) a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting farnesene synthase activity.

[0103] 7. The method according to any one of the preceding embodiments 5 or 6, wherein the recombinant nucleic acid molecule further comprises regulatory sequences suitable for driving expression of the nucleic acid molecule in the host cell.

[0104] 8. The method according to the preceding embodiment, wherein the regulatory sequences comprise T7, Ptac, Ptrc, Plac, PBAD, Prha, Ptet, or sigma70-based promoter sequences; and / or T7term, or rrnBterm terminator sequences.

[0105] 9. The method according to any one of the preceding embodiments 4 to 8, wherein said cultivating in step B) comprises cultivating the host cell in an aqueous fermentation medium at a temperature between 5 °C to 60 °C, preferably between 20 °C and 40 °C and a pH in the range of 3 to 10.

[0106] 10. The method according to any one of the preceding embodiments 4 to 9, wherein said obtaining alpha-farnesene comprises at least one or more of the following steps: precipitation, adsorption, solid-liquid separation, filtration, centrifugation, chromatography, extraction using a hydrophobic solvent; phase separation, solvent evaporation, distillation, preferably solvent evaporation and distillation.

[0107] 11. The method according to any one of the preceding embodiments 4 to 10, wherein the method further comprises a step of purifying the desired product, preferably purifying the obtained alpha-farnesene or composition comprising alpha-farnesene.

[0108] 12. The method according to the preceding embodiment, wherein the purifying comprises distillation of the composition comprising alpha-farnesene.

[0109] 13. A composition obtained by and / or obtainable by the method according to any of the preceding two embodiments, comprising alpha-farnesene in an amount of at least 40 % (w / v); more preferably at least 50 % (w / v) still more preferably at least 60 % (w / v).

[0110] 14. A composition comprising alpha-farnesene in an amount of at least 40 % (w / v) and melonal; preferably in an amount of at least 1 pg / L; or alpha-farnesene in an amount of at least 40 % (w / v) and melonal.

[0111] 15. Use of the composition according to any one of embodiments 13 or 14 for producing vitamins such as vitamin A or vitamin E, and / or aviation fuels and / or flavor and fragrance compositions, preferably flavor and fragrance compositions comprising sinensal.

[0112] 16. A recombinant nucleic acid molecule comprising at least: i) a nucleotide sequence encoding an alpha-farnesene synthase, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:1 ; c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; ii) regulatory sequences suitable for driving expression of the nucleic acid molecule in a host cell.

[0113] 17. A vector comprising the nucleic acid molecule according to any of embodiment 16.

[0114] 18. A host cell comprising the nucleic acid molecule according embodiment 16 or the vector according to the preceding embodiment 17.

[0115] 19. The host cell according to the preceding embodiment, wherein the host cell is a microbial cell, such as a fungal cell, an archaea cell, an algae cell, a protozoa cell or a bacterial cell; more preferably a fungal or a bacterial cell, even more preferably a bacterial cell, still more preferably a cell of a Rhodobacter species or an Escherichia species, still more preferably a Rhodobacter sphaeroides cell.

[0116] 20. A non-human transgenic organism comprising the nucleic acid molecule according to embodiment 16, or the vector according to embodiment 17, or the host cell according to embodiment 18.

[0117] 21 . Use of the nucleic acid molecule according to embodiment 16, or the vector according to 17, or the host cell according to embodiment 18, or the non-human transgenic organism according to embodiment 20 in the production of alpha-farnesene or in the production of a composition comprising alpha-farnesene.

[0118] 22. Use according to claim 21 , wherein the production of alpha-farnesene is a fermentation process comprising the cultivation of a recombinant host cell under conditions suitable for the expression of alpha-farnesene synthase and alpha-farnesene synthesis.

[0119] All references are herewith incorporated by reference in their entireties as well as with respect to the disclosure content specifically mentioned in this specification.

[0120] FIGURES

[0121] Fig. 1 : Alpha-farnesene synthase (AFS) catalyzed conversion of farnesyl diphosphate (FPP) to alpha-farnesene.

[0122] Fig. 2: Multiwell plate screening of alpha-farnesene synthases (AFSs) in combination with the isopentenol utilization pathway in E. coii. Shown are various alpha-farnesene synthases (AFS), Md - Malus domestica, At - Arabidopsis thah'ana, Gm - Glycine max, Pa - Picea abies, Pt - Populus trichocarpa, Aa - Artemisia annua, Sa - Santaium album, Sd - Scoparia duicis, Rc - Rosa chinensis, Ls - Lactuca sativa; (for UniProt databse IDs see Table 1 ).

[0123] Fig. 3: Gas chromatogram of (a) authentic a-farnesene standard (Merck) in dodecane and (b) organic extraction of E. coZZcultivation. E. coZZ carried the genes for the isopentenol utilization pathway and SdAFS.

[0124] Fig. 4: Mass spectra of main peak (RT 1.73 min, Fig. 3) in organic extract of extraction of E. coii cultivation. E. coZZhost cells carried the genes for the isopentenol utilization pathway and SdAFS. Mass spectra confirms the formation ofalpha-farnesene.

[0125] Fig. 5: Relative fermentation titers of selected AFSs in R. sphaeroides a large scale (1 L). - MdAFS reference; SdAFS. EXAMPLES

[0126] The Examples shall merely illustrate the invention. They shall by no means be construed as limiting the scope.

[0127] Example 1. Cloning of synthase genes in E. coli

[0128] The amino acid sequences of the synthases were identified by a diversity search from public databases (Table 1

[0015] ). The respective DNA sequences were derived thereof using standard codon usage of Escherichia coli; for example SEQ ID NO:3 and SEQ ID NO:7. The plasmid pETDuet1-idi-ispA was constructed by ligation of the DNA string idi ispA (synthesized by BioCat GmbH, SEQ ID NO:8) into pETDuetl (Novagen) using the restriction sites Ncol and Notl. The DNA sequences for the putative synthases were synthesized (Twist Bioscience or BioCat GmbH) and cloned into the plasmid pETDuetl -idi-ispA using the restriction sites Ndel and XhoL The resulting plasmids were used to transform competent cells

[0016] of the E. coli strain BL21- Gold(DE3) (Agilent) harboring the second plasmid pCDFDuetl JPK_thiM, which encodes the genes for isopentenyl phosphate kinase from Methanocaldococcus Jannaschii (\PK, UniProt ID: Q60352,

[0017] ) and hydroxyethlythiazole kinase from Lactiscaeilbacillus case / (thiM, UniProt ID: B3W782; WO2021229106) . The plasmid was obtained by cloning the codon-optimized genes for IPK and thiM (synthesized by BioCat GmbH) into the Ncol I Notl, and Ndel I Xhol restriction sites of pCDFDuetl (Novagen), respectively.

[0129] Table 1. List of tested putative alpha-farnesene synthases

[0130] Example 2. In vivo enzyme screening for the conversion of isoprenol toalpha-farnesene A single colony of the transformants was transferred into a well of a 96-well microplate with 900 pL LB medium supplemented with 100 pg / mL spectinomycin (Sigma Aldrich) and 50 pg / mL carbenicillin (Sigma Aldrich) and incubated overnight at 37 °C at an agitation of 1 ,000 rpm. Twenty pL of the overnight culture was used to inoculate 820 pL BASF preculture medium (WO2023 / 105080) containing 100 pg / mL spectinomycin and 50 pg / mL carbenicillin in a well of a 48-well FlowerPlate (m2p labs). Subsequently, the culture was incubated at 30 °C, 1 ,000 rpm, for 5 hours before gene expression was induced by the addition of 0.12 mM isopropyl-R>-D- thiogalactopyranosid. After 2 hours of incubation, isoprenol (BASF ) was added at a final concentration of 58.1 mM and the culture was overlaid with 150 pL dodecane (Merck). After additional 17 hours, 150 pL dodecane containing 0.1 % tetradecane as an internal standard were added. The plate was centrifuged at 18,000 g for 15 min and the organic phase was collected for GC measurement (example 3). The resulting alpha-farnesene concentrations are shown in Fig. 2

[0131] Example 3. Quantification of alpha-farnesene

[0132] The quantification of alpha-farnesene in dodecane was carried out with GC using an Agilent 7890 GC, equipped with an Agilent DB-5 column (10 m x 0.1 mm x 0.1 pm) and a flame ionization detector. Hydrogen was used as a carrier gas with a flow rate of 0.3 mL / min and the injection volume was set to 1 pL with a split ratio of 1 :100 (120 °C; 120 240 °C, 40 °C / min). The quantification of a farnesene was performed using a standard curve of an authentic alpha- farnesene standard (Merck), see Fig. 3.

[0133] Example 4. Identification of compounds via GC-MS a-Farnesene was identified by GC-MS using an Agilent 6890N GC coupled to a Quadrupole MS and equipped with a VF-1 ms column (50 m x 0.25 mm x 0.25 pm). Helium was used as a carrier gas with a flow rate of 1 .2 mL / min and the injection volume was set to 1 pL with a split ratio of 1 :20 (50 °C, 2 min; 50 280 °C, 10 °C / min; 280 °C, 45 min), as depicted in Fig. 4.

[0134] Example 5. Cloning of MdAFS and SdAFS for the expression in Rhodobacter sphaeroides

[0135] For the expression of the MdAFS (SEQ ID NO:6) and SdAFS gene (SEQ ID NO:2) in combination with and without MBP solubility tag under the regulation of promoter SPppa, the constructs SPppa-MBP and SPppa, and the MdAFS or SdAFS gene were synthesized by Genscript USA Inc. (Piscataway, N.J., USA) and cloning in a pUC57 modD plasmid. All the sequences were codon optimized for the expression in R. sphaeroides (e.g. SEQ ID NO:4). The plasmids with the AFS sequences were assembled with other pUC57 modD plasmids which contain the terpene pathway machinery by Golden gate cloning using standard procedures. Therefore, the final plasmid contains the gene for expressing one of the AFS’ and genes for expressing the enzymes for catalysing the reaction steps of the mevalonate pathway or another metabolic pathway enabling the production of the C5 prenyl diphosphates isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP), which are the universal isoprenoid building blocks (US 2015 / 0079649 A1). Reaction mixtures were transformed into E. coli S17-1 cells. Transfer of the plasmids from S17-1 to R. sphaeroides Rs265-9c by conjugation was performed using standard procedures (U.S. Pat. No. 9,260,709B2).

[0136] Example 6. Growth conditions shake flask cultivation of R. sphaeroides

[0137] Seed cultures were performed in 100 ml shake flasks without baffles with 20 ml RS102 medium (U.S. Pat. No. 9,260,709B2) with 100 mg / L neomycin and a loop of glycerol stock. The flasks were grown for 48 hours at 30° C. in a shaking incubator with an orbit of 50 mm at 110 rpm. Then the OD600 of the culture was assessed to calculate the exact volume of culture to be transferred to the larger flasks. Shake flask experiments were performed in 300 ml shake flasks with 2 bottom baffles. Twenty ml of RS102 medium and neomycin to a final concentration of 100 mg / L were added to the flask together with 2 ml of sterile n-dodecane. The volume of the inoculum was adjusted to obtain a final OD600 value of 0.05 in 20 ml medium. The flasks were kept for 72 hours at 30° C. in a shaking incubator with an orbit of 50 mm at 110 rpm.

[0138] Example 7. Sample preparation for analysis of isoprenoid content in organic phase

[0139] Cultures were collected 72 hours after inoculation in pre-weighted 50 ml PP tubes which were then centrifuged at 4500xg for 20 minutes. The n-dodecane layer was transferred to a microcentrifuge tube in acetone solution (1 :9) for later GC analysis.

[0140] Example 8. Gas Chromatography Flame Ionization Detector (GC-FID)

[0141] Gas chromatography was performed on a Shimadzu GC2010 Plus equipped with a Restek RTX- 5sil MS capillary column (30 mx0.25 mm, 0.5 pm). The injector and FID detector temperatures were set to 280° C. and 300° C., respectively. Gas flow through the column was set at 1 .3 mL / min. The oven initial temperature was 70° C., increased to 180° C. at a rate of 5° C. / min, further increased to 300° C. at a rate of 50° C. / min, and held at that temperature for 3 min. Injected sample volume was 1 pL with a 1 :50 split-ratio, and the nitrogen makeup flow was 30 ml / min.

[0142] Example 9. Growth conditions for R. sphaeroides cultivation in 1-L fermenters

[0143] 1 L fermentation was carried out for 144 hours using standard procedures as described in WO 2023 / 012111 A2. Example 10. Large scale production using R.sphaeroides

[0144] Seed medium and main fermentation medium, including an organic second phase, were prepared as described in the patent EP 3017050 B1. The main fermentation was performed in a 35 m3vessel that is charged with 10000 kg medium containing 22 g / L initial glucose at a temperature of 30°C, a pH of 7.0 (controlled with 28 wt% NH3 solution), an aeration of 2 Nm3 / min and an overpressure of 0.5 bar. At 0 hours the main fermentation was inoculated using 1 m3 seed culture. The dissolved oxygen (DO) is kept constant at 35% by adjusting the stirrer speed between 60 and 90 RPM and adjusting the aeration between 0.2 and 1.8 vvm. After 12 hours of batch fermentation the pO2 value decreased strongly to below 15%. Also the pH increase rapidly from pH 7.0 to pH 7.7. Analysis shows that all glucose was consumed and glucose feeding was started, keeping the pH at 7.0. Surprisingly, when the product concentration is increased above 1 g / L and the pO_,2 is maintained below 15%, >1 ug / L of melonal (2,6-dimethyl-5-heptenal) is spontaneously formed. Melonal has a powerful green melon odor, and a composition containing natural alpha-farnesene and natural melonal can be obtained. This composition was deemed organoleptically favorable by flavorists (see example 12).

[0145] Example 11. Analysis of Terpene Production by R. sphaeroides Sxa\r\s

[0146] The titer obtained with the strain expressing SdAFS without solubility tag was 91 % more of that obtained with the MBP-MdAFS strain in shake flask experiments. AFS01-037 yielded 73% more alpha-farnesene than MBP-MdAFS in 120h fermentation; results depicted in Fig. 5.

[0147] Example 12. Organoleptic analysis of alpha-farnesene compositions

[0148] A panel of three flavor analysts (panelists) were given samples of different alpha-farnesene compositions for odor and taste comparison. The results are compiled below in Tables 2 and 3 below.

[0149] Table 2. Sensorial analysis of alpha-farnesene compositions with 7 ppm melonal

[0150] Table 3. Sensorial analysis of alpha-farnesene compositions with 73 ppm melonal

[0151] Already at 7 ppm melonal, the sensory perception was improved in comparison to an alpha- farnesene composition without melonal. In an alpha-farnesene composition with 73 ppm melonal, the sensory improvement was even more pronounced.

[0152] In summary, the presence of melonal improved the organoleptic characteristics, i.e. olfactory and gustatory characteristics, of the compositions.

[0153] Cited literature

[0154] 1. Huang, Y., Z. Ye, X. Wan, G. Yao, J. Duan, J. Liu, M. Yao, X. Sun, Z. Deng, K. Shen, H. Jiang, and T. Liu, Systematic Mining and Evaluation of the Sesquiterpene Skeletons as High Energy Aviation Fuel Molecules. Adv Sci (Weinh), 2023. 10(23): p. e2300889

[0155] 2. Ye, Z., B. Shi, Y. Huang, T. Ma, Z. Xiang, B. Hu, Z. Kuang, M. Huang, X. Lin, Z. Tian, Z. Deng, K. Shen, and T. Liu, Revolution of vitamin E production by starting from microbial fermented farnesene to isophytol. Innovation (Camb), 2022. 3(3): p. 100228

[0156] 3. WO2021 / 130058, Bonrath, W., A. Mueller, and B. Wuestenberg, Funtionalisation of 1 ,3- Alpha-Dienes (I), DSM, 2021

[0157] 4. WO2021 / 197889, Bonrath, W., A. Mueller, and B. Wuestenberg, Process for Production of New Sulfolenic Intermediates, DSM, 2021

[0158] 5. Liu, Y., Z. Wang, Z. Cui, Q. Qi, and J. Hou, Progress and perspectives for microbial production of farnesene. Bioresour Technol, 2022. 347: p. 126682

[0159] 6. Meadows, A.L., K.M. Hawkins, Y. Tsegaye, E. Antipov, Y. Kim, L. Raetz, R.H. Dahl, A. Tai, T. Mahatdejkul-Meadows, L. Xu, L. Zhao, M.S. Dasika, A. Murarka, J. Lenihan, D. Eng, J.S. Leng,

[0160] C.L. Liu, J.W. Wenger, H. Jiang, L. Chao, P. Westfall, J. Lai, S. Ganesan, P. Jackson, R. Mans,

[0161] D. Platt, C.D. Reeves, P.R. Saija, G. Wichmann, V.F. Holmes, K. Benjamin, P.W. Hill, T.S. Gardner, and A.E. Tsong, Rewriting yeast central carbon metabolism for industrial isoprenoid production. Nature, 2016. 537(7622): p. 694-697

[0162] 7. Chatzivasileiou, A.O., V. Ward, S.M. Edgar, and G. Stephanopoulos, Two-step pathway for isoprenoid synthesis. Proc Natl Acad Sci U S A, 2019. 116(2): p. 506-511

[0163] 8. Clomburg, J.M., S. Qian, Z. Tan, S. Cheong, and R. Gonzalez, The isoprenoid alcohol pathway, a synthetic route for isoprenoid biosynthesis. Proc Natl Acad Sci U S A, 2019. 116(26): p. 12810-12815

[0164] 9. Lund, S., R. Hall, and G.J. Williams, An Artificial Pathway for Isoprenoid Biosynthesis Decoupled from Native Hemiterpene Metabolism. ACS Synth Biol, 2019. 8(2): p. 232-238

[0165] 10. WQ2012 / 106405, Zhao, L., L. Xu, P. Westfall, and A. Main, Methods of Developing Terpene Synthase Variants, Amyris, 2012

[0166] 11. Tong, Y., T. Hu, L. Tu, K. Chen, T. Liu, P. Su, Y. Song, Y. Liu, L. Huang, and W. Gao, Functional characterization and substrate promiscuity of sesquiterpene synthases from Tripterygium wilfordii. Int J Biol Macromol, 2021. 185: p. 949-958

[0167] 12. Whitehead, J.N., N.G.H. Leferink, L.O. Johannissen, S. Hay, and N.S. Scrutton, Decoding Catalysis by Terpene Synthases. ACS Catal, 2023. 13(19): p. 12774-12802

[0168] 13. Tang, R., Q. Wen, M. Li, W. Zhang, Z. Wang, and J. Yang, Recent Advances in the Biosynthesis of Farnesene Using Metabolic Engineering. J Agric Food Chem, 2021. 69(51): p. 15468-15483

[0169] 14. Yamamura, Y., F. Kurosaki, and J.B. Lee, Elucidation of terpenoid metabolism in Scoparia dulcis by RNA-seq analysis. Sci Rep, 2017. 7: p. 43311

[0170] 15. UniProt, https: / / www.uniprot.org

[0171] 16. Chung, C.T. et al., Proc Natl Acad Sci U S A, 1989, 86, 2172

[0172] 17. DOI: 10.1128 / JB.188.9.3192-3198.2006

[0173] 18. Nature, volume 537, pages 694-697 (2016), https: / / doi.org / 10.1038 / nature19769 19. Advanced Science, VolumelO, Issue 23, 2023, 2300889, https: / / doi.org / 10.1002 / advs.

[0174] 202300889

[0175] 20. Needleman, Saul B. & Wunsch, Christian D. (1970). "A general method applicable to the search for similarities in the amino acid sequence of two proteins". Journal of Molecular Biology 48 (3): 443-453

[0176] 21. WO 2023 / 105080

[0177] 22. WO 2008 / 000632

[0178] 23. Altschul, S.F., Gish, W., Miller, W., Myers, E.W. & Lipman, D.J. (1990) "Basic local alignment search tool." J. Mol. Biol. 215:403-410

[0179] 24. Altschul, Stephen F., Thomas L. Madden, Alejandro A. Schaffer, Jinghui Zhang, Zheng Zhang, Webb Miller, and David J. Lipman (1997), "Gapped BLAST and PSI-BLAST: a new generation of protein database search programs", Nucleic Acids Res. 25:3389-3402

[0180] 25. Rice, P., et al. Trends in Genetics (2000) 16: ' l'o-' n

[0181] 26. Sambrook et aL, "Molecular Cloning”, Cold Spring Harbor Laboratory, 1989

[0182] 27. Hames and Higgins (Ed.) 1985, ’’Nucleic Acids Hybridization: A Practical Approach”, IRL Press at Oxford University Press, Oxford

[0183] 28. Brown (Ed.) 1991 , "Essential Molecular Biology: A Practical Approach”, IRL Press at Oxford University Press, Oxford29. WO2014 / 014339

[0184] 30. EP 3017050 B1

[0185] 31. W02023 / 212400

[0186] 32. Lee-Yoon Low et aL, Transgenic Plants: Gene constructs, vector and transformation method. 2018. DOI.10.5772 / intechopen.79369

[0187] 33. Pinkert, C. A. (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky G. M.

[0188] 34. Robl, J. M. (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington D.C); Sambrook, loc.cit, Ausubel, loc.cit

[0189] 35. Methods in Molecular Biology, 1995, Vol. 44, Agrobacterium protocols, Ed.: Gartland and Davey, Humana Press, Totowa, New Jersey

[0190] 36. Hellens et al, Trends in Plant Science (2000) 5, 446—451

[0191] 37. Plant Molecular Biology and Biotechnology (CRC Press, Boca Raton, Florida), chapter 6 / 7, pp. 71-119 (1993)

[0192] 38. F.F. White, Vectors for Gene Transfer in Higher Plants; in: Transgenic Plants, vol. 1 , Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press, 1993, 15-38

[0193] 39. B. Jenes et aL, Techniques for Gene Transfer, in: Transgenic Plants, vol. 1 , Engineering and Utilization, Ed.: Kung and R. Wu, Academic Press (1993), 128-143

[0194] 40. Potrykus 1991 , Annu. Rev. Plant Physiol. Plant Molec. BioL 42, 205225

[0195] 41. Smith 1988, Gene 67:31-40

[0196] 42. Amann 1988, Gene 69:301-315

[0197] 43. Studier 1990, Methods in Enzymology 185, 60-89

[0198] 44. Baldari 1987, Embo J. 6:229-234

[0199] 45. Kurjan 1982, Cell 30:933-943

[0200] 46. Schultz 1987, Gene 54:113-123 47. van den Hondel, C.A.M.J.J., & Punt, P.J. (1991) “Gene transfer systems and vector development for filamentous fungi, in: Applied Molecular Genetics of fungi, J.F. Peberdy et aL, Ed., pp. 1-28, Cambridge University Press: Cambridge

[0201] 48. More Gene Manipulations in Fungi (J.W. Bennett & L.L. Lasure, Ed., pp. 396-428: Academic Press: San Diego

[0202] 49. Smith 1983, Mol. Cell Biol. 3:2156-2165)

[0203] 50. Lucklow 1989, Virology 170:31-39.

[0204] Table 4. Sequence Listing

Claims

Claims1 . A method for producing alpha-farnesene, the method comprising at least the following steps:A) providing at least one polypeptide exhibiting alpha-farnesene activity, wherein the polypeptide comprises an amino acid sequence selected from the group consisting of: a) an amino acid sequence encoded by the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) an amino acid sequence encoded by a nucleotide sequence having at least 70% sequence identity to the nucleic acid sequence of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; c) an amino acid sequence as set forth in SEQ ID NO:1 ; d) an amino acid sequence having at least 70% sequence identity to the amino acid sequence of SEQ ID NO:1 ; e) an amino acid sequence of a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting farnesene synthase activity, or exhibiting farnesene synthase activity; andB) converting farnesyl pyrophosphate to alpha-farnesene by the alpha-farnesene synthase activity of the polypeptide.

2. The method according to claim 1 , wherein the step of converting farnesyl pyrophosphate into alpha-farnesene is carried out in a host cell.

3. The method according to the preceding claim 2, wherein the host cell is a microbial cell, such as a fungal cell, an archaea cell, an algae cell, a protozoa cell or a bacterial cell; more preferably a fungal or a bacterial cell.

4. The method according to the preceding claims further comprising: cultivating the host cell in a fermentation broth under conditions and for a time suitable for alpha-farnesene production by the alpha-farnesene synthase activity of the polypeptide; and obtaining a desired product from the fermentation broth, preferably obtaining alpha- farnesene or a composition comprising alpha-farnesene or a composition derived from alpha-farnesene from the fermentation broth.

5. The method according to any one of the preceding claims, wherein the polypeptide exhibiting alpha-farnesene synthase activity is expressed from a recombinant nucleic acid molecule present in the host cell.

6. The method according to any one of the preceding claims, wherein the recombinant nucleic acid molecule comprises a nucleotide sequence selected from the group consisting of:a) a nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence having at least 70 % of sequence identity to any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4, c) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of SEQ ID NO:1 ; d) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; e) a fragment of any one of (a) to (d), said fragment encoding a polypeptide exhibiting farnesene synthase activity; preferably further comprising regulatory sequences suitable for driving expression of the nucleic acid molecule in the host cell.

7. A composition obtained by and / or obtainable by the method according to any one of the preceding claims, comprising alpha-farnesene in an amount of at least 40 % (w / v).

8. A composition comprising alpha-farnesene in an amount of at least 40 % (w / v) and melonal (2,6-dimethyl-5-heptenal) preferably in an amount of at least 1 pg / L.

9. Use of the composition according to any one of claims 7 or 8 for producing vitamins, such as vitamin A or vitamin E, and / or aviation fuels, and / or flavor and fragrance compositions, preferably flavor and fragrance compositions comprising sinensal.

10. A recombinant nucleic acid molecule comprising at least: i) a nucleotide sequence encoding an alpha-farnesene synthase, wherein said nucleotide sequence is selected from the group consisting of: a) the nucleotide sequence as set forth in any of SEQ ID NO:2, SEQ ID NO:3, or SEQ ID NO:4; b) a nucleotide sequence that encodes a polypeptide comprising the amino acid sequence of any of SEQ ID NO:1 ; c) a nucleotide sequence that encodes a polypeptide comprising an amino acid sequence having at least 70% sequence identity to the amino acid sequence of any of SEQ ID NO:1 ; ii) regulatory sequences suitable for driving expression of the nucleic acid molecule in a host cell.

11. A vector comprising the nucleic acid molecule according to any of claim 10.

12. A host cell comprising the nucleic acid molecule according claim 10 or the vector according to the preceding claim 11 .

13. The host cell according to the preceding claim 12, wherein the host cell is a microbial cell, such as a fungal cell, an archaea cell, an algae cell, a protozoa cell or a bacterial cell; more preferably a fungal or a bacterial cell, even more preferably a bacterial cell.

14. A non-human transgenic organism comprising the nucleic acid molecule according to claim 10, or the vector according to claim 11 , or the host cell according to claim 12.

15. Use of the nucleic acid molecule according to claim 10, or the vector according to claim11 , or the host cell according to claim 12 in the production of alpha-farnesene or in the production of a composition comprising alpha-farnesene.

Images