An artificial alkane oxidation system for the allylic oxidation of terpene substrates.

Abstract

An artificial alkane oxidation system comprising components a. an oxidase enzyme, and b. one or more enzymes to provide one or more alkanes. The artificial alkane oxidation system optionally comprises c. an electron transfer compound suitable for transferring at least one electron to the oxidase enzyme, and further optionally an electron transfer compound regenerating enzyme suitable for reducing the electron transfer compound of dc when it is in an oxidized state. The oxidase enzyme is an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or fragments or variants thereof.

Description

[Technical field]

[0001] The present invention relates to an artificial alkane oxidation system, its synthetic nucleic acid sequence, expression cassettes, methods for the oxidation of at least one alkane, preferably an alkene, more preferably a terpene, oxidized terpene products produced therefrom, and uses of the artificial alkane oxidation system. [Background technology]

[0002] Terpenes are widely used in food processing, personal care, and care chemicals. They are extracted from natural sources such as citrus fruits, oils from bark, etc. Terpenes are also produced by chemical as well as biosynthetic pathways.

[0003] Many citrus oils, including orange and mandarin oils, contain sesquiterpene aldehydes, commonly referred to as terpenes, more specifically α-sinensal and β-sinensal. Sinensal is part of the aldehyde fraction of orange oil and is considered a quality parameter, especially with regard to organoleptic properties. The odor of β-sinensal is described as orange, sweet, fresh, waxy, and juicy, while the odor of α-sinensal is described as citrus, orange, and mandarin. Sinensal is used as a flavoring agent in citrus products.

[0004] The chemical synthesis routes of the aldehyde fraction of this oil are known in the art. The synthesis of α-sinensal is described by Buchi (1974; JACS 96, 7573-4) starting from (E)-3-methyl-2,4-pentadiene-1-ol and using pyridine and phosphorus tribromide. The synthesis of β-sinensal is described, inter alia, by Bertele & Schudel (US Pat. No. 3,699,169; US Pat. No. 3,974,226) starting from β-farnesene and using ozonolysis, and by Hiyama (1978, Tetrahedron letters 19, 3051-4) starting from trienaldehyde.

[0005] However, chemically synthesized compounds or compounds prepared by synthetic routes are classified separately from the naturally occurring aldehyde fraction. Compounds obtained by biosynthesis are generally labeled as natural and have a price advantage. However, so far, not all terpene compounds have been disclosed as being applicable for biosynthetic production.

[0006] For example, the biosynthesis of sinensal is not known in the art. Structurally, α-sinensal and β-sinensal are related to the sesquiterpene hydrocarbons α-farnesene and β-farnesene. Both sinensals retain an aryl aldehyde group compared to farnesene. Both α-farnesene and β-farnesene are well-known components of citrus oils. However, the biosynthetic pathway by which farnesene is allylated to aryl alcohol by oxidative enzymes such as monooxygenases, which is further oxidized to sinensal by alcohol dehydrogenases, to produce sinensal, is not known in the art. Also, monooxygenases or any other oxidative enzymes that perform the appropriate aryl oxidation of farnesene have not been described in the art.

[0007] Arfmann (Biocatalys, 1988, Vol. 2, pp. 59-67) has demonstrated that sinensal can be produced from nerolidol. Microorganisms incubated with trans-nerolidol give 12-hydroxy-trans-nerolidol, which upon further oxidation gives the 12-carboxylic acid of trans-nerolidol. The use of microorganisms for the allylic oxidation (or "omega oxidation") of different stereoisomers of nerolidol to 12-hydroxynerolidol has been disclosed (Hrdlicka, Biotechnol. Prog. 2004, 20, 368-376). However, further transformation of 12-hydroxynerolidol has not been disclosed in the art.

[0008] Abraham (Z. Naturforsch. 47c, 851-858, 1992) discloses sulfonated derivatives of farnesene in which the diallyl group is oxidized by Nocardia sp. DSM 43130 and Pseudomonas lapsa DSM 50274. However, the activity of the strains used on the non-sulfonated form of farnesene has not been disclosed in the art.

[0009] Iurescia Sandra et al. (Applied and Environmental Microbiology, American Society For Microbiology, US, vol. 65, no. 7, 1 July 1999 (1999-07-01), pages 2871-2876) disclose an isolated and biologically transformed Pseudomonas sp. M1 and four open reading frames (ORFs) named myrA (aldehyde dehydrogenase), myrB (alcohol dehydrogenase), myrC (acyl-Coenzyme A (CoA) synthetase), and myrD (enoyl-CoA hydratase). Iurescia et al. present a 13-myrcene catabolic pathway in Pseudomonas sp. strain M1 involving these four proteins. According to Iurescia and co-workers, these four enzymes initiate the catabolism by myrcene alcohol. However, the enzyme that catalyzes the first step in converting myrcene to myrcene alcohol was not disclosed by Iurescia and coworkers at that time.

[0010] In another research project, the authors reported the results of a shotgun sequencing project of Pseudomonas strain M1 (Iurescia et al 1999, Soares-Castro&Santos 2013). The authors disclosed more than 6000 provisional genes of Pseudomonas sp. M1. Among the 6000 provisional genes, there is one that encodes a protein designated as fatty acid desaturase, with the UniProt database identifier W5IZV3. This gene is identical to the protein of SEQ ID NO: 1 of the present invention. It should be noted that this database entry is flagged to warn that the sequence is preliminary data. Furthermore, the four genes myrA-myrD identified in the initial study on myrcene catabolism have UniProt database entries Q9XD59, Q9XD58, Q9XD57 and Q9XD60, respectively, and therefore this fatty acid desaturase is not related to the authors' other work reported in Applied And Environmental Microbiology, 1 July 1999, pages 2871-2876. Moreover, these four sequences related to myrcene catabolism share less than 35% sequence identity with SEQ ID NO: 1 of the present invention.

[0011] The aryl oxidation of other terpenes such as amorphadiene (Ro et al 2006, Nature 440:940-943), germacrene A (https: / / doi.org / 10.1104 / pp.19.00629), and santalene (Diaz-Chavez PLoS One. 2013 Sep 18;8(9):e75053.) has been disclosed to be mediated by plant cytochrome P450 enzymes. However, no enzymes have been disclosed to mediate the aryl oxidation of farnesene to sinensal.

[0012] DEGENHARDT J ET AL, PHYTOCHEMISTRY, ELSEVIER, AMSTERDAM, NL, vol. 70, no. 15-16, 1 October 2009 (2009-10-01), pages 1621-1637 provides a review of monoterpene and sesquiterpene synthases.

[0013] EP 2706111 A1 discloses pathways and mechanisms for imparting the production of desired carbon-based products, such as ethanol, ethylene, chemicals, polymers, n-alkanes, isoprenoids, pharmaceuticals or intermediates, in photoautotrophic organisms such that these organisms efficiently convert carbon dioxide and light into the desired carbon-based products, and the use of such organisms, inter alia, for the commercial production of ethanol, ethylene, chemicals, polymers, n-alkanes, isoprenoids, pharmaceuticals or intermediates.

[0014] In Williams Shoshana C Et Al,Journal Of Inorganic Biochemistry,Elsevier Inc,US,vol.219,16 March 2021(2021-03-16),ISSN:0162-0134, the authors report a rubredoxin-fused alkane monooxygenase gene from Dietzia cinnamea that can oxidize long-chain fully saturated alkanes such as heptane, octane, nonane, decane, undecane, and tridecane in cell lysates. The authors perform a relational analysis of the class of rubredoxin-fused alkane monooxygenases and the role of this class of alkane monooxygenases in cell wall biosynthesis. Furthermore, introduction of point mutations into the full-length protein of Dietzia cinnamea resulted in the production of less epoxide products than the wild type with the fully saturated alkanes heptane, octane, nonane, decane, undecane, and tridecane as substrates. The mutant produced approximately equal amounts of aldehyde and epoxide products. Summary of the Invention [Problem to be solved by the invention]

[0015] The object of the present invention was to provide a complete biosynthetic process for terpene-based aldehyde and / or alcohol products. A further object of the present invention was to provide a fermentation system for the production of terpene-based aldehyde and / or alcohol products, such as the first fermentation-based production of sinensal. [Means for solving the problem]

[0016] Surprisingly, it has been found that the above objectives are achieved by providing an artificial alkane oxidation system, its synthetic nucleic acid sequence, expression cassette, a method for the oxidation of at least one alkane, preferably an alkene, more preferably a terpene, the oxidized terpene products produced therefrom, and uses of the artificial alkane oxidation system.

[0017] Thus, in one aspect, the present invention provides a method for producing a composition comprising: a. an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment thereof; b. one or more enzymes, preferably terpene synthase proteins, which provide at least one alkane; Optionally, c. an electron transfer compound suitable for transferring at least one electron to an oxidase enzyme, and optionally and an electron transport compound regenerating enzyme suitable for reducing the electron transport compound of the dc when it is in an oxidized state, The at least one alkane-providing oxidase enzyme and the one or more enzymes are not found in combination in nature and constitute an artificial alkane oxidation system.

[0018] The artificial alkane oxidation system is maintained under conditions suitable for producing at least one alkane and / or contacted with at least one alkane under conditions suitable for oxidizing said at least one alkane. In the latter case, in one embodiment, the alkane oxidation system comprises components a., optionally supplemented with c. and optionally with d., but not including b.

[0019] In another aspect, the presently claimed invention relates to a synthetic nucleic acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:44, wherein the synthetic nucleic acid sequence comprises a nucleic acid sequence encoding an oxidase enzyme as described above, and optionally comprises a rubredoxin peptide and / or a rubredoxin reductase peptide.

[0020] In another aspect, the presently claimed invention comprises: A first isolated nucleic acid sequence encoding a protein having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; a second isolated nucleic acid sequence that is not physically linked to the first nucleic acid encoding an electron transfer protein; Optionally, a third nucleic acid sequence encoding an electron transfer protein reductase, which third nucleic acid sequence may be fused to the first and / or second nucleic acid sequence.

[0021] In another aspect, the presently claimed invention comprises: a synthetic nucleic acid sequence, or A nucleic acid sequence encoding an alkane oxidation system, or A nucleic acid sequence for an alkane oxidation system, a. has at least 70% sequence identity to SEQ ID NO:20; or A nucleic acid sequence of an oxidase enzyme encoding an oxidase enzyme having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. has at least 70% sequence identity to SEQ ID NO:21; or A nucleic acid sequence of a rubredoxin peptide encoding rubredoxin of SEQ ID NO: 2 or SEQ ID NO: 45, and optionally c. has at least 70% sequence identity to SEQ ID NO:22; or The present invention relates to an expression cassette comprising a nucleic acid sequence of an alkane oxidation system comprising the nucleic acid sequence of a rubredoxin reductase peptide, which encodes the rubredoxin reductase peptide of SEQ ID NO:3.

[0022] In another aspect, the presently claimed invention relates to an isolated expression cassette comprising a nucleic acid sequence of an alkane oxidation system, the nucleic acid sequence comprising: a. a nucleic acid sequence of an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO: 20, or a nucleic acid sequence encoding an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. a nucleic acid sequence of a rubredoxin peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:21, or a nucleic acid sequence encoding a rubredoxin having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:2, and optionally c. A nucleic acid sequence of a rubredoxin reductase peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO: 22, or a combination of a, b and c.

[0023] In another aspect, the presently claimed invention relates to a method for the oxidation of at least one alkane substrate, preferably an alkene substrate, more preferably a terpene substrate, the method comprising: a. for the oxidation of at least one alkane, preferably an alkene, more preferably a terpene substrate, under conditions suitable for the oxidation of the terpene substrate; i. an artificial alkane oxidase system as disclosed above, and / or ii. Expression of the synthetic nucleic acid sequence disclosed above, or the expression cassette disclosed above, and / or iii. Expression of a nucleic acid sequence encoding an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO:1, SEQ ID NO:10, SEQ ID NO:11, SEQ ID NO:23-43, or fragments or variants thereof; and iv. optionally providing at least one alkane, preferably an alkene, more preferably a terpene substrate, if not produced by an alkane oxidation system; b. allylic oxidation of an alkane, preferably an alkene, more preferably a terpene substrate to produce at least one oxidized alkane, preferably an alkene, more preferably a terpene product; and c. optionally extracting at least one oxidized alkane, preferably an alkene, more preferably a terpene product.

[0024] In another aspect, the presently claimed invention relates to a method for producing at least one oxidized alkane product, preferably an alkene product, more preferably a terpene product, as defined herein, comprising the steps of: a. for the oxidation of at least one alkane, preferably alkene, more preferably terpene substrate, under conditions suitable for the oxidation of the alkane, preferably alkene, more preferably terpene substrate; i. an artificial alkane oxidase system as disclosed above, and / or ii. Expression of the synthetic nucleic acid sequence disclosed above, or the expression cassette disclosed above, and / or iii. Expression of a nucleic acid sequence encoding an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; and iv. optionally providing at least one alkane, preferably an alkene, more preferably a terpene substrate, if not produced by an alkane oxidation system; b. the allylic oxidation of an alkane, preferably an alkene, more preferably a terpene substrate to produce at least one oxidized terpene product; c. optionally extracting at least one oxidized alkane, preferably an alkene, more preferably a terpene product.

[0025] If the alkane oxidase system of the invention produces one or more alkane, preferably alkene, more preferably terpene substrates, or is present with a system producing one or more substrates, or is contained within a host cell producing at least one substrate, then no substrate needs to be added. In any case, one or more substrates may still be added in such circumstances, for example to make more substrate available or to provide a desired substrate not present prior to the alkane oxidation system or in the method of the invention.

[0026] In another aspect, the invention claimed herein relates to the use of an alkane oxidation system as disclosed above, or a non-human host cell of the invention, a fermentation composition as disclosed herein, or the expression of a nucleic acid sequence as disclosed above, or an expression cassette as disclosed above, for the oxidation of a terpene substrate, thus producing one or more oxidized terpene products.

[0027] In another aspect, the presently claimed invention relates to the production of alkane oxidation products by artificial alkane oxidation systems, synthetic nucleic acids, non-human host cells comprising the expression cassettes, or methods as described.

[0028] In another aspect, the invention claimed herein relates to a composition produced by the method as described above or by a non-human host cell, the composition comprising myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderasin and bicyclo-octanediol, oxidized α-guaiene, oxidized β-guaiene, or combinations thereof, and optionally one or more terpene substrates, and optionally an oxidase enzyme as described.

[0029] In another aspect, the presently claimed invention comprises: a non-human host cell cultured in a culture medium; and at least one oxidized terpene product as a major compound produced from a non-human host cell. The fermentation composition includes a terpene substrate and optionally one or more by-products are trace compounds.

[0030] In one embodiment, the fermented composition is an intermediate product that is subjected to further processing, including but not limited to extraction, concentration, purification, drying, heat treatment, pressure treatment, vacuum treatment, or combinations thereof.

[0031] In another embodiment, the fermentation composition is the end product that is not subjected to further processing or that is subjected to further processing.

[0032] In another aspect, the presently claimed invention relates to a method for the fermentative production of at least one oxygenated terpene product, the method comprising: Providing an oxidase enzyme system in a non-human host cell; and culturing the non-human host cell in the culture medium to produce at least one oxidized terpene product.

[0033] In another aspect, the present invention relates to a method for preparing a mutant polypeptide having oxidase enzymatic activity, the method comprising: a) selecting a synthetic nucleic acid as described herein (having at least 70% sequence identity to any one of SEQ ID NOs: 4, 12, 17, 18, 19, or 44, wherein the synthetic nucleic acid sequence comprises a nucleic acid sequence encoding an oxidase enzyme as defined herein, and optionally an electron transfer protein and / or an electron transfer protein reductase), or a nucleic acid encoding an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43 or a fragment thereof; b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c) transforming a non-human host cell with the mutated nucleic acid sequence and expressing the oxidase enzyme encoded by the mutated nucleic acid sequence; d) screening the oxidase enzyme for at least one altered property in addition to the function of the oxidase enzyme encoded by the selected nucleic acid of a); e) optionally repeating process steps (a)-(d) to obtain an oxidase enzyme with the desired mutant function; f) optionally isolating the mutated nucleic acid sequence from the transformed non-human host cell obtained in step (c). [Brief description of the drawings]

[0034] [Figure 1a]The embodiment shows the conversion of myrcene by E. coli BL21 containing pET-DUET as a control and pET-M1-alkB-rr-rrr carrying an alkane oxidation system. [Figure 1b] Soares-Castro P, Montenegro-Silva P, Heipieper HJ, Santos PM. 2017. Functional characterization of a 28-kilobase catabolic island from Pseudomonas sp. strain M1 involved in biotransformation of β-myrcene and related plant-derived volatiles. Appl Environ Microbiol 83:e03112-16. Copied for demonstration purposes mass spectra as found in Figure S2. [Figure 1c] The mass spectrum identified as myrcene aldehyde from the conversion of myrcene shown in Figure 1a after comparison with the literature reference in Figure 1b is shown. [Diagram 2] 1 shows an embodiment of the conversion of β-farnesene by E. coli BL21 containing pET-DUET as a control and pET-M1-alkB-rr-rrr carrying an alkane oxidation system. [Figure 3a] FIG. 1 shows an embodiment with sinensal produced by the Rhodobacter strain Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr harboring an alkane oxidation system. [Figure 3b] FIG. 1 shows an embodiment with sinensal produced by Rhodobacter strain Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr, confirmed by comparison to a sinensal standard. [Figure 3c] Mass spectrum of sinensal produced by Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr. [Figure 3d]The mass spectrum of sinensal standard is shown. [Figure 4] 1 shows a mass spectrum demonstrating the production of α-sinensal using an alkane oxidation system, see the Examples section for details. [Diagram 5] 1 shows mass spectra demonstrating the production of bisabolene and lantheolaldehyde using an alkane oxidation system. See the Examples section for details. [Figure 6] 1 shows mass spectra demonstrating that santalene and santalol were found when using the alkane oxidation system, see the Examples section for details. [Figure 7a] FIG. 1 shows an alignment of polypeptides of the invention with conserved amino acids at the indicated positions, in which the positions of conserved amino acids are indicated by white font letters on a black background. [Figure 7b] FIG. 1 shows an alignment of polypeptides of the invention with conserved amino acids at the indicated positions, in which the positions of conserved amino acids are indicated by white font letters on a black background. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0035] Before describing the compositions and formulations of the present invention, it is to be understood that the invention is not limited to the particular compositions and formulations described, as such compositions and formulations may, of course, vary. It is also to be understood that the terms used herein are not intended to be limiting, since the scope of the present invention will be limited only by the appended claims.

[0036] The terms "comprising," "comprises," and "comprised of," as used herein, are synonymous with "including," "includes," or "containing," and are inclusive or open-ended and do not exclude additional, unrecited members, elements, or method steps. The terms "comprising," "comprises," and "comprised of," as used herein, should be understood to include the terms "consisting of," "consists," and "consists of." More specifically, the term "comprise," as used herein, means that the claim includes all recited elements or method steps, but may also include additional, unspecified elements or method steps. For example, a method including steps a), b), and c) in the narrowest sense includes a method consisting of steps a), b), and c). The phrase "consisting of" means that the composition (or device or method) has the recited elements (or steps) and no others. In contrast, the term "comprises" can also encompass methods that include additional steps, such as steps d) and e) in addition to steps a), b) and c).

[0037] Furthermore, terms such as "first", "second", "third" or "(a)", "(b)", "(c)", "(d)" in the specification and claims are used to distinguish between similar elements and do not necessarily describe a sequential or chronological order. It is to be understood that terms so used are interchangeable under appropriate circumstances and that the embodiments of the invention described herein can be performed in other orders than those described or illustrated herein. When terms such as "first", "second", "third" or "(A)", "(B)" and "(C)" or "(a)", "(b)", "(c)", "(d)", "i", "ii" etc. relate to steps of a method or use or assay, unless otherwise indicated in the application, as described hereinbefore or hereinafter, there is no time or consistency between the steps, i.e., the steps may be performed simultaneously or there may be a time interval of seconds, minutes, hours, days, weeks, months or even years between such steps.

[0038] In the following text, the different aspects of the invention are defined in more detail. Each aspect so defined can be combined with any other aspect or aspects, unless expressly indicated otherwise. In particular, any feature indicated as being preferred or advantageous can be combined with any other feature or features indicated as being preferred or advantageous.

[0039] References throughout this specification to "one embodiment" or "an embodiment" mean that a particular feature, structure, or characteristic described in connection with the embodiment is included in at least one embodiment of the present invention. Thus, the appearances of the phrase "in one embodiment" or "in an embodiment" in various places throughout this specification do not necessarily all refer to the same embodiment, but may refer to the same embodiment. Furthermore, particular features, structures, or characteristics can be combined in any suitable manner in one or more embodiments, as would be apparent to one of ordinary skill in the art from this disclosure. Furthermore, although some embodiments described herein include some features but not other features included in other embodiments, it is understood by one of ordinary skill in the art that combinations of features of different embodiments are within the scope of the present invention and form different embodiments. For example, in the appended claims, any of the claimed embodiments may be used in any combination.

[0040] Furthermore, ranges defined throughout this specification are inclusive, i.e., a range of 1 to 10 means that both 1 and 10 are included in the range. For the avoidance of doubt, Applicant reserves the right to any equivalents pursuant to applicable law.

[0041] As used herein, the term "about" when used to qualify the value of a stated item, number, percentage or term refers to a range of plus or minus 10 percent, 9 percent, 8 percent, 7 percent, 6 percent, 5 percent, 4 percent, 3 percent, 2 percent, or 1 percent of the value of the stated item, number, percentage or term. A range of plus or minus 10 percent is preferred.

[0042] When a numerical range is used herein, such as "at a concentration of 1 to 5 micromolar," the range includes not only 1 micromolar and 5 micromolar, but also all values ​​between 1 and 5 micromolar, for example, 2, 3, and 4 micromolar.

[0043] The term "in vitro" as used herein refers to outside or external to the animal or human body. The term "in vitro" as used herein should be understood to include "ex vivo." The term "ex vivo" generally refers to tissues or cells that have been removed from an animal or human body and maintained or grown outside the body, for example in a culture vessel. The term "in vivo" as used herein refers to inside or internal to the animal or human body.

[0044] The terms "protein" or "polypeptide" or "(poly)peptide" or "peptide" (all terms used interchangeably unless otherwise indicated) as used herein encompass isolated and / or purified and / or recombinant (poly)peptides essentially free of other host cell polypeptides. The term "peptide" as referred to herein comprises at least 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 26, 27, 28, 29, 30, 31, 32, 33, 34, 35, 36, 37, 38, 39, 40, 45, 50, 60, 70, 80, 90, 100, 150, 200, 250, 300 or even more amino acid residues in which one α-carboxyl group is linked to another α-amino group. Post-translational modification of a protein or peptide as used and contemplated herein refers to modifications of the newly formed protein or peptide and may include deletion, substitution or addition of amino acids, chemical modification of specific amino acids such as amidation, acetylation, phosphorylation, glycosylation, formation of pyroglutamic acid, oxidation / reduction of the sulfa group in methionine, or addition of similar small molecules to specific amino acids.

[0045] "Homolog" means bacterial, fungal, plant or animal homologs, preferably plant homologs, of the oxidase enzyme or rubredoxin or rubredoxin reductase useful in the present invention, but also includes truncated sequences, single stranded DNA or RNA of the coding and non-coding DNA sequences.

[0046] Sequence identity, homology or similarity is defined herein as the relationship between two or more amino acid sequences or two or more nucleic acid sequences as determined by comparing these sequences. Sequence identity or similarity is usually compared over the entire length of the sequences, but may only be compared over a portion of the sequences aligned with each other. Preferably, sequence identity or similarity is compared herein over the entire length of the sequences. In the art, "identity" or "similarity" also refers to the degree of sequence relatedness between such sequences, as may be determined by the match between polypeptide sequences or nucleic acid sequences, as the case may be.

[0047] Sequence alignments can be generated using 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 sequence acid of two proteins”. Journal of Molecular Biology 48(3):443-453.

[0048] This algorithm is implemented, for example, in the "NEEDLE" program, which performs a global alignment of two sequences, and is included, for example, within 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, for example, based on alignment of conserved regions 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 of the many BLOSUMs is "BLOSUM62", which is often the "default" setting in many programs when aligning protein sequences. -BLAST (Basic Local Alignment Search Tool) - consists of several individual programs (BlastP, BlastN) that are primarily used to search for similar sequences in large sequence databases. BLAST programs also generate local alignments. Usually, the "BLAST" interface provided by NCBI (National Centre for Biotechnology Information) is used, which is an improved version ("BLAST2"). “Original” BLAST: Altschul, SF, Gish, W., Miller, W., Myers, EW & Lipman, DJ (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.

[0049] 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 using the NOBRIEF option ('Brief identity and similarity' to NO) which calculates the "longest identity" (version 2.8.0 and later, EMBOSS: The European Molecular Biology Open Software Suite-Rice, P., et al. Trends in Genetics (2000) 16:276-277; http: / / emboss.bioinformatics.nl). The identity, homology or similarity between two aligned sequences is calculated as follows: the number of corresponding positions in the alignment that show identical amino acids in both sequences is divided by the total length of the alignment after subtracting 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.

[0050] Sequence identity is usually given as "% sequence identity" or "% identity". In a first step, to determine the percent identity between two amino acid sequences, a pairwise sequence alignment is generated between these two sequences, aligning the two sequences over their entire length, full length or full length (i.e., pairwise global alignment). The alignment is generated using the programs or software described herein. The preferred alignment for the purposes of the present invention is the alignment that allows the maximum sequence identity to be determined.

[0051] In one aspect, the present invention relates to an artificial alkane oxidation system, comprising at least one alkane, such as but not limited to an alkene, such as but not limited to a terpene, and as a further component: a. an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; b. one or more enzymes, preferably terpene synthase proteins, which provide at least one alkane; Optionally, c. an electron transfer compound suitable for transferring at least one electron to an oxidase enzyme, and optionally and an electron transfer compound regenerating enzyme suitable for reducing the electron transfer compound of dc when it is in an oxidized state.

[0052] As component a., it is possible to have more than one type of oxidase enzyme.

[0053] The artificial alkane oxidation system is maintained under conditions suitable for producing at least one alkane and / or contacted with at least one alkane under conditions suitable for oxidizing said at least one alkane.

[0054] In the latter case, in one embodiment, the alkane oxidation system comprises component a. plus c. and optionally d., but does not include b.

[0055] An electron transfer compound suitable for transferring at least one electron to the oxidase enzyme (see c. above) is not intentionally included in the artificial alkane oxidation system, but may be present in the vicinity of the alkane oxidation system so as to be able to functionally interact with the alkane oxidation system. For example, when the alkane oxidation system is contained within a host cell, the host cell can provide a suitable electron transfer compound without the need to have an additional electron transfer compound included in the alkane oxidation system. As another example, it is possible to disrupt a cell with a suitable electron transfer compound and then combine a portion of the cell membrane containing a suitable electron transfer compound with an alkane oxidation system of the invention that does not itself contain a suitable electron transfer compound.

[0056] Generally, as has been demonstrated, it is advantageous to include a suitable electron transport compound as part of the alkane oxidation system.

[0057] Artificial alkane oxidation system: Alkanes should be understood to include alkenes. In one embodiment, alkane oxidation systems are referred to interchangeably as alkene oxidation systems.

[0058] "Alkene" is to be understood as a hydrocarbon, preferably a terpene compound, containing a carbon-carbon double bond. In one aspect of the invention, the alkene is a monoterpene, sesquiterpene or diterpene having one or more allyl groups. In a further aspect of the invention, the alkene is a diallylalkene, for example a diallylsesquiterpene.

[0059] Thus, one aspect of the present invention refers to an artificial alkane oxidation system, a method of the present invention, and an expression cassette and a host cell useful in the method of the present invention, wherein at least one alkane is made of at least 5 carbon atoms, or at least one C5, which is an integer between 1 and 6, has 5, 10, 15, 20, 25 or 30 carbon atoms per alkane molecule, respectively. The at least one alkane includes a linear alkene and a non-linear alkene. In one embodiment, the at least one alkane is a non-linear alkene. In a preferred embodiment, the non-linear alkene is a terpene. It should be understood that references to an alkane preferably refer to an alkene, more preferably a terpene. Thus, any reference to an alkane substrate preferably refers to an alkene substrate, more preferably a terpene substrate, and any reference to an alkane product preferably refers to an alkene product, more preferably a terpene product.

[0060] In one embodiment, the alkane oxidation system of the present invention should therefore be understood to be a terpene oxidation system.

[0061] Component a: Oxidase enzyme The disclosed alkane oxidation system comprises one or more oxidase enzymes suitable for oxidizing at least one alkane, at least one heterologous sesquiterpene synthase protein, optionally a rubredoxin peptide, or / and a rubredoxin reductase peptide.

[0062] In another aspect of the invention, the oxidase enzyme is an enzyme suitable for oxidizing at least one alkane, preferably having a PFAM domain A_desaturase (PF00487) in its central part (analyzed using PFAM version 34.0, for details of PFAM see "Pfam: The protein families database in 2021: J. Mistry, S. Chuguransky, L. Williams, M. Qureshi, GA Salazar, ELL Sonnhammer, SCE Tosato, L. Paladin, S. Raj, LJ Richardson, RD Finn, A. Bateman Nucleic Acids Research (2020) doi: 10.1093 / nar / gkaa913 and http: / / pfam.xfam.org / ).

[0063] In one aspect of the invention, the oxidase enzyme is suitable for oxidizing at least one alkane and is an oxidoreductase of the enzyme class 1.14.19.x. In a further aspect of the invention, the oxidase enzyme is an enzyme of the enzyme class 1.14.19.1.

[0064] In one embodiment, the oxidase enzyme or monooxygenase enzyme or alkane oxidase is defined as an integral membrane diiron protein. In a further embodiment, the oxidase enzyme is based on an enzyme from bacteria, for example and not limited to those further described in Smits 2002, J Bacteriol 184, 1733-1742. Alkane-oxidizing alkB enzymes have been described from a wide range of bacteria (Smits 2002, J Bacteriol 184, 1733-1742). However, activity of the alkB enzymes has not been demonstrated on farnesene or any other sesquiterpene.

[0065] In one embodiment, the oxidase enzyme is an alkene monooxygenase from a subclass that is not naturally fused to rubredoxin. Non-limiting examples are those shown as SEQ ID NOs: 1 and 43. One of skill in the art can easily determine, for example, by the length of the protein, whether it is a class of alkene monooxygenase that is naturally fused to rubredoxin or not.

[0066] Preferably, the oxidase enzyme is an alkane oxidase having at least 50% identity to the known Pseudomonas M1 alkB hydroxylase (Soares-Castro 2017 Appl Environ Microbiol 83:e03112-16.; SEQ ID NO:1), such as any one of SEQ ID NOs:23-43 or any of the engineered proteins set forth in SEQ ID NOs:10, 11, or fragments or variants thereof. In one embodiment, the oxidase enzyme is sourced from or based on these enzymes forming Pseudomonas species, such as, but not limited to, Pseudomonas M1 bacteria. This M1 is capable of growing on myrcene, a monoterpene structurally similar to farnesene but lacking one of the isoprene units.

[0067] More preferably, the oxidase is an alkane oxidizing alk B enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of the sequences from Acinetobacter as exemplified by SEQ ID NOs: 1, 10, 11 or 23-42 (PM1_0216370), e.g., the Acinetobacter guillouiae protein disclosed under the name Uniprot entry A0A077KZY1, or the Acinetobacter sp. enzyme disclosed as A0A2S9EQI7 (SEQ ID NOs: 23 and 24, respectively). In another more preferred embodiment, the oxidase enzyme of the alkane oxidation system is an alkane oxidation alk B enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO: 43 which encodes the CMR5c oxidase protein / AlkB oxidase enzyme (Pseudomonas sp. CMR5c). SEQ ID NO: 44 encodes the rubredoxin of SEQ ID NO: 43 and SEQ ID NO: 45.

[0068] More preferably, the oxidase enzyme is a protein having an amino acid sequence that has at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity at the amino acid level to any one of SEQ ID NOs: 1, 10, 11, 23-43, or fragments thereof.

[0069] In one embodiment, the variant of the oxidase enzyme is a polypeptide of any of SEQ ID NOs: 10, 11, 23-43 or an amino acid sequence with at least 70%, 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43 and has oxidase activity. Preferably, the variant is a conservatively modified variant. More preferably, the variant has conserved amino acids as in Figures 7a, 7b for SEQ ID NOs: 1, 33, 36, 37, 39, 40, 41 and 42.

[0070] A fragment of an oxidase enzyme, as referred to herein, may be a polypeptide consisting of the amino acid sequence of any of the above-mentioned sequences and sequence variants, which has a sufficient length to exhibit alkane oxidase enzyme activity as defined herein.

[0071] Typically, a fragment will consist 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 a sequence or sequence variant referred to herein. In preferred embodiments, the fragment has at least 20%, or at least 30%, or at least 40%, or at least 50%, or at least 60%, or at least 70%, or at least 80%, or at least 90%, or 100% of the activity of the full-length sequence.

[0072] In one embodiment of the present invention, a fragment of an oxidase enzyme is a polypeptide exhibiting alkane oxidase enzyme activity, as defined herein, capable of converting at least one terpene substrate into an oxidized terpene product. It is therefore envisaged that the above-mentioned biologically active fragment of a polypeptide preferably comprises the PFAM domain A_desaturase (PF00487), preferably in its central part.

[0073] Terpenes: By definition, the term "terpene" includes only hydrocarbons composed of carbon and hydrogen. The term "terpenoid" refers to terpenes that contain further functional groups that give rise to derivatives such as alcohols, aldehydes, ketones, and acids: see e.g., Flavors and Fragrances: Chemistry, Bioprocessing and Sustainability RG Berger; Black et al., Terpenoids and their role in wine flavour: recent advances. Australian Journal of Grape and Wine Research 21, 582-600, 2015; Zhou & Pichersky, More is better: the diversity of terpene metabolism in plants. Current Opinion in Plant Biology 2020, 55:1-10; Degenhardt J, Kollner TG, Gershenzon J (2009) Monoterpene and sesquiterpene synthases and the origin of terpene skeletal diversity in plants. Phytochemistry 70(15):1621-1637). Depending on the number of isoprene units in their structure that are connected by head-tail addition, terpenes are classified according to the number of carbon atoms or sesquiterpenoid moieties, respectively: monoterpenes (C10), sesquiterpenes (C15), diterpenes (C20), triterpenes (C30), or polyterpenes with up to 30,000 connected isoprene units. Similar to terpenes, terpenoids are also classified according to the number of isoprene units, and are structured as monoterpenoids (C10) or sesquiterpenoids (C15), and are further named with the suffix "-oids". In scientific literature, the term terpene is often used interchangeably with the term terpenoid, although they have different meanings. As used herein, the term "terpene" includes both hydrocarbons and their functional derivatives, preferably hydrocarbons.Typically, the terpenes when used as substrates in the alkane oxidation system of the present invention will not be in an oxidized form and therefore may not include oxidized terpene products as defined elsewhere herein, although there may be some exceptions, such as DETA, as defined herein.

[0074] As used herein, a "monoterpene" is a C10 terpene. Monoterpenes are usually prepared from C10 geranyl diphosphate (GPP) as an intermediate and may be cyclic or linear.

[0075] "Sesquiterpenes" are C15 terpenes constructed from three isoprene units. Like monoterpenes, sesquiterpenes can be acyclic or contain rings, including many unique combinations. They are found in many other biological systems, particularly in higher plants, as well as marine organisms and fungi. In nature, they occur as hydrocarbons or in oxidized forms, including lactones, alcohols, acids, aldehydes, and ketones. Sesquiterpenes also include essential oils and aroma compounds that have some pharmacological activity.

[0076] "Diterpenes" are C20 terpenes that occur naturally in plants and microorganisms. Diterpene molecules have commercial value because they can be converted into amber notes that find application in the fragrance industry. Typically, sesquiterpenes when used as substrates in the alkane oxidation system of the present invention are not in oxidized form and therefore may not include oxidized terpene products as defined elsewhere herein, although there may be some exceptions, such as DETA, as defined herein.

[0077] Component b: Terpene synthase protein: Terpene synthase proteins include terpene synthase proteins that have the ability to form one or several terpenes from a single substrate or multiple substrates.

[0078] The terpene synthase proteins referred to below include monoterpene synthases, diterpene synthases and / or sesquiterpene synthases. Monoterpene synthases are involved in a general carbocation reaction mechanism that is initiated by divalent metal ion-dependent ionization of the substrate. The resulting cationic intermediate undergoes a series of cyclizations, hydride shifts, or other rearrangements before the reaction is terminated by loss of a proton or addition of a nucleophile. Monoterpene synthases are further described in Jorg Degenhardt (Phytochemistry 70 (2009) 1621-1637). Generally, monoterpene synthase proteins promote the formation of monoterpenes such as myrcene, pinene, camphene, phellandrene, terpinolene, limonene, ocimene, linalool, cineole, geraniol, terpinene, terpineol, fenchol, carene, sabinene, bornyl diphosphate, and the like, and structural variants, isomers, derivatives, and the like, thereof.

[0079] Genes encoding diterpene synthases have been extensively described (Zerbe, Trends Biotechnol 2015 Jul;33(7):419-28.), and microbial production of these compounds has been demonstrated (e.g., Schalk J. Am. Chem. Soc. 2012, 134, 18900-18903).

[0080] Sesquiterpene synthase protein refers to a protein that facilitates the conversion of acyclic prenyl diphosphates and squalene to multiple cyclic and acyclic forms.

[0081] Sesquiterpene synthase proteins catalyze the formation of sesquiterpenes from farnesyl diphosphate using a carbocation-based reaction mechanism similar to that of monoterpene synthases. In general, sesquiterpene synthase proteins facilitate the formation of sesquiterpenes (hereinafter interchangeably referred to as sesquiterpene substrates), which are terpenes having 15 carbon atoms (3 isoprene units), such as longifolene, farnesene, bisabolene, curcumene, germacrene A / D / D-4-ol, patchoulol, valencene, sesquithujene, macrocarpene, caryophyllene, humulene, eudesmol, nerolidol, barbatene, amorpha-4,11-diene, 8-epi-cedrol, 5-epi-aristolochene, cadinene, cadinenebetispiradiene, bergamotene, elemene, cubene, cubebol, muurola-3,5-diene, selinene, zingiberene, farnesol, sinenal, santalene, valencene, guayene, diterpenes, and the like, and structural variants, isomers, derivatives, and the like, thereof.

[0082] Terpene Substrates: Terpene substrates should be understood to include monoterpenes, sesquiterpenes, and / or diterpenes, including but not limited to terpene hydrocarbon molecules as substrates for oxidation reactions. In one aspect of the present invention, the one or more terpene substrates are one or more sesquiterpene substrates. For example, the one or more sesquiterpene substrates include α-farnesene, β-farnesene, sinensal, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, α-guayene, diterpene, monoterpene, geraniol, nerol monoterpene, linalyl acetate, limonene, β-pinene, or geranyl lactone, or mixtures thereof. Typically, the terpene substrate is not an oxidized terpene product as defined elsewhere herein, although there may be some exceptions, such as DETA, as defined herein.

[0083] Component c: Electron transport compound: The electron transport compound transfers at least one electron to the oxidase enzyme.

[0084] Preferably, the electron transport compounds include a) proteins having soluble electron transfer agents, b) membrane-bound components of the electron transport chain, c) metalloenzymes, and the like.

[0085] In one embodiment, the electron transport compound is an electron transport protein, preferably an F1-S0 type protein.

[0086] More preferably, the electron transport compound is a rubredoxin peptide comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:2 or the sequence known from UniProt database entry A0A3G7A099 (SEQ ID NO:45) or a fragment thereof.

[0087] Rubredoxin peptides are soluble, low molecular weight iron-containing peptides required for electron transfer. Rubredoxins contain functional iron atoms strongly bound to sulfur, like other non-heme iron proteins such as ferredoxins, hemerythrins, aconitases, etc., but not porphyrins. The single iron atom is connected to the rest of the protein through four tetrahedrally arranged sulfur atoms. Electron transfer is handled by a single Fe redox center coordinated to four cysteinyl thioates. The two redox states are formally Fe(II) and Fe(III).

[0088] Preferably, the rubredoxin peptide is an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:2, or the polypeptide known as A0A3G7A099 from the UniProt database, or a fragment thereof.

[0089] More preferably, the rubredoxin peptide is an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity at the amino acid level to SEQ ID NO: 2 or 45, or a fragment thereof.

[0090] A fragment of a rubredoxin peptide, as referred to herein, may be a polypeptide consisting of any of the amino acid sequences of the above sequences and sequence variants that have a length sufficient to exhibit electron transfer activity capable of reducing and regenerating an oxidized alkane oxidase enzyme.

[0091] Typically, fragments of rubredoxin peptides consist of a stretch of at least 20, at least 25, at least 30, at least 40, at least 45 or at least 50 contiguous amino acids from the above sequences or sequence variants.

[0092] Component d: Electron transport compound regenerating enzyme: The electron transport compound regenerating enzyme is one suitable for reducing an electron transport compound as described herein when the electron transport compound is in an oxidized state and typically regenerating it.

[0093] The electron transfer compound regenerating enzyme is an electron transfer protein reductase, preferably a rubredoxin reductase protein.

[0094] Rubredoxin reductase is normally NADH-dependent and is required to recycle rubredoxin after electron transfer. Rubredoxin is reduced to the ferrous state by NADH during catalysis by rubredoxin reductase and reoxidized to the ferric form by ω-hydroxylase during the catalytic cycle.

[0095] Preferably, the rubredoxin reductase peptide is an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:3, or a fragment thereof.

[0096] More preferably, the rubredoxin peptide is an amino acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity at the amino acid level to SEQ ID NO:3 or a fragment thereof.

[0097] A fragment of a rubredoxin reductase peptide, as referred to herein, may be a polypeptide consisting of any of the amino acid sequences of the above-mentioned sequences and sequence variants, which has a length sufficient to exhibit electron transfer activity capable of reducing and regenerating oxidized rubredoxin.

[0098] Typically, fragments of rubredoxin reductase peptides are 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 derived from the above sequences or sequence variants.

[0099] In a preferred embodiment, the artificial alkane oxidation system comprises one or more electron transfer proteins as electron transfer compounds, preferably of the F1-S0 type, more preferably one or more rubredoxin peptides, comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity with SEQ ID NO:2 or SEQ ID NO:45 or a fragment or variant thereof, and / or the at least one electron transfer compound regenerating enzyme is an electron transfer protein reductase, preferably a rubredoxin reductase, more preferably a rubredoxin reductase peptide, having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity with SEQ ID NO:3 or a fragment thereof.

[0100] In a more preferred embodiment, the alkane oxidation system comprises an oxidase enzyme encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:20, a rubredoxin peptide encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:21, and / or a rubredoxin reductase peptide encoded by a nucleic acid sequence having at least 70% sequence identity to SEQ ID NO:22.

[0101] Preferably, the oxidase enzyme is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:20.

[0102] Preferably, the rubredoxin peptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:21.

[0103] Preferably, the rubredoxin reductase peptide is encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:22, or a fragment thereof.

[0104] The alkane oxidation system of the present invention can be applied to host cells, but can also be applied in a cell-free environment. For example, cell membrane parts derived from disrupted cells, artificial membrane systems, reconstituted cell membranes, etc. may be used in combination with the alkane oxidation system of the present invention.

[0105] Another aspect of the invention relates to a synthetic nucleic acid having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, or SEQ ID NO:19, or SEQ ID NO:44, wherein the synthetic nucleic acid sequence comprises a nucleic acid sequence encoding an oxidase enzyme, and optionally a rubredoxin peptide and / or a rubredoxin reductase peptide.

[0106] Preferably, the synthetic nucleic acid sequence encodes an artificial alkane oxidation system, the components listed in brackets being encoded by the nucleic acid sequence of SEQ ID NO:4 (components a, c and d), SEQ ID NO:12 (a, c and d), SEQ ID NO:17 (a, c and d), SEQ ID NO:18 (a, c and d) or SEQ ID NO:19 (only a and c).

[0107] Preferably, the artificial alkane oxidation system has components a, c and d encoded by a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:4, SEQ ID NO:12, or SEQ ID NO:17, SEQ ID NO:18.

[0108] Preferably, the artificial alkane oxidation system has only components a and c encoded by nucleic acid sequences comprising nucleic acid sequences having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:19.

[0109] In one embodiment, at least one polypeptide of components a to d is fused to a tag peptide. In one embodiment, one or more of the polypeptides used in components a to d are composed of a first segment comprising a tag peptide and a second segment comprising the respective polypeptide of each component according to the present invention. A polypeptide composed of the first segment and the second segment may be referred to herein as a "tagged polypeptide".

[0110] The tag peptide is preferably selected from the group of nitrogen utilization protein (NusA), thioredoxin (Trx), maltose binding protein (MBP), glutathione S-transferase (GST), ubiquitin-like factor (SUMO) or calcium binding protein (Fh8), and functional homologs thereof. As used herein, a functional homolog of a tag peptide is a tag peptide that has at least about the same effect on the solubility of the tagged enzyme compared to the non-tagged enzyme. Typically, a homolog differs in that one or more amino acids are inserted, substituted, deleted or extended in the peptide of the homolog. A homolog may in particular include one or more substitutions of a hydrophilic amino acid for another hydrophilic amino acid, or one or more substitutions of a hydrophobic amino acid for another hydrophobic amino acid. A homologue may in particular have at least 40%, more particularly at least 50%, preferably at least 55%, more preferably at least 60%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 98% or at least 99% sequence identity with the sequence of NusA, Trx, MBP, GST, SUMO or Fh8.

[0111] In a preferred embodiment, the tag peptide is maltose binding protein from Escherichia coli, or a functional homologue thereof.

[0112] The use of tagged polypeptides according to the invention is particularly advantageous in that it may contribute to increased production, and in particular increased cellular production, of at least one oxidized terpene product.

[0113] To improve the solubility of the tagged polypeptide (compared to an untagged polypeptide), it is preferred that a first segment of the polypeptide is linked at its C-terminus to the N-terminus of a second segment, or alternatively, that a first segment of the tagged polypeptide is linked at its N-terminus to the C-terminus of a second segment.

[0114] The present disclosure further relates to a nucleic acid comprising a nucleotide sequence encoding a polypeptide, the polypeptide being composed of a first segment comprising a tag peptide, preferably MBP, NusA, Trx, GST, SUMO or Fh8-tag, or a functional homologue of any of these, and a second segment comprising the polypeptide of any of components a to d. The second segment may comprise, for example, an amino acid sequence as set forth in SEQ ID NO: 1, 10, 11, or 23 to 43, or a functional analogue thereof.

[0115] Furthermore, the disclosure relates to a host cell comprising said nucleic acid encoding said tagged polypeptide. Particular nucleic acids according to the invention encoding tagged polypeptides are shown in SEQ ID NO: 4, 12, 17, 18, 19, or 44. The host cell may in particular comprise a gene comprising any of these sequences or a functional analogue thereof.

[0116] Furthermore, the present disclosure relates to a polypeptide comprising a first segment comprising a tag peptide and a second segment comprising any one of the polypeptides of components a to d, wherein the tag peptide is preferably selected from the group consisting of MBP, NusA, Trx or SET.

[0117] The nucleic acid (or polynucleotide) of the present invention includes a nucleic acid sequence encoding an alkane oxidation system of the present invention. The nucleic acid sequence encoding an alkane oxidation system of the present invention is preferably a recombinant and / or isolated and / or purified nucleic acid sequence. The nucleic acid sequence encoding an alkane oxidation system of the present invention can be produced and isolated using standard techniques of molecular biology, the sequence information and organisms provided herein. The term "nucleic acid" as used herein includes reference to deoxyribonucleotide or ribonucleotide polymers, i.e., polynucleotides, in either single-stranded or double-stranded form, and includes known analogs that have the basic properties of natural nucleotides in that they hybridize to single-stranded nucleic acids in the same way as natural nucleotides (e.g., peptide nucleic acids), unless otherwise limited. A polynucleotide can be a full-length sequence or a subsequence of a natural or heterologous structural or regulatory gene. Unless otherwise indicated, the term includes reference to the sequence specified as well as its complementary sequence. Thus, DNA or RNA with backbones modified for stability or for other reasons are "polynucleotides" as that term is intended herein. Furthermore, DNA or RNA containing 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 understood that a wide 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 used herein encompasses such chemically, enzymatically or metabolically modified forms of polynucleotides, as well as chemical forms characteristic of viral and cellular DNA and RNA, including simple and complex cells, among others. All nucleic acid sequences herein that code for polypeptides such as oxidase enzymes or rubredoxin or rubredoxin reductase also refer to all possible silent variations of the nucleic acid by reference to the genetic code. The term "conservatively modified variants" applies to both amino acid and nucleic acid sequences.With respect to a particular nucleic acid sequence, the term "conservatively modified variants", when used, may refer to nucleic acids that code for identical or conservatively modified variants of amino acid sequence 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 code for any given protein. For example, the codons GCA, GCC, GCG, and GCU all code for the amino acid alanine. Thus, at every position where alanine is specified by a codon, the codon can be changed to any of the corresponding codons described without changing the encoded polypeptide. Such nucleic acid variations are "silent variations" and refer to one type of conservatively modified variation. The terms "polypeptide", "peptide" and "protein" are used interchangeably herein to refer to a polymer of amino acid residues.

[0118] The terms "polypeptide", "peptide" and "protein" also apply to natural amino acid polymers, as well as to amino acid polymers in which one or more amino acid residues are artificial chemical analogues of the corresponding natural amino acids. An essential property of such analogues of natural amino acids is that, when incorporated into a protein, the protein is specifically reactive with antibodies elicited against the same protein, but a protein entirely composed of natural amino acids. The terms "polypeptide", "peptide" and "protein" also include modifications, including but not limited to glycosylation, lipid attachment, sulfation, gamma-carboxylation of glutamic acid residues, hydroxylation and ADP-ribosylation. Within the context of the present application, oligomers (oligonucleotides, oligopeptides, etc.) are considered as a type of group of polymers. Oligomers have a relatively small number, generally 2-100, in particular 6-100, of monomer units, including, for example, primer sequences such as those used in the examples to clone the oxidase enzyme or rubredoxin or rubredoxin reductase useful in the present invention.

[0119] The term "heterologous" when used in reference to a nucleic acid (DNA or RNA) or protein of the invention refers to a nucleic acid or protein that does not naturally occur as part of the organism, cell, genome or DNA or RNA sequence in which it is found, or that is found in one or more locations in a cell or genome or DNA or RNA sequence that is different from that in which it is found in nature. Heterologous nucleic acids or proteins of the invention are not endogenous to the cell in which they are introduced, but are obtained from another cell, or are synthetically or recombinantly produced. Generally, but not necessarily, such nucleic acids encode proteins that are not normally produced by the cell in which the DNA is expressed. Genes that are endogenous to a particular host cell, but that have been modified from their native form, for example, by using DNA shuffling, are also referred to as heterologous. The term "heterologous" also includes multiple copies of a naturally occurring DNA sequence that do not occur in nature. Thus, the term "heterologous" can refer to a DNA segment that is foreign or heterologous to the cell, or that is homologous to the cell, but is present in a location and / or number within the host cell nucleic acid in which that segment is not normally found. The foreign DNA segment is expressed to obtain a foreign polypeptide.

[0120] A "homologous" DNA sequence of the present invention is a DNA sequence that is naturally associated with the host cell into which it is introduced. Any nucleic acid or protein that one of skill in the art would recognize as heterologous or foreign to the cell in which it is expressed is encompassed herein by the term heterologous nucleic acid or heterologous protein.

[0121] When used herein with respect to a protein or polypeptide that is compared to another protein or polypeptide, the terms "modified", "modified", "mutated" or "mutation" are applied mutatis mutandis to what is to be changed in the nucleotide or nucleic acid sequence. The mentioned terms are used to indicate that the modified nucleotide or nucleic acid sequence encoding the protein or polypeptide has at least one difference in the nucleotide or nucleic acid sequence compared to the nucleotide or nucleic acid sequence of the protein or polypeptide to which it is compared. This term is used regardless of whether the modified or mutated protein was in fact obtained by mutagenesis of the nucleic acid encoding these amino acids, or by modification of the polypeptide or protein, or by another method, such as artificial gene synthesis methods. Mutagenesis is a method well known in the art and includes, for example, site-directed mutagenesis by PCR or via oligonucleotide-mediated mutagenesis, as described in Sambrook, J., and Russell, DW Molecular Cloning: A Laboratory Manual. 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001). The terms "modified", "modification", "mutated" or "mutation" as used herein in reference to a gene are used to indicate that at least one nucleotide in the nucleotide sequence of the gene or its regulatory sequence differs from the nucleotide sequence to which it is compared. A modification or mutation may in particular be a substitution of a nucleotide with a different nucleotide, a deletion of a nucleotide or an insertion of a nucleotide.

[0122] kit: Another aspect of the present invention relates to a kit for an artificial alkane oxidation system, the kit comprising: a first isolated nucleic acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO: 1, 10, 11, 23-43, comprising a nucleic acid sequence encoding an oxidase enzyme; a second isolated nucleic acid sequence encoding an electron transfer protein, preferably not physically associated with the first nucleic acid; Optionally, a third nucleic acid sequence encoding an electron transfer protein reductase, which may be fused to the first and / or second nucleic acid sequence.

[0123] In one embodiment, the organism to which the kit is applied comprises a nucleic acid sequence encoding an alkane synthase, preferably a terpene synthase, more preferably a sesquiterpene synthase as described herein.

[0124] In another embodiment, the kit comprises four nucleic acid sequences encoding an alkane synthase, preferably a terpene synthase, more preferably a sesquiterpene synthase, as described herein, and a fourth nucleic acid sequence if the organism does not produce the corresponding alkane synthase.

[0125] In one embodiment, the kit is used to identify or detect the presence of an artificial alkane oxidation system.

[0126] In an alternative embodiment, the kit is used to prepare an artificial alkane oxidation system.

[0127] In another aspect, the present invention provides a method for producing a composition comprising: a synthetic nucleic acid sequence, or A nucleic acid sequence encoding an alkane oxidation system, or A nucleic acid sequence for an alkane oxidation system, a. has at least 70% sequence identity to SEQ ID NO:20; or A nucleic acid sequence of an oxidase enzyme encoding an oxidase enzyme having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. has at least 70% sequence identity to SEQ ID NO:21; or A nucleic acid sequence of a rubredoxin peptide encoding rubredoxin of SEQ ID NO: 2 or SEQ ID NO: 45, and optionally c. has at least 70% sequence identity to SEQ ID NO:22; or The present invention relates to an expression cassette comprising a nucleic acid sequence of an alkane oxidation system comprising the nucleic acid sequence of a rubredoxin reductase peptide, which encodes the rubredoxin reductase peptide of SEQ ID NO:3.

[0128] Another aspect of the invention relates to an expression cassette comprising a synthetic nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 12, 17, 18 or 19, wherein the synthetic nucleic acid sequence comprises a nucleic acid sequence encoding an oxidase enzyme, a terpene synthase protein if necessary if the desired terpene synthase function is not available in the intended host cell, and optionally a rubredoxin peptide and / or a rubredoxin reductase peptide.

[0129] In another embodiment, the present invention relates to a nucleic acid sequence encoding an alkane oxidation system as disclosed above.

[0130] In another embodiment, the present invention relates to an expression cassette comprising a nucleic acid sequence for an alkane oxidation system, the nucleic acid sequence comprising: a. a nucleic acid sequence of an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 20, or a nucleic acid sequence encoding an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. a nucleic acid sequence of a rubredoxin peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 21; or c. A nucleic acid sequence of a rubredoxin reductase peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO: 22, or a combination of a, b and c.

[0131] Preferably, the nucleic acid sequence of the oxidase enzyme is a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:20.

[0132] Preferably, the nucleic acid sequence of the rubredoxin peptide is a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:21.

[0133] Preferably, the nucleic acid sequence of the rubredoxin reductase peptide is a nucleic acid sequence having at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, at least 99%, or even 100% sequence identity to SEQ ID NO:22.

[0134] In one embodiment, the artificial alkane oxidation system is a sesquiterpene oxidation system comprising one or more oxidase enzymes having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to any one of SEQ ID NOs:1, 10, 11, 23-43, one or more sesquiterpene synthases, and optionally a rubredoxin peptide comprising an amino acid sequence having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:2, and optionally a rubredoxin reductase peptide having at least 70, 75, 80, 85, 90, 91, 92, 93, 94, 95, 96, 97, 98 or 99% sequence identity to SEQ ID NO:3.

[0135] Another aspect of the present invention relates to a method for the oxidation of one or more alkane substrates, preferably terpene substrates, more preferably sesquiterpene substrates, the method comprising: a. for the oxidation of an alkane substrate, preferably a terpene substrate, more preferably a sesquiterpene substrate; i. artificial alkane oxidation system components a and c, and optionally b and / or d, as disclosed above; or ii. Expression of a synthetic nucleic acid sequence as disclosed above encoding an oxidase enzyme and optionally a rubredoxin peptide and / or a rubredoxin reductase peptide, or an expression cassette thereof as disclosed above, or an expression cassette comprising an artificial oxidation system as disclosed above; iii. Expression of a nucleic acid sequence encoding an oxidase enzyme comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; and iv. optionally providing at least one alkane substrate, preferably a terpene substrate, if not produced by an alkane oxidation system, and in the case of a terpene synthase, preferably a terpene substrate, if a sesquiterpene synthase is not present or is not functional, preferably a sesquiterpene substrate; b. allylic oxidation of an alkane substrate, preferably a terpene substrate, more preferably a sesquiterpene substrate, with an alkane oxidation system to produce at least one oxidized alkane product, preferably a terpene product, including one or more aldehyde and / or alcohol products; c. Optionally, extracting at least one oxidized alkane substrate, preferably a terpene product, including one or more aldehyde and / or alcohol products.

[0136] In another preferred embodiment of the method of the present invention, at least one oxidized alkane, preferably alkene, more preferably terpene product is prepared in a host cell or non-human transgenic organism, heterologously expressing the alkane oxidation system components a and c, and optionally b (if no endogenous terpene synthase is present) and / or d, of the present invention.

[0137] In another embodiment, a host cell according to the invention can be used industrially in the fermentative production of one or more of the oxygenated terpene products described above.

[0138] For example, the host cells or non-human transgenic organisms of the invention can be used in the fermentative production of one or more oxygenated terpene products.

[0139] Preferably, at least one oxidized terpene product is produced in a fermentation process, i.e., a method comprising culturing a microbial host cell, such as a Rhodobacter host cell, in a culture medium under conditions in which the alkane oxidation system of the present invention is expressed. The actual reaction of allylic oxidation of one or more terpene substrates by the alkane oxidation system to produce one or more oxidized terpene products usually occurs intracellularly. It should be noted that the term "fermentation" is used in a broad sense herein in any process that uses the culture of an organism to synthesize a compound from a suitable feedstock (e.g., carbohydrate, amino acid source, fatty acid source). Thus, the fermentation process as meant herein is not limited to anaerobic conditions, but extends to processes under aerobic conditions. Suitable feedstocks are generally known to Rhodobacter host cells. Suitable conditions may be based on methods known for Rhodobacter host cells, for example as described in WO 2011 / 074954 or WO 2014 / 014339.

[0140] At least one oxidized terpene product produced by oxidation of one or more terpene substrates can be isolated or extracted from the host cell or non-human transgenic organism by methods known in the art (for plants, see, e.g., Jiang et al., Curr Protoc Plant Biol. 2016;1:345-358. Doi:10.1002 / cppb.20024; for Rhodobacter, see, e.g., WO 2014 / 014339).

[0141] In general, the method includes the steps of: 1) disrupting cells to release their chemical components, including the oxidized terpene products; 2) extracting a sample containing the oxidized terpene products using a suitable solvent (or by distilling or trapping the compounds); 3) separating the desired oxidized terpene products from other undesirable extract contents that would confound analysis and quantification; and 4) using an appropriate analytical method (e.g., thin layer chromatography (TLC), gas chromatography (GC) or liquid chromatography (LC) or another method as described herein).

[0142] In one embodiment, the oxidized terpene products are produced extracellularly and their extraction is facilitated by extraction of the sample with a suitable solvent.

[0143] Terpene Substrates: The terpene substrate is defined above.

[0144] Preferably, the one or more terpene substrates include α-farnesene, β-farnesene, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, α-guayene, a diterpene, a monoterpene, geraniol, nerol monoterpene or geranyl lactone, linalyl acetate, limonene, β-pinene, or mixtures thereof.

[0145] In one embodiment, the at least one terpene substrate is a sesquiterpene substrate selected from α-farnesene, β-farnesene, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, or α-guayene.

[0146] In one embodiment, the terpene substrate comprises one or more distal end terpene alcohols. Distant end terpene alcohols (DETAs) have an alcohol group at the end of the carbon chain that is considered the distal end of the terpene before it is oxidized to the alcohol. Non-limiting examples of these include the compound of formula I for the terpene myrcene, and the compounds of formulas II and III below for the terpene β-farnesene. [ka] [ka] [ka]

[0147] Oxidized terpene products: At least one oxidized terpene product is produced by oxidation, preferably allylic oxidation, of a terpene substrate.

[0148] Preferably, at least one oxidized terpene product is based on terpene and is produced by oxidizing one or more terpene substrates. These may include aldehyde, alcohol, and / or allyl alcohol groups introduced by the action of alkane oxidation system components a and c, and optionally d. In one embodiment, the oxidized terpene product includes at least one of the following: myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderalasin and bicyclo-octanediol, oxidized α-guayene, oxidized β-guayene.

[0149] In one embodiment, the oxidized terpene product comprises the distal terminal terpene alcohol DETA as defined above. DETA can be an intermediate in the oxidation of the corresponding terpene to an oxidized terpene product, such as, but not limited to, myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, or 9,10-epoxygeranylacetone. DETA can itself be an oxidized terpene product when oxidation does not continue to a more highly oxidized terpene product, such as an aldehyde. As described above, DETA can have a dual function, since the alkane oxidation system of the present invention can use DETA as a terpene substrate to generate a more highly oxidized terpene substrate, or DETA can be a product of the alkane oxidation system of the present invention, in one embodiment.

[0150] In one embodiment, the monoterpene substrate myrcene is used to produce myrcene aldehyde.

[0151] In another embodiment, the sesquiterpene substrates α-farnesene and β-farnesene are used to produce α-sinensal and β-sinensal, respectively.

[0152] In another embodiment, the sesquiterpene substrate santalene is used to produce santalol.

[0153] In another embodiment, the sesquiterpene substrate valencene is used to produce nootkatone or vetivone.

[0154] In another embodiment, the sesquiterpene substrate, α-guayene, is used to produce rotundone.

[0155] In another embodiment, the sesquiterpene substrate, a diterpene, is used to produce rebaudioside.

[0156] In another embodiment, the sesquiterpene substrates geraniol or nerol monoterpene are used to produce 8-hydroxygeraniol or 8-hydroxynerol.

[0157] In another embodiment, geranyl acetone is used to produce 9,10-epoxygeranyl acetone.

[0158] In a preferred embodiment, the at least one alkane substrate is selected from a diterpene, monoterpene, or sesquiterpene, including α-farnesene, β-farnesene, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, α-guayene, the monoterpene geraniol, the nerol monoterpene, geranyl lactone, linalyl acetate, limonene, β-pinene, or combinations thereof.

[0159] In another aspect, the present invention relates to a method for producing at least one oxidized alkane product by oxidizing an alkane substrate.

[0160] Another aspect of the invention relates to a non-human host cell. The non-human host cell comprises: Artificial alkane oxidation system, or Synthetic nucleic acid, expression cassette, or Oxidation of alkane substrates, or This includes the production of alkane oxidation products.

[0161] The non-human host cell is suitable for oxidizing terpene substrates as disclosed above.

[0162] The non-human host cell is a non-mammalian cell, preferably a non-vertebrate cell, more preferably an isolated host cell.

[0163] Preferably, the transgenic non-human organism of the invention is a bacterium, a yeast, a fungus, a protist, an algae or a cyanobacterium, a non-human animal or a non-human mammal, or a plant.

[0164] Non-host cells include host cells such as bacteria and fungi.

[0165] The present invention also relates to a vector or genetic construct comprising a nucleic acid of the invention.

[0166] The nucleic acid of the present invention is operably linked to an expression control sequence that allows expression in a prokaryotic or eukaryotic host cell, or an isolated fraction thereof, in a vector or genetic construct. Thus, in one embodiment, the vector is an expression vector. Expression of the nucleic acid of the present invention includes transcribing the polynucleotide into a translatable mRNA. Regulatory elements that ensure expression in a prokaryotic or eukaryotic host cell are well known in the art. In one embodiment, the regulatory element includes a regulatory sequence that ensures initiation of transcription and / or a polyA signal that ensures termination of transcription and stabilization of the transcript. Further regulatory elements may include transcriptional and translational enhancers. Operable regulatory elements allowing expression in prokaryotic host cells include, for example, the lac, trp or tac promoters in E. coli, or the Rhodobacter promoter (https: / / doi.org / 10.1073 / pnas.2010087117), and examples of regulatory elements allowing expression in eukaryotic host cells include the AOX1 or GAL1 promoters in yeast, or the CMV, SV40, RSV (Rous Sarcoma Virus), CMV enhancer, SV40 enhancer or globin intron in mammalian and other animal cells. Plant promoters are described, for example, in Plant Biotechnology: Principles and Applications, pp117-172, 2017. Furthermore, inducible expression control sequences may be used in the expression vector. Such inducible vectors may include tet or lac operator sequences, or sequences inducible by heat shock or other environmental factors. Suitable expression control sequences are well known in the art. Besides elements involved in transcription initiation, such regulatory elements may also comprise transcription termination signals, such as the SV40-poly-A site or the tk-poly-A site downstream of the polynucleotide.In this context, suitable expression vectors are known in the art, for example Okayama-Berg cDNA expression vectors pcDV1 (Pharmacia), pBluescript (Stratagene), pCDM8, pRc / CMV, pcDNA1, pcDNA3 (Invitrogen) or pSPORT1 (Invitrogen). Expression vectors derived from viruses, such as retroviruses, vaccinia viruses, adeno-associated viruses, herpes viruses or bovine papilloma viruses, may be used to deliver the polynucleotide or vector to the target cell population.

[0167] To construct a vector or gene construct containing the nucleic acid of the present invention, methods well known to those skilled in the art may be used, see for example the techniques described in Sambrook, Molecular Cloning A Laboratory Manual, Cold Spring Harbor Laboratory (2001) NY and Ausubel, Current Protocols in Molecular Biology, Green Publishing Associates and Wiley Interscience, NY (1994).

[0168] The term "gene" as used herein is used broadly to refer to any segment of a nucleic acid associated with a biological function, such as the nucleic acid of the present invention. Thus, genes include coding sequences and / or regulatory sequences required for their expression. For example, genes refer to nucleic acid fragments that express mRNA or functional RNA or code for a particular protein, including regulatory sequences. Genes also include non-expressed DNA segments that, for example, form recognition sequences for other proteins. Genes can be obtained from a variety of sources, including cloning from a source of interest or synthesis from known or predicted sequence information, and can include sequences designed to have desired parameters.

[0169] The term "chimeric gene," as used herein, refers to any gene that contains 1) a DNA sequence that includes regulatory and coding sequences that are not found together in nature, or 2) a sequence that encodes portions of a protein that are not contiguous in nature, or 3) a portion of a promoter that is not contiguous in nature. Thus, a chimeric gene may contain regulatory and coding sequences that are derived from different sources, or it may contain regulatory and coding sequences that are derived from the same source but that are organized differently than found in nature.

[0170] "Genetic constructs", as used herein, can vary in complexity depending on the intended insertion. Constructs can be designed to be randomly inserted into the genome of an organism (called genetic recombination by addition) or can be designed to be inserted at the correct location into the genome at a specific target site on a determined chromosome (called genetic recombination by homologous recombination). In either case, the construct must be complete, with structures to control gene expression, such as a promoter, a transcription start site, a polyadenylation site, and a transcription end site. That is, the information inserted into the recipient genome has a start, a middle, and an end, thus avoiding the problem of uncontrolled expression in the host cell or organism.

[0171] The terms "open reading frame" and "ORF" as used herein refer to the amino acid sequence encoded between the translation initiation and termination codons of a coding sequence. The terms "initiation codon" and "termination codon" refer to adjacent units of three nucleotides ("codons") within a coding sequence that specify the initiation and chain termination of protein synthesis (mRNA translation), respectively.

[0172] "Coding sequence," as used herein, refers to a DNA or RNA sequence that codes for a specific amino acid sequence and does not contain non-coding sequences. A coding sequence may constitute an "uninterrupted coding sequence," i.e., it may be free of introns, such as cDNA, or it may contain one or more introns bound by appropriate splice junctions. "Introns" are sequences of RNA that are contained in the primary transcript but are removed by cleavage and religation of the RNA within the cell to produce a mature mRNA that can be translated into protein.

[0173] "Regulatory sequence," as used herein, refers to a nucleotide sequence located upstream (5' non-coding sequences), within, or downstream (3' non-coding sequences) of a coding sequence that influences the transcription, RNA processing or stability, or translation of the associated coding sequence. Regulatory sequences include enhancers, promoters, translation leader sequences, introns, and polyadenylation signal sequences. These include natural and synthetic sequences, as well as sequences that may be combinations of synthetic and natural sequences. As noted above, the term "suitable regulatory sequence" is not limited to promoters. Examples of regulatory sequences include promoters (transcriptional promoters, constitutive promoters, inducible promoters, etc.), operators, enhancers, mRNA ribosomal binding sites, and appropriate sequences that control transcription and translation initiation and termination. A nucleic acid sequence is "operably linked" when the regulatory sequence is functionally associated with the DNA or cDNA sequence of the invention. As used herein, the terms "operably linked" or "operably linked" refer to the juxtaposition of the components so described in a relationship that allows them to function in the intended manner. A regulatory sequence "operably linked" to another regulatory sequence and / or coding sequence is linked in such a way that transcription and / or expression of the coding sequence is achieved under conditions compatible with the regulatory sequences. Generally, operably linked means that the nucleic acid sequences being linked are contiguous, and, where necessary to join two protein coding regions, contiguous and in the same reading frame. Each of the regulatory sequences can be independently selected from heterologous and homologous regulatory sequences.

[0174] A "promoter", as used herein, refers to a nucleotide sequence that is usually upstream (5') to its coding sequence, and controls the expression of said coding sequence by providing RNA polymerase recognition and other factors required for proper transcription. A "promoter" includes a minimal promoter, a short DNA sequence consisting of a TATA box and other sequences that serve to specify the transcription start site, to which control elements are added to control expression. A "promoter" also refers to a nucleotide sequence that includes a minimal promoter plus control elements that can control the expression of a coding sequence or functional RNA. This type of promoter sequence consists of proximal and more distal upstream elements, the latter elements often referred to as enhancers. Thus, an "enhancer" is a DNA sequence that can stimulate promoter activity and can be an intrinsic element of the promoter or a heterologous element inserted to enhance the level or tissue specificity of the promoter. It can operate in both orientations (normal or inverted) and can function when moved either upstream or downstream from the promoter. Both enhancers and other upstream promoter elements bind to sequence-specific DNA-binding proteins that mediate their effects. Promoters can be derived in their entirety from a native gene, or can be composed of different elements from different promoters found in nature, or even composed of synthetic DNA segments. Promoters can also contain DNA sequences that are involved in the binding of protein factors that control the effectiveness of transcription initiation in response to physiological or developmental conditions.

[0175] "Expression cassette" as used herein means a DNA sequence capable of inducing the expression of a particular nucleotide sequence, for example a nucleotide sequence encoding an oxidase enzyme useful in the present invention, and optionally rubredoxin, and optionally rubredoxin reductase, in a suitable host cell as defined herein, comprising a promoter operably linked to a nucleotide sequence of interest operably linked to a termination signal. It usually includes sequences required for the proper translation of the nucleotide sequence. The coding region usually codes for a protein of interest, but may also code for a functional RNA of interest, for example an antisense RNA or a non-translated RNA, in the sense or antisense orientation. The expression cassette containing the nucleotide sequence of interest may be chimeric, meaning that at least one of its components is heterologous to at least one of the other components. The expression cassette may also be naturally occurring, but may also be obtained in a recombinant form useful for heterologous expression. The expression of the nucleotide sequence of the expression cassette may be under the control of a constitutive promoter or an inducible promoter that initiates transcription only when the host cell is exposed to some specific external stimulus. In the case of multicellular organisms, promoters can also be specific to particular tissues or organs, or to developmental stages, for example, in plant development.

[0176] The term "vector" as used herein refers to a structure composed of genetic material designed to induce transformation of a target cell. A vector contains multiple genetic elements that are positionally and sequentially oriented, i.e. operatively linked with other required elements, so that the nucleic acid in the nucleic acid cassette can be transcribed and, if necessary, translated in the transformed cell. In particular, the vector may be selected from the group of viral vectors, (bacteri)phages, cosmids or plasmids. The vector may also be a yeast artificial chromosome (YAC), a bacterial artificial chromosome (BAC) or an Agrobacterium binary vector. The vector may or may not be self-transmissible or mobilizable, and may be in double-stranded or single-stranded, linear or circular form, capable of transforming a host organism, such as, for example, Rhodobacter, either by being integrated into the cell genome or by being present on an additional chromosome (e.g., a self-replicating plasmid with an origin of replication). In particular, shuttle vectors are included, which mean DNA vehicles capable of replicating naturally or by design in two different host organisms as defined herein. Preferably, the nucleic acid in the vector is under the control of and operably linked to a promoter or other regulatory element suitable for transcription in a host cell as specified herein. The vector may be a bifunctional expression vector that functions in multiple hosts. In the case of genomic DNA, the bifunctional expression vector may contain its own promoter or other regulatory element, and in the case of cDNA, it may be under the control of a promoter or other regulatory element suitable for expression in a host cell. The vector containing the nucleic acid can be prepared based on methods known in the art. For example, a cDNA sequence encoding an oxidase enzyme or rubredoxin or rubredoxin reductase useful in the present invention can be used, operably linked to a suitable regulatory element, such as a transcriptional or translational regulatory nucleic acid sequence.

[0177] The term "vector", as used herein, includes reference to vectors for standard cloning procedures ("cloning vectors") as well as more specific types of vectors such as (autosomal) expression vectors and cloning vectors that are used to integrate into a host cell chromosome ("integrating vectors").

[0178] A "cloning vector" usually contains one or a few restriction endonuclease recognition sites into which foreign DNA sequences can be inserted in an identifiable manner without losing the essential biological functions of the vector, as well as a marker gene suitable for use in identifying and selecting cells transformed with the cloning vector.

[0179] The term "expression vector" as used herein refers to a linear or circular DNA molecule that contains a segment encoding a polypeptide of interest under the control (i.e., operably linked) of an additional nucleic acid segment that provides for its transcription. Such additional segments may include promoter and termination sequences, and may optionally include one or more origins of replication, one or more selectable markers, enhancers, polyadenylation signals, and the like. Expression vectors are generally derived from plasmid or viral DNA, or may contain elements of both. In particular, an expression vector comprises, in a 5' to 3' direction, a nucleotide sequence that contains and is operably linked to (a) a transcriptional and translational initiation region recognized by the host organism, (b) a coding sequence for a polypeptide of interest, and (c) a transcriptional and translational termination region recognized by the host organism. A "plasmid" refers to an autonomously replicating extrachromosomal DNA that is not integrated into the genome of a microorganism and is usually circular in nature.

[0180] "Integrating vector" refers to a linear or circular DNA molecule that can be integrated into the genome of a microorganism, e.g., a bacterial genome, to provide for stable inheritance of a gene encoding a polypeptide of interest, such as an alkane oxidation system useful in the present invention, e.g., an oxidase enzyme, and optionally a terpene synthase, and optionally a rubredoxin and rubredoxin reductase. Integrating vectors generally contain one or more segments that consist of a genetic sequence encoding a polypeptide of interest under the control (i.e., operably linked) of an additional nucleic acid segment that provides for its transcription.

[0181] Such further segments may include promoter and termination sequences, as well as one or more segments that induce the integration of the gene of interest into the genome of the target cell, usually by a process of homologous recombination. Usually, an integrating vector is one that can be introduced into the target cell, but has a replicon that is non-functional in that organism. Integration of the segment containing the gene of interest can be selected if an appropriate marker is contained within the segment. One or more nucleic acid sequences encoding suitable signal peptides that are not naturally associated with the polypeptide to be expressed in the host cell of the invention can be incorporated into the (expression) vector. For example, a DNA sequence of a signal peptide leader may be fused in frame to the nucleic acid of the invention, such that the oxidase enzyme or rubredoxin or rubredoxin reductase useful in the invention is initially translated as a fusion protein containing the signal peptide. Depending on the nature of the signal peptide, the expressed polypeptides are targeted differently. Secretory signal peptides that are functional in the intended host cell promote, for example, extracellular secretion of the expressed polypeptide. Other signal peptides direct the expressed polypeptide to specific organelles, such as chloroplasts, mitochondria and peroxisomes. The signal peptide may be cleaved from the polypeptide upon transport into the intended organelle or upon transport out of the cell. It is possible to effect fusion of additional peptide sequences at the amino or carboxyl terminus of the polypeptide.

[0182] Additionally, the present invention relates to a host cell comprising a vector or a genetic construct of the invention.

[0183] The host cell is transformed by the vector or genetic construct of the present invention. Those skilled in the art are well aware of the genetic elements that must be present on the genetic construct in order to successfully transform, select and grow host cells containing the vector or genetic construct of the present invention. The host cell of the present invention is capable of expressing the polypeptide of the alkane oxidation system contained in the vector or genetic construct of the present invention.

[0184] "Transformation" and "transforming" as used herein refer to the introduction of a heterologous nucleotide sequence, such as a nucleotide sequence encoding an alkane oxidation system useful in the present invention, e.g., an oxidase enzyme, and optionally, rubredoxin, and optionally, rubredoxin reductase, into a host cell, regardless of the method used for insertion, e.g., direct uptake, transduction, conjugation with F factor, or electroporation. The exogenous polynucleotide may be maintained as a non-integrated vector, e.g., a plasmid, or alternatively, may be integrated into the host cell genome.

[0185] Host cells according to the invention can be produced according to standard genetic and molecular biology techniques generally known in the art, for example as described in Sambrook, J., and Russell, DW "Molecular Cloning: A Laboratory Manual" 3d ed., Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY, (2001); and FM Ausubel et al, eds., "Current protocols in molecular biology", John Wiley and Sons, Inc., New York (1987), and subsequent supplements.

[0186] The host cell may be any cell selected from a microbial cell, such as a bacterial cell, an archaeal cell, a fungal cell, such as a yeast cell, and a protist cell. The host cell may also be an algal or cyanobacterial cell, a non-human or mammalian animal cell, or a plant cell.

[0187] In particular, the host cell may be selected from any one of the following organisms:

[0188] Bacteria: The bacterial host cell may be selected, for example, from the group consisting of Escherichia, Klebsiella, Helicobacter, Bacillus, Lactobacillus, Streptococcus, Amycolatopsis, Rhodobacter, Pseudomonas, Paracoccus, Pantoea, or Lactococcus.

[0189] Gram positive: Bacillus, Streptomyces: Useful gram-positive bacterial host cells include Bacillus cells, such as Bacillus alkalophius, Bacillus amyloliquefaciens, Bacillus brevis, Bacillus circulans, Bacillus clausii, Bacillus coagulans, Bacillus firmus, Bacillus Jautus, Bacillus lentus, Bacillus licheniformis, Bacillus megaterium, Bacillus pumilus, and the like. The most preferred prokaryotic organisms include, but are not limited to, Bacillus pumilus, Bacillus stearothermophilus, Bacillus subtilis and Bacillus thuringiensis. The most preferred prokaryotic organisms are Bacillus cells, preferably Bacillus subtilis, Bacillus pumilus, Bacillus licheniformis or Bacillus lentus Bacillus cells.

[0190] Some other preferred bacteria include strains of the order Actinomycetales, preferably the genus Streptomyces, preferably Streptomyces spheroides (ATTC23965), Streptomyces thermoviolaceus (IFO12382), Streptomyces lividans or Streptomyces murinus or Streptoverticillum verticillium ssp. Verticillium. Other preferred bacteria include Rhodobacter sphaeroides, Rhodomonas palustri, and Streptococcus lactis. Further preferred bacteria include strains belonging to the genus Myxococcus, such as M. virescens.

[0191] Gram-negative: E. coli, Pseudomonas, Rhodobacter, Paracoccus or Pantoea sp. Preferred gram-negative bacteria are Escherichia coli, Pseudomonas sp., preferably Pseudomonas purrocinia (ATCC15958) or Pseudomonas fluorescens (NRRL B-11), Rhodobacter capsulatus or Rhodobacter sphaeroides, Paracoccus carotinifaciens or Paracoccus zeaxanthinifaciens, or Pantoea ananatis.

[0192] fungi: Aspergillus, Fusarium, Trichoderma The host cell may be a fungal cell. "Fungi" as used herein includes Ascomycota, Basidiomycota, Chytridiomycota and Zygomycota as well as Oomycota and Deuteromycotina and all vegetative spore-forming fungi. Representative groups of Ascomycota include, for example, Neurospora, Eupenicillium (=Penicillium), Emericella (=Aspergillus), Eurotium (=Aspergillus) and the true yeasts listed below. Examples of Basidiomycota include mushrooms, rusts and smuts. Representative groups of Chytridiomycota include, for example, Allomyces, Blastocladiella, Coelomomyces and aquatic fungi. Representative groups of Oomycota include, for example, Saprolegniomycetous aquatic fungi (water molds), such as Achlya. Examples of vegetative spore-forming fungi include Aspergillus, Penicillium, Candida and Alternaria. Representative groups of the phylum Zygomycota include, for example, the genera Rhizopus and Mucor.

[0193] Some preferred fungi include those from the subdivision Deuteromycotina, class Hyphomycetes, such as the genera Fusarium, Humicola, Tricoderma, Myrothecium, Verticillum, Arthromyces, Caldariomyces, Ulocladium, Embellisia, Cladosporium or Dreschlera, in particular Fusarium oxysporum (DSM2672), Humicola insolens, Trichoderma resii, and the like. resii, Myrothecium verrucana (IFO6113), Verticillum alboatrum, Verticillum dahlie, Arthromyces ramosus (FERM P-7754), Caldariomyces fumago, Ulocladium chartarum, Embellisia alli or Dreschlera halodes.

[0194] Other preferred fungi include strains belonging to the subdivision Basidiomycotina, class Basidiomycetes, such as the genera Coprinus, Phanerochaete, Coriolus or Trametes, in particular Coprinus cinereus f. microsporus (IFO8371), Coprinus macrorhizus, Phanerochaete chrysosporium (e.g. NA-12) or Trametes (formerly called Polyporus), such as T. versicolor (e.g. PR4 28-A).

[0195] Further preferred fungi include strains belonging to the subdivision Zygomycotina, class Mycoraceae, such as the genera Rhizopus and Mucor, in particular Mucor hiemalis.

[0196] yeast: Genus Pichia Saccharomyces The fungal host cell may be a yeast cell. Yeast, as used herein, includes ascosporogenous yeast (Endomycetales), basidiosporogenous yeast, and yeast belonging to the Fungi imperfecti (Blastomycetes). Ascosporogenous yeast are divided into the families Spermophthoraceae and Saccharomycetaceae. The latter is composed of four subfamilies, Schizosaccharomycoideae (e.g., Schizosaccharomyces), Nadsonioideae, Lipomycoideae, and Saccharomycoideae (e.g., Kluyveromyces, Pichia, and Saccharomyces). Basidiosporogenous yeasts include the genera Leucosporidim, Rhodosporidium, Sporidiobolus, Filobasidium, and Filobasidiella. Yeasts belonging to the Fungi imperfecti are divided into two families, Sporobolomycetaceae (e.g., the genera Sporobolomyces and Bullera) and Cryptococcaceae (e.g., the genus Candida).

[0197] Eukaryotes: Eukaryotic host cells further include, but are not limited to, non-human animal cells, non-human mammalian cells, avian cells, reptilian cells, insect cells or plant cells.

[0198] In a preferred embodiment, the host cell is a host cell selected from the following: a) bacterial cells of the group of gram-negative bacteria, such as the genera Rhodobacter (e.g. Rhodobacter sphaeroides or Rhodobacter capsulatus), Paracoccus (e.g. P. carotinifaciens, P. zeaxanthinifaciens), Escherichia or Pseudomonas; b) bacterial cells selected from the group of gram-positive bacteria, such as Bacillus, Corynebacterium, Brevibacterium, Amycolatopis; c) fungal cells selected from the group of Aspergillus, Blakeslea, Peniciliium, Phaffia (Xanthophyllomyces), Pichia, Saccharamoyces, Kluyveromyces, Yarrowia and Hansenula; d) transgenic plant cells or cultures comprising transgenic plant cells, wherein the cells are cells of a transgenic plant selected from Arabidopsis spp., Nicotiana spp., Cichorum intybus, lacuca sativa, Mentha spp., Artemisia annua, tuber-forming plants, oil crops such as Brassica spp. or Brassica napus, fruit-producing flowering plants (angiosperms) and trees; or e) a transgenic mushroom or a culture comprising transgenic mushroom cells, wherein the microorganism is selected from the genera Schizophyllum, Agaricus and Pleurotisi.

[0199] More preferred host cells from organisms are host cells of microorganisms belonging to the genera Escherichia, Saccharomyces, Pichia, Rhodobacter, Pseudomonas, Pantoea or Paracoccus (e.g. Paracoccus carotinifaciens, Paracoccus zeaxanthinifaciens), and even more preferred are host cells of the species E. coli, S. cerevisae, Rhodobacter sphaeroides, Rhodobacter capsulatus, and the like. capsulatus, Pantoea ananatis or Amycolatopis sp.

[0200] Preferably, the vector or genetic construct encodes and is suitable for producing the alkane oxidation system components a and c, and optionally b and / or d, of the invention in a microbial cell as defined herein.

[0201] In one embodiment, the host cell is a Rhodobacter host cell selected from the group of Rhodobacter capsulatus and Rhodobacter sphaeroides.

[0202] The transgenic non-human organism of the present invention comprises a nucleic acid of the present invention, a vector or gene construct of the present invention, or a host cell of the present invention. In a preferred embodiment, the transgenic non-human organism of the present invention is used to prepare one or more oxidized terpene products as defined herein, such as, but not limited to, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, or 9,10-epoxygeranylacetone, as described in more detail elsewhere herein. The one or more oxidized terpene products are prepared by allylic oxidation of a terpene substrate by the alkane oxidation system of the present invention.

[0203] Preferably, the transgenic non-human organism of the invention is a bacterium, a yeast, a fungus, a protist, an algae or a cyanobacterium, a non-human animal or a non-human mammal, or a plant. In particular, the organisms mentioned in connection with the host cell of the invention can also be used to generate the transgenic non-human organism of the invention.

[0204] The bacterium is preferably a Gram-negative bacterium, preferably of the genus Rhodobacter, Escherichia, Pseudomonas, Pantoea or Paracoccus.

[0205] The term "transgenic" as used herein with respect to a transgenic organism or cell refers to an organism or cell (which may be the organism itself or a cell of an isolated multicellular organism) that contains a nucleic acid that does not naturally occur in that organism or cell, and which nucleic acid has been introduced into that organism or cell using recombinant DNA techniques known in the art (i.e., introduced into the organism or cell itself, or into an ancestor of that organism, or into an ancestor organism of the organism from which the cell was isolated). Stated another way, the nucleic acid is heterologous to the transgenic organism or transgenic cell.

[0206] "Transgene" refers to a gene, such as an oxidase enzyme gene or a rubredoxin gene or a rubredoxin reductase gene, that is introduced into the genome by transformation and preferably stably maintained. Preferably, a transgene comprises a gene that is heterologous to the genes of the particular cell or organism to be transformed. Additionally, a transgene can comprise a native gene that is inserted into a non-native organism or a chimeric gene. The term "endogenous gene" refers to a native gene in its natural location in the genome of an organism. A "foreign" gene refers to a gene that is not normally found in the host organism but is introduced by gene transfer.

[0207] Methods for generating 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, CA (ed.) 1994. Transgenic animal technology: A laboratory handbook. Academic Press, Inc., San Diedo, Calif.; Monastersky GM and Robl, JM (ed.) (1995) Strategies in Transgenic Animal Science. ASM Press. Washington DC); Sambrook, loc.cit, Ausubel, loc.cit).

[0208] Another aspect of the invention relates to a composition produced by a method or by a non-human host cell, the composition comprising: an oxidase enzyme as defined herein; at least one oxidized terpene product selected from myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol, trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderasin, bicyclo-octanediol, α-guaiene oxide, or β-guaiene oxide, or a combination thereof; and optionally one or more terpene substrates.

[0209] Another aspect of the invention relates to compositions produced by the methods as disclosed above.

[0210] Preferably, the composition comprises at least one oxidized terpene product, such as α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, or 9,10-epoxygeranylacetone.

[0211] Preferably, the oxidized terpene products are selected from α-sinensal, β-sinensal, trans-α-santalol, trans-β-santalol, nootkatone and farnesol.

[0212] Another aspect of the present invention is a method for producing a A non-human host cell as defined above, cultured in a culture medium; and at least one oxidized terpene product produced from the non-human host cell as a major compound, the fermentation composition comprising a terpene substrate, and optionally one or more by-products are minor compounds.

[0213] The term "fermentation" is used to refer to the cultivation of microorganisms that utilize a carbon source, such as sugar, as an energy source to produce a desired product.

[0214] The term "culture medium" refers to a medium that allows the growth of biomass and the production of metabolic products of microorganisms. The culture medium includes a carbon source and may further include a nitrogen source, a phosphorus source, a vitamin source, a mineral source, etc.

[0215] As used herein, the term "fermentation medium" may be used synonymously with "culture medium." In general, the term "fermentation medium" may be used to refer to a medium suitable for culturing a microorganism for an extended period of time to produce a desired compound from the microorganism.

[0216] The term "medium" refers to a culture medium and / or a fermentation medium. A "medium" may be liquid or semi-solid. A given medium may be both a culture medium and a fermentation medium.

[0217] The term "whole cell broth" refers to the entire contents of a vessel (e.g., flask, plate, fermenter, etc.) including cells, the aqueous phase, the hydrocarbon phase, and / or compounds produced in an emulsion. Thus, whole cell broth includes a mixture of water, a carbon source (e.g., sugars), minerals, vitamins, other dissolved or suspended matter, microorganisms, metabolites, and compounds produced by the microorganisms, as well as one or more terpene substrates, and / or a culture medium containing all other components of the material held in the vessel in which oxidized terpene products, including, for example, oxidized sesquiterpene products, are produced by the microorganisms.

[0218] The term "fermentation composition" is used interchangeably with "whole cell broth." The fermentation composition can also include an overlay, if one is added to the vessel during fermentation.

[0219] The fermentation process may be carried out in two stages: a build phase and a production phase. The build phase is carried out for a period of time sufficient to produce a quantity of cellular biomass capable of supporting the production of the terpene substrate and the resulting oxidized terpene products during the production phase. The build phase is carried out for a period of time sufficient to allow several doublings of the population present at the time of inoculation to occur until the desired cell density is reached.

[0220] A method for producing a terpene substrate, and the resulting oxidized terpene products, may include carrying out a fermentation of the genetically modified host cells under sufficiently aerobic conditions to allow the genetically modified host cells to grow and be maintained, then providing microaerobic fermentation conditions sufficient to induce production of the terpene substrate (such as myrcene or other terpenes, by-products, etc.), and maintaining the microaerobic conditions throughout the fermentation run. Microaerobic conditions may be used throughout the fermentation run. An inducer may be added during the production stage to activate promoters or relieve repression of transcriptional regulators to promote production of the terpene substrate and / or oxidized terpene products.

[0221] The method for producing a terpene substrate and an alkane oxidation system and the resulting oxidized terpene products can include culturing at least one microbial host cell in separate construction and production culture media. For example, the method can include culturing at least one genetically modified microbial host cell in a construction stage, where the cells are cultured under non-production conditions (e.g., under non-induction conditions) to produce an inoculum, and then transferring the inoculum to a second fermentation medium under conditions suitable for inducing the terpene substrate product (e.g., under induction conditions), and maintaining stationary conditions in the second fermentation stage to produce a cell culture comprising the terpene substrate and an alkane oxidation system and the resulting oxidized terpene products.

[0222] In another embodiment, a terpene substrate is produced in one host cell while an artificial alkane oxidase system is provided in another host cell.

[0223] In one embodiment, the terpene substrate produced by one host cell and the artificial alkane oxidase system provided by another host cell belong to the same species, for example, the two host cells are from Rhodobacter species.

[0224] In another embodiment, the two host cells are provided in a mixed fermentation process.

[0225] During the fermentation process, an engineered alkane oxidase system within a host cell provides an oxidase enzyme, an optional terpene synthase, an optional electron transfer compound, and an optional electron transfer compound regenerating enzyme, and allylic oxidizes at least one terpene substrate to produce an oxidized terpene product.

[0226] Culture media and conditions for maintaining and growing microbial cultures are well known to those skilled in the art of microbiology or fermentation science (see, e.g., Bailey et al., Biochemical Engineering Fundamentals, second edition, McGraw Hill, New York, 1986). Appropriate culture media, pH, temperature, and aerobic, microaerobic, or anaerobic conditions requirements can be selected depending on the particular requirements of the microbial host cell, fermentation, and process.

[0227] The culture medium used in the methods of producing oxidized terpene products as provided herein can include any culture medium in which a genetically modified microorganism capable of producing a terpene can survive, i.e., can support and maintain the growth and survival. The culture medium can also promote the biosynthetic pathways necessary to produce the desired terpene substrate and the resulting oxidized terpene product.

[0228] The culture medium may be an aqueous solution containing assimilable carbon, nitrogen and phosphate sources. Such a medium may also contain appropriate salts, minerals, metals and other nutrients. The carbon source and each of the essential cellular nutrients may be added to the fermentation medium sequentially or continuously, with the required nutrients being maintained at a minimum level essentially required for efficient absorption by the growing cells according to a predetermined cell growth curve based, for example, on the metabolic or respiratory functions of the cells to convert the carbon source into biomass.

[0229] The carbon source can be a monosaccharide (simple sugar), a disaccharide, a polysaccharide, a non-fermentable carbon source, or a combination of one or more thereof. Non-limiting examples of suitable monosaccharides include glucose, galactose, mannose, fructose, ribose, and combinations thereof. Non-limiting examples of suitable disaccharides include sucrose, lactose, maltose, trehalose, cellobiose, and combinations thereof. Non-limiting examples of suitable polysaccharides include starch, glycogen, cellulose, chitin, and combinations thereof. Non-limiting examples of suitable non-fermentable carbon sources include acetate and glycerol. Carbon sources can be obtained from a wide variety of crops and sources. Some non-limiting examples of suitable crops or sources include sugarcane, bagasse, miscanthus, sugar beet, sorghum, grain sorghum, switchgrass, barley, hemp, kenaf, potato, sweet potato, cassava, sunflower, fruit, molasses, whey or skim milk, corn, forage, grain, wheat, wood, paper, straw, cotton, many types of cellulosic waste, and other biomass. Suitable crops or sources can include sugarcane, sugar beet, and corn. The sugar source can be sugarcane juice or molasses. Any combination of the above carbon sources can be used.

[0230] A suitable medium may be supplemented with one or more additional agents, such as, for example, an inducer (e.g., when one or more nucleotide sequences encoding the gene product are under the control of an inducible promoter), a repressor (e.g., when one or more nucleotide sequences encoding the gene product are under the control of a repressible promoter), or a selection agent (e.g., an antibiotic for selecting for microorganisms containing the genetic modification).

[0231] A liquid organic overlay may be added to the medium during the production stage of the fermentation. The liquid organic overlay may be an immiscible organic liquid in contact with the aqueous culture medium, in which the terpene substrate and other by-products, as well as the oxidized terpene products secreted from the microorganisms, may be trapped. The liquid organic overlay may not only reduce the evaporation of volatile monoterpenes from the fermentation vessel, but may also reduce the toxicity of the terpene substrate and / or the oxidized terpene products to potential microorganisms. Examples of overlays include, but are not limited to, isopropyl myristate (IPM) or other hydrocarbon liquids, such as white mineral oil or polyalphaolefins.

[0232] The fermentation method may be carried out in a suitable container or vessel, including but not limited to a cell culture plate, flask, or fermenter. The fermentation may be carried out in a closed system to capture the terpenes in the gas phase. For example, the closed system may include a series of vessels connected together to capture the exhaust gas containing the monoterpenes in the gas phase. For example, a first vessel may contain a culture medium containing an aqueous medium and a genetically modified microorganism. A second vessel containing an organic overlay may be connected in series with the first vessel to capture the volatile terpenes. One or more additional vessels may be connected in series and / or in parallel with the first vessel to capture the gas composition containing the terpene substrate and other terpene by-products, as well as the oxidized terpene products.

[0233] Additionally, the method can be performed at any scale of fermentation known in the art to support industrial production of microbial products. Any suitable fermenter can be used, including stirred tank fermenters, airlift fermenters, bubble fermenters, or any combination thereof.

[0234] Furthermore, the method allows fermentation to be carried out in any fermentation volume, for example, from lab scale (eg, 10 ml to 20 L) to pilot scale (eg, 20 L to 500 L) to industrial scale (eg, 500 L to 500,000 L or more).

[0235] Disclosed herein are fermentation compositions comprising the genetically modified microbial host cells described herein, culture media, and terpene substrates, as well as oxidized terpene products produced from the genetically modified microbial host cells. In the fermentation compositions provided herein, the oxidized terpene products are typically the major compounds, with minor compounds including the reserve terpene substrate and one or more by-products (produced simultaneously with the terpene).

[0236] In one embodiment, the fermentation composition comprises, with respect to terpenes, at least about 50% major compounds and less than about 50% minor compounds relative to the total amount of terpene substrate, based on the relative area percentage of the terpene peak shown in a GC chromatogram of monoterpenes.

[0237] In another embodiment, the fermentation composition comprises at least 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, or 95% of the major compounds with respect to terpenes compared to the total amount of terpene substrates in the culture medium.

[0238] In another embodiment, the fermentation composition comprises at least 45% or 40% or 35% or 30% or 25% or 20% or 15% or 10% or 5% of trace compounds with respect to terpenes compared to the total amount of terpene substrates in the culture medium.

[0239] In another embodiment, the primary compound comprises at least two oxidized terpene products. In a further embodiment, the at least two oxidized terpene products comprise trans-α-santalol and trans-β-santalol.

[0240] In another embodiment, by using a terpene synthase that produces two or more terpenes, e.g., a mixture of α-guaiene and β-guaiene, at least two oxidized terpene products are also formed, such that a mixture of oxidized terpene products, e.g., a mixture of oxidized α-guaiene and β-guaiene, is formed as the predominant compound.

[0241] Another aspect of the present invention relates to a method for the fermentative production of at least one oxygenated terpene product, the method comprising: Providing a non-human host cell with an oxidase enzyme system or expression cassette as described herein; Optionally, providing the host cell with at least one alkane synthase for the production of an alkane substrate; Cultivating the non-human host cell in a culture medium under conditions for the host cell to produce the encoded polypeptide in an active form; Optionally, generating and / or providing one or more alkane substrates to a host cell; Producing at least one oxidized alkane product; and optionally purifying the at least one oxidized alkane product.

[0242] In one embodiment, a method for the fermentative production of at least one oxygenated terpene product comprises providing a bacterial host cell, preferably a Rhodobacter strain.

[0243] In another embodiment, the fermentation process for the production of at least one oxygenated terpene product includes the production of sinensal.

[0244] In another embodiment, a method for the fermentative production of at least one oxidized terpene product comprises: a. an oxidase enzyme; b. one or more enzymes for providing one or more alkanes; Optionally, c. an electron transfer compound suitable for transferring at least one electron to an oxidase enzyme, and optionally and an electron transport compound regenerating enzyme suitable for reducing the electron transport compound of the dc when it is in an oxidized state, The oxidase enzymes include oxidase enzyme systems, which are amino acid sequences having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or fragments or variants thereof.

[0245] Another aspect of the invention relates to the use of an alkane oxidation system as disclosed or expression of a nucleic acid sequence, wherein the synthetic nucleic acid sequence comprises an oxidase enzyme and optionally a nucleic acid sequence encoding a rubredoxin peptide and / or a rubredoxin reductase peptide or an expression cassette thereof, or an expression cassette of an alkane oxidation system for oxidizing a terpene substrate to produce one or more oxidized terpene products.

[0246] Another aspect of the present invention relates to a method for producing an alkane oxidation product or composition, the oxidized alkane product being selected from alcohols or aldehydes, or both, including myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderasin and bicyclo-octanediol, α-guaiene oxide, β-guaiene oxide, or combinations thereof. Preferably, the oxidation product is selected from α-sinensal, β-sinensal, trans-α-santalol, trans-β-santalol, nootkatone and farnesol.

[0247] Another aspect of the invention relates to a composition produced by the alkane oxidation system of the invention, the method of the invention, or the non-human host cell of the invention, comprising one or more oxidized terpene products, such as, but not limited to, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, or 9,10-epoxygeranylacetone, or combinations thereof, and optionally one or more terpene substrates, preferably in a weight ratio of 3:1 to 1:10000 relative to their corresponding oxidized terpene products, and optionally an oxidase enzyme as defined above. In one aspect of the invention, the composition of the invention also comprises one or more electron transfer compounds, preferably comprising rubredoxin, and optionally at least one electron transfer compound regenerating enzyme, preferably rubredoxin reductase, suitable for reducing the electron transfer compound when it is in an oxidized state.

[0248] In one embodiment, the weight ratio of terpene substrate to oxidized terpene product is 250 to 1, or 220 to 1, or 200 to 1, or 180 to 1, 170 to 1, or 160 to 1. In preferred embodiments, the weight ratio is 150 to 1, 130 to 1, or 120 to 1, 110 to 1, or 100 to 1. In more preferred embodiments, the weight ratio is 99 to 1 or less, but greater than 0.1:1, for example, the weight ratio of farnesene to oxidized terpene product is between 99:1 and 10:1, preferably 31 to 1 or less, for example 20, 18, 17, or 14 to 1.

[0249] In one embodiment, at least 0.3% (w / w) of oxidized terpene products are formed. In a preferred embodiment, 0.6% (w / w) of sinensal is formed as an oxidized terpene product.

[0250] For example, when two or more oxidized terpene products are produced by providing both α-farnesene and β-farnesene, the ratio of the first oxidized terpene product to the second oxidized terpene product is, in one embodiment, 0.8:1 to 1:0.8, preferably 0.9:1 to 1:0.9, and more preferably 1:1.

[0251] Another aspect of the invention relates to a method for preparing a mutant polypeptide having oxidase enzymatic activity, the method comprising the steps of: a) selecting a nucleic acid encoding a synthetic nucleic acid sequence as disclosed herein, or an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43 or a fragment thereof, preferably SEQ ID NOs: 1, 23, 24, 43; b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c) transforming a non-human host cell with the mutated nucleic acid sequence and expressing the oxidase enzyme encoded by the mutated nucleic acid sequence; d) screening the oxidase enzyme for at least one altered property in addition to the function of the oxidase enzyme encoded by the selected nucleic acid of a); e) optionally repeating process steps (a)-(d) to obtain an oxidase enzyme with the desired mutant function; f) optionally isolating the mutated nucleic acid sequence from the transformed non-human host cell obtained in step (c).

[0252] This function may be an oxidase enzyme of any of SEQ ID NOs: 1, 10, 11, 23, 24, or SEQ ID NOs: 25-43, preferably SEQ ID NOs: 1, 10, 11 or 25-43.

[0253] As referred to herein, "polypeptide variant" / "variant polypeptide" means a polypeptide that has oxidase enzyme activity and is substantially homologous to a polypeptide according to any of the above embodiments relating to an oxidase enzyme, but has an amino acid sequence that differs from that encoded by any of the nucleic acid sequences of the invention by virtue of one or more deletions, insertions or substitutions.

[0254] Variants may include conservatively substituted sequences, meaning that a given amino acid residue is replaced with a residue having similar physiochemical properties. Examples of conservative substitutions include the substitution of one aliphatic residue for another, such as He, VaI, Leu, or Ala, or the substitution of one polar residue for another, such as Lys for Arg, GIu for Asp, or GIn for Asn. See Zubay, Biochemistry, 1983, Addison-Wesley Pub. Co. The impact of such substitutions can be calculated using substitution score matrices such as PAM-120, PAM-200, and PAM-250, as discussed in Altschul, J.MoI.Biol., 1991, 219, 555-565. Other such conservative substitutions, such as the substitution of entire regions with similar hydrophobic properties, are well known. Natural peptide variants are also encompassed by the present invention. Examples of such variants include proteins resulting from alternative mRNA splicing events or proteolytic cleavage of the polypeptides described herein. Proteolytic changes include, for example, differences at the N- or C-terminus when expressed in different types of host cells due to proteolytic removal of one or more terminal amino acids from the polypeptides encoded by the sequences of the invention.

[0255] Table: The following variants refer to SEQ ID NO:1 and indicate the positions of conserved amino acids with permitted exchanges.

[0256] TIFF2024529026000004.tif230170

[0257] TIFF2024529026000005.tif44170

[0258] Variants of the polypeptides of the invention can be used, for example, to achieve a desired enhancement or reduction in enzymatic activity, modification of regiochemistry or stereochemistry, or alteration of substrate utilization or product distribution, increased affinity for substrates, improved specificity for the production of one or more desired compounds, increased rate of enzymatic reaction, higher activity or stability in a particular environment (pH, temperature, solvent, etc.), or improved expression levels in a desired expression system. Variants or site-directed mutants can be made by any method known in the art. Variants and derivatives of native polypeptides can be obtained by isolating the nucleotide sequence of naturally occurring variants or variants of other or identical plant lineages or species, or by artificially programming mutations in the nucleotide sequence encoding the polypeptides of the invention. Modification of the native amino acid sequence can be achieved by any of a number of conventional methods.

[0259] Polypeptide variants resulting from the fusion of additional peptide sequences at the amino and carboxyl termini of the polypeptides of the invention can be used to enhance the expression of the polypeptide, can be useful for protein purification, or can improve the enzymatic activity of the polypeptide in a desired environment or expression system. Such additional peptide sequences can be, for example, signal peptides. Thus, the invention encompasses variants of the polypeptides of the invention, such as those obtained by fusion with other oligopeptides or polypeptides and / or those linked to signal peptides. Fusion polypeptides encompassed by the invention also include fusion polypeptides resulting from the fusion of other functional proteins, such as other proteins from the terpene biosynthetic pathway.

[0260] Thus, in one embodiment, the present invention provides a method for preparing a mutant polypeptide having oxidase enzymatic activity as described in any of the above embodiments, comprising the steps of: (a) selecting a nucleic acid encoding an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to a synthetic nucleic acid sequence as disclosed herein, or to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment thereof; (b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; (c) transforming a host cell or a unicellular organism with the mutated nucleic acid sequence and expressing the polypeptide encoded by the mutated nucleic acid sequence; (d) screening the polypeptide for at least one altered property; (e) optionally, if the polypeptide does not have the desired mutant oxidase enzymatic activity, repeating process steps (a) to (d) until a polypeptide having the desired mutant oxidase enzymatic activity is obtained; (f) optionally, if a polypeptide having the desired mutant oxidase enzyme activity is identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c).

[0261] According to a preferred embodiment, the prepared mutant polypeptide is capable of producing oxidized terpene products as the major compound.

[0262] According to an even more preferred embodiment, a mixture of major and minor compounds can be produced, with the oxidized terpene product being the major compound and one or more by-products being the minor compounds, the oxidized terpene product being at least 60%, preferably at least 80%, preferably at least 90% of the mixture.

[0263] In step (b), a large number of mutant nucleic acid sequences can be generated, for example, by random mutagenesis, site-directed mutagenesis, or DNA shuffling. Detailed procedures for gene shuffling can be found in Stemmer, DNA shuffling by random fragmentation and reassembly: in vitro recombination for molecular evolution. Proc Natl Acad Sci USA., 1994, 91(22):10747-1075. Briefly, DNA shuffling refers to the process of random recombination of known sequences in vitro, involving at least two nucleic acids selected for recombination. For example, mutations can be introduced at specific loci by synthesizing oligonucleotides containing the mutant sequence and flanking them with restriction sites that allow ligation to fragments of the native sequence. After ligation, the resulting reconstructed sequence encodes an analog with the desired amino acid insertion, substitution, or deletion. Alternatively, oligonucleotide-directed site-directed mutagenesis procedures can be used to provide mutant genes, and predetermined codons can be mutated by substitution, deletion, or insertion.

[0264] Thus, a polypeptide comprising SEQ ID NO:1 may be combined with any other oxidase enzyme encoding nucleic acid isolated, for example, from an organism other than Pseudomonas sp. Thus, mutant nucleic acids may be obtained and isolated, and the mutant nucleic acids may be used to transform host cells, for example, according to standard procedures as disclosed in the Examples.

[0265] In step (d), the polypeptides obtained in step (c) are screened for at least one altered property, such as a desired altered enzymatic activity. Examples of desirable enzymatic activities for which the expressed polypeptides can be screened include enhanced or decreased enzymatic activity as measured by KM or Vmax values, regiochemical or stereochemical modifications, and altered substrate utilization or product distribution.

[0266] Screening for enzymatic activity can be carried out according to procedures well known to those skilled in the art and disclosed in the present examples. Step (e) provides for the repetition of process steps (a) to (d), which can preferably be carried out in parallel. Thus, by generating a significant number of mutant nucleic acids, it is possible to simultaneously transform many host cells with different mutant nucleic acids, and then to screen a large number of polypeptides. In this way, the chances of obtaining the desired mutant polypeptide can be increased at the discretion of the skilled person.

[0267] advantage: Producing oxidized terpene products such as sinensal from glucose-derived microbial cultures is a challenging task, and no enzymes are known to mediate the oxidation of the diallyl position of terpenes, preferably sesquiterpenes. The disclosed method is applicable to a variety of terpene substrates bearing a diallyl group, such as santalene, valencene, etc.

[0268] Method A Gas Chromatography and Mass Spectrometry (GC and MS) The cultures are extracted with 1 ml of dichloromethane (DCM). The DCM phase is collected, dried over anhydrous Na2SO4, and injected into a 7890A gas chromatograph (Agilent) equipped with a mass selective detector (model 5975C, Agilent) and scanned in the range 45-450 m / z. Splitless injections of 1 μl of sample were performed at 250 °C on a Zebron ZB-5 MS column (30 m × 0.25 mm, 0.25 μm thickness; Phenomenex) with a helium flow rate of 1 ml / min. The temperature program was 45 °C for 2.25 min, then 40 °C / min to 300 °C, then 3 min at 300 °C. To avoid saturation, the MS detector was turned off at room temperature for 8 min for myrcene conversion experiments and 14 min for farnesene experiments. Compounds were identified by comparing their retention indices and mass spectra with the NIST 8 database or literature data.

[0269] The present invention will now be described in more detail by the following embodiments and combinations of embodiments taken together with reference to and association with the corresponding dependencies.

[0270] I. An artificial alkane oxidation system comprising: a. an oxidase enzyme; b. one or more enzymes, preferably terpene synthase proteins, which provide at least one alkane; Optionally, c. an electron transfer compound suitable for transferring at least one electron to an oxidase enzyme, and optionally and an electron transport compound regenerating enzyme suitable for reducing the electron transport compound of the dc when it is in an oxidized state, The artificial alkane oxidation system, wherein the oxidase enzyme is an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof.

[0271] II. The artificial alkane oxidation system of embodiment I, wherein the alkane is made of five carbon building blocks, contains 1 to 5 such building blocks, and is preferably a terpene, and at least one alkane is a terpene, and at least one terpene synthase protein as component b.

[0272] III. The artificial alkane oxidation system of embodiment I or II, wherein the electron transfer compound is an electron transfer protein, preferably of the F1-S0 type, more preferably a rubredoxin peptide, comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity with any one of SEQ ID NO: 2 or 45 or a fragment or variant thereof, and / or the electron transfer compound regenerating enzyme is an electron transfer protein reductase, preferably a rubredoxin reductase, more preferably a rubredoxin reductase peptide, having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity with SEQ ID NO: 3 or a fragment thereof.

[0273] IV. A synthetic nucleic acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO:4, SEQ ID NO:12, SEQ ID NO:17, SEQ ID NO:18, SEQ ID NO:19, or SEQ ID NO:44, comprising a nucleic acid sequence encoding an oxidase enzyme as defined in embodiment I, and optionally comprising a rubredoxin peptide and / or a rubredoxin reductase peptide of embodiment II.

[0274] V. An expression cassette comprising the synthetic nucleic acid sequence of embodiment IV.

[0275] VI. An expression cassette comprising a nucleic acid sequence encoding an alkane oxidation system of embodiment I, II or III.

[0276] VII. An expression cassette comprising a nucleic acid sequence for an alkane oxidation system, the nucleic acid sequence comprising: a. a nucleic acid sequence of an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 20, or a nucleic acid sequence encoding an oxidase enzyme having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. a nucleic acid sequence of a rubredoxin peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NO: 21, and optionally c. An expression cassette comprising a nucleic acid sequence of a rubredoxin reductase peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:22, or a combination of a, b and c.

[0277] VIII. A method for the oxidation of at least one terpene substrate, comprising: a. for the oxidation of at least one terpene substrate, i. an artificial alkane oxidase system according to any one of embodiments I to III; or ii. Expression of the synthetic nucleic acid sequence of embodiment IV, or the expression cassette of embodiments V-VII; or iii. Expression of a nucleic acid sequence encoding an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; and iv. optionally providing at least one terpene substrate if not produced by an alkane oxidation system; b. allylic oxidizing a terpene substrate to produce at least one oxidized terpene product; c. extracting the at least one oxidized terpene product.

[0278] IX. The method of embodiment VIII, wherein the terpene substrate comprises a diterpene, monoterpene or sesquiterpene, such as, but not limited to, α-farnesene, β-farnesene, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, α-guayene, the monoterpene geraniol, nerol monoterpene, geranyl lactone, or a combination thereof.

[0279] X. The method of embodiment VIII or IX, wherein the oxidized terpene product is an alcohol or an aldehyde, or both.

[0280] XI. The method of any one of embodiments VIII-X, wherein the at least one oxidized terpene product is selected from myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderasin, and bicyclo-octanediol, oxidized α-guayene, oxidized β-guayene, or a combination thereof.

[0281] XII. A non-human host cell comprising an artificial alkane oxidation system according to any of embodiments I-III, a synthetic nucleic acid according to embodiment IV, an expression cassette according to embodiments V-VII, or suitable for the oxidation of a terpene substrate according to the methods of embodiments VIII-XI.

[0282] XIII. The non-human host cell of embodiment XII, wherein the host cell is a Rhodobacter species.

[0283] XIV. A composition produced by the method of embodiments VIII-XI or the non-human host cell of embodiment XII or XIII, comprising α-sinensal, β-sinensal, trans-α-santalol and trans-β-santalol, lanceol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, or 9,10-epoxygeranylacetone, or a combination thereof, and optionally one or more terpene substrates, and optionally an oxidase enzyme as defined in any of the embodiments of embodiments I-XIII.

[0284] XV. A fermentation composition comprising a non-human host cell of embodiment XII or XIII cultured in a culture medium and at least one oxidized terpene product produced from the non-human host cell as a major compound, wherein the fermentation composition comprises a terpene substrate and optionally one or more by-products are minor compounds.

[0285] XVI. Use of the alkane oxidation system of embodiments I-III, or the expression of the nucleic acid sequence of embodiment IV, or the expression cassette of embodiments V-VII, the non-human host cell of embodiment XII or the fermentation composition of embodiment XV for the oxidation of a terpene substrate.

[0286] XVII. The alkane oxidation system of embodiments I-III, wherein the oxidase enzyme is encoded by SEQ ID NO:20, the rubredoxin peptide is encoded by SEQ ID NO:21, and / or the rubredoxin reductase peptide is encoded by SEQ ID NO:22.

[0287] XVIII. An alkane oxidation system comprising: a. an oxidase enzyme comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; b. a heterologous sesquiterpene synthase protein, and optionally c. a rubredoxin peptide comprising an amino acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:2 or any one of its fragments; d. An alkanoic acid family comprising a rubredoxin reductase peptide having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to SEQ ID NO:3 or a fragment thereof, or comprising a combination of a, b and c.

[0288] XIX. An expression cassette comprising a nucleic acid sequence encoding the alkane oxidation system of embodiments XVII and XVIII.

[0289] XX. A method for the oxidation of at least one terpene substrate, preferably a sesquiterpene substrate, comprising: a. for the hydroxylation of terpene, preferably sesquiterpene, substrates; i. An alkane oxidation system of embodiment I or II or XVII or XVIII; or ii. Expression of the nucleic acid sequence of embodiment IV, or the expression cassette of embodiment V or VII; or iii. Expression of a nucleic acid sequence encoding an oxidase enzyme comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43 (alkB) or a fragment or variant thereof; and iv. optionally providing a terpene, preferably a sesquiterpene substrate; b. allylic oxidation of a terpene, preferably a sesquiterpene substrate, by an alkane oxidation system to produce one or more oxidized terpene products; c. extracting the at least one oxidized terpene product.

[0290] XXI. A method of producing at least one oxidized terpene product by the method of any one of embodiments VIII-XII.

[0291] XXII. Use of the non-human host cell of embodiment XII or the fermentation composition of embodiment XV for the production of oxidized terpene.

[0292] XXIII. A fermentative method for the production of at least one oxygenated terpene product, comprising: Providing a non-human host cell with an oxidase enzyme system according to any one of embodiments I-XXII; and culturing the non-human host cell in a culture medium to produce at least one oxidized terpene product.

[0293] XXIV. A kit for an artificial alkane oxidation system comprising: A first isolated nucleic acid sequence having at least 62%, 66%, 69%, 70%, 75%, 80%, 85%, 90%, 94%, 95%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, comprising a nucleic acid sequence encoding an oxidase enzyme as defined in embodiment I; a second isolated nucleic acid sequence that is not physically linked to the first nucleic acid encoding an electron transfer protein; Optionally, a third nucleic acid sequence encoding an electron transfer protein reductase, which can be fused to the first and / or second nucleic acid sequence.

[0294] XXV. Methods for preparing mutant polypeptides having the functions required for their use as any of the components a-d, comprising: a) selecting a nucleic acid of the invention or a nucleic acid encoding a polypeptide of the invention; b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c) transforming a host cell or a unicellular organism with the mutated nucleic acid sequence and expressing the polypeptide encoded by the mutated nucleic acid sequence; d) screening the polypeptides encoded by the selected nucleic acids of a) for at least one altered property in addition to the function of the polypeptide; e) optionally, if the polypeptide does not have the desired polypeptide function, repeating process steps (a) to (d) until a polypeptide is obtained having the desired mutant function as any of components a to d; f) optionally, if a polypeptide having the desired mutant function is identified in step (d), isolating the corresponding mutant nucleic acid obtained in step (c). EXAMPLES

[0295] The present invention will be further described in conjunction with the following examples. These examples are provided to illustrate the present invention and are not intended to limit the scope of the invention claimed herein in any way. The terms and abbreviations in the examples have their usual meanings. For example, "%", "Eq.wt.", "Eq.", "°C", "wt.%", "%w / w", "%w / v", and "gm" represent "Percentage", "Equivalent Weight", "Equivalents", "Celsius", "Weight Percent", "Weight / Weight Percent", "Weight / Volume Percent", and "Gram", respectively.

[0296] The present invention includes digital sequence information of biological materials published by third parties. For example, for the sequences of SEQ ID NOs: 1-3 and 20-21, it is disclosed that the biological materials as the source of the published digital sequence information were supplied from the Netherlands (Soares-Castro 2017 Appl Environ Microbiol 83:e03112-16.).

[0297] TIFF2024529026000006.tif68170

[0298] TIFF2024529026000007.tif214170

[0299] TIFF2024529026000008.tif217170

[0300] TIFF2024529026000009.tif35170

[0301] TIFF2024529026000010.tif77170

[0302] TIFF2024529026000011.tif56170

[0303] TIFF2024529026000012.tif34170

[0304] Example 1: Construct for expressing M1-alkB in E. coli Constructs were designed to express M1-alkB (PM1_0216370), M1-rubredoxin (PM1_0216365), and M1-rubredoxin reductase (SEQ ID NOs: 20 to 22, respectively) in Escherichia coli. M1 alkB is located at positions 3472570 to 3471680 on contig 2 of the Pseudomonas M1 genome, and M1-rubredoxin is encoded immediately downstream of M1-alkB. M1-rubredoxin reductase, which does not colocalize with M1-alkB, is located at positions 438956–437814 on contig 2 of the Pseudomonas M1 genome (Soares-Castro & Santos, 2014, Genome Biol. Evol. 7(1):1-17) and was identified by blast using rubredoxin reductase from Pseudomonas fluorescens (accession SUD34760.1) as bait.

[0305] The protein sequences of M1-alkB, M1-rubredoxin and M1-rubredoxin reductase are SEQ ID NO:1, SEQ ID NO:2, and SEQ ID NO:3, respectively.

[0306] To express the M1 protein in E. coli, the open reading frame was codon optimized for in silico expression in Rhodobacter sphaeroides using standard tools, and fragments of artificial DNA encoding M1-alkB, M1-rubredoxin, and M1-rubredoxin reductase were synthesized using standard service providers.

[0307] The synthetic nucleic acid sequence of SEQ ID NO:4 shows the M1-alkB-rubredoxin-rubredoxin reductase construct.

[0308] SEQ ID NO:4 was cloned into vector pET-DUET1 using XbaI and NotI restriction sites to obtain pET-M1-alkB-rr-rrr for expression.

[0309] In alternative example 1a, the CMR5c protein shown in SEQ ID NO: 43 represents the CMR5 oxidase / AlkB oxidase enzyme. The synthetic DNA sequence of SEQ ID NO: 44 encoded the protein of SEQ ID NO: 43 (component a) and the rubredoxin peptide shown in SEQ ID NO: 45 (component c). SEQ ID NO: 44 overlaps the region of "ATGA" and overlaps with TG to cover both ATG and TGA. The ATG in the overlap acts as the start codon for the rubredoxin peptide (component c) at bp 888.

[0310] Example 2: Verification of the ability of M1-alkB to convert to myrcene and β-farnesene Constructs pET-DUET-1 and pET-M1-alkB-rr-rrr were transformed into E. coli BL21-DE3 and selected on LB agar containing 1% glucose and 100 μg / ml ampicillin. Transformants carrying pET-M1-alkB-rr-rrr and pET-DUET1 were inoculated into LB containing 100 μg / ml ampicillin and grown overnight at 37° C. at 225 rpm. The culture was then diluted 1:20 in 10 ml of 2xYT+Amp liquid medium and incubated at 37° C. at 225 rpm for 2 hours until the A600 was 0.6. 1 mM IPTG was then added and the culture was incubated at 30° C. at 225 rpm for 2 hours. Subsequently, 3 ml of the culture was mixed with 10 μl of myrcene or 10 μl of β-farnesene or without product, sealed in a 4 ml glass vial with screw cap and incubated at 225 rpm at 30° C. for 3 hours. Compounds were subsequently identified from the culture after extraction by method A.

[0311] Myrcene conversion by E. coli BL21 containing pET-DUET comparison strain (CS1) and pET-M1-alkB-rr-rrr, i.e., strain with an artificial alkane oxidation system or invention strain (IS1), is shown throughout FIG. 1a.

[0312] When comparing bacterial cultures expressing pET-DUET1 (CS1) and pET-M1-alkB-rr-rrr (IS1), oxidation products of myrcene were observed. Of note, a compound eluting at 10.7 min (compound 2, see Figure 1c) was observed, which was identified as myrcene aldehyde by its mass spectrum when compared to compound 1 in the literature (see Figure 1b). In addition, several oxidation products of limonene were identified, likely resulting from the 5% limonene contamination of the myrcene batch. The peak intensity of the limonene product was in the same range as that of myrcene aldehyde, suggesting that limonene is a preferred substrate over myrcene. The formation of myrcene aldehyde was observed, indicating that M1-alkB can mediate the oxidation of myrcene at the diallyl position when coexpressed with M1-rubredoxin and M1-rubredoxin reductase.

[0313] Referring to FIG. 2, when β-farnesene was used as a substrate in bacterial cultures expressing pET-DUET1 (CS1) and pET-M1-alkB-rr-rrr (IS1), certain conversion products were also observed, but much less prominently (see FIG. 2). The products corresponded to hexadecenal (14.1 min), farnesol (14.8 min), denderalasin (15.3 min), and bicyclo-octanediol (16.0 min). In particular, the observation of farnesol and denderalasin suggests that farnesene is utilized as a substrate by M1-alkB, but is modified at other positions than the diallyl position, indicating its broad terpene oxidation capability.

[0314] Example 3: Construct for expressing alkB in Rhodobacter To synthesize custom DNA, the following synthetic DNA fragments were synthesized by a standard service provider and cloned into the vector pUC57:

[0315] DNAs encoding the protein sequences of SEQ ID NO:6, SEQ ID NO:7, SEQ ID NO:8, and SEQ ID NO:9 are examples of alkane oxidation system component b: (AaBFS) β-farnesene synthase, (MdAFS) α-farnesene synthase, (ZoBBS) ginger (Zingiber officinale)-(S)-β-bisabolene synthase, and (CiCaSSy) plant terpene synthase derived from camphor tree (Cinnamomum camphora).

[0316] SEQ ID NO: 10 and SEQ ID NO: 11 are the sequences of alkane oxidation system components a, mutated alkane oxidation system components a, namely, alkB having a point mutation of F93S introduced by the present inventors (M1-alkB-F93S) and alkane oxidation system components a, having a point mutation of W189S introduced by the present inventors (M1-alkB-W189S), respectively, as examples of further components a.

[0317] The nucleic acid sequences of SEQ ID NO:5, SEQ ID NO:12, SEQ ID NO:13, corresponding to (AaBFS) Artemisia annua β-farnesene synthase, (M1-alkB-rr-rrr) alkane oxidation system components a, c and d, and (D000) blank sequence, respectively, were synthesized by a standard service provider.

[0318] These sequences were delivered as part of pUC57.

[0319] Plasmid pUC57-AaBFS was then digested using restriction enzymes EcoRI and HindIII. Plasmids pUC57-M1-alkB-rr-rrr and pUC57-D000 were digested with BamHI and HindIII, respectively, and combined in a ligation reaction with BamHI-EcoRI digested pm-LPppa-CiCaSSy-mpmii alt (described in US Patent Publication No. 2020 / 0010822) following standard procedures. The ligation was transformed into E. coli S17-1 and selected on LB containing 100 μg / ml neomycin.

[0320] The resulting E. coli S17-1 colonies carried the plasmids pBBR-AaBFS-M1-alkB-rr or pBBR-MVA-AaBFS-D000. These plasmids were conjugated to Rhodobacter sphaeroides strain Rs265-9c using the methods disclosed in WO2011074954 (see pages 64-67) and trans-form conjugates were selected using plates containing Ra medium and 100 μg / ml neomycin. Rhodobacter colonies carrying the plasmids pBBR-MVA-AaBFS-M1-alkB-rr or pBBR-MVA-AaBFS-D000 were selected. The resulting strains were named after the plasmids.

[0321] Strains Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr (IS2) carrying β-farnese synthase, oxidase enzyme alkB, and rubredoxin and rubredoxin reductase, as well as Rs265-9c-pBBR-MVA-AaBFS-D000 (CS2) as a control, were cultivated in 20 ml of RS102 medium using 2 ml of n-dodecane as an overlay essentially as described in WO2018160066A1. After 72 hours of cultivation, the n-dodecane layer was harvested and analyzed by GC-MS essentially as described in WO2018160066A1 (see FIG. 3a).

[0322] The n-dodecane of culture Rs265-9c-pBBR-MVA-AaBFS-D000 (CS2) contained β-farnesene as the predominant peak eluting at 16.81 min. The dodecane of Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr (IS2) showed an additional peak at 22.23 min, which was absent in (CS2) Rs265-9c-pBBR-MVA-AaBFS-D000 (see Figure 3b). Comparison of the mass spectrum and retention time of the peak eluting at 22.23 min with that of the standard sinensal (Aroma Uden) revealed that this peak represented β-sinensal (see Figure 3c for the (IS2)Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr-based peak and Figure 3d for the sinensal standard).

[0323] Quantitative analysis revealed that 15.2 g of farnesene was produced per kg of dodecane, while 0.38 g of sinensal was produced per kg of dodecane.

[0324] Example 4: We verified whether the M1-alkB module could oxidize α-farnesene to α-sinensal. First, a synthetic construct expressing α-farnesene synthase from Malus domesticus was ordered from a standard service provider and delivered as the plasmid pUC57.

[0325] The nucleic acid sequence of SEQ ID NO:14 corresponds to the nucleic acid of (MdAFS) alpha-farnesene synthase.

[0326] Plasmid pUC57-MdAFS was then digested using restriction enzymes EcoRI and HindIII. Plasmids pUC57-M1-alkB-rr-rrr and pUC57-D000 were digested with BamHI and HindIII, respectively, and combined with BamHI-EcoRI digested pm-LPppa-CiCaSSy-mpmii alt pDEST4 in a ligation reaction following standard procedures. The ligation was transformed into E. coli S17-1 and selected on LB containing 100 μg / ml neomycin.

[0327] The resulting E. coli S17-1 colonies carried the plasmid pBBR-MVA-MdAFS-M1-alkB-rr-rrr, or pBBR-MVA-MdAFS-D000. These plasmids were conjugated to Rhodobacter sphaeroides strain Rs265-9c using standard procedures, for example those described in WO2011074954, and trans-form conjugates were selected using plates containing Ra medium and 100 μg / ml neomycin. Rhodobacter colonies carrying the plasmid pBBR-MVA-MdAFS-M1-alkB-rr-rrr, or pBBR-MVA-MdAFS-D000 were selected. The resulting strains were named after the plasmids.

[0328] Strains (IS3) Rs265-9c-pBBR-MVA-MdAFS-M1-alkB-rr-rrr and enzyme-free (CS3) Rs265-9c-pBBR-MVA-MdAFS-D000 were cultivated in 20 ml of RS102 medium with 2 ml of n-dodecane as an overlay essentially as described in WO2018160066A1. After 72 hours of cultivation, the n-dodecane layer was harvested and analyzed by GC-MS essentially as described in WO2018160066A1.

[0329] The n-dodecane of the control culture (CS3) Rs265-9c-pBBR-MVA-MdAFS-D000 contained α-farnesene as the predominant peak eluting at 19.28 min. The dodecane of (IS3) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-rr-rrr showed an additional peak at 24.88 min, which was absent in (CS3) Rs265-9c-pBBR-MVA-MdAFS-M1-D000 (see Figure 4). Comparison of the mass spectrum and retention time of the peak eluting at 24.883 min with that of the standard sinensal (Aroma Uden) revealed that this peak was indicative of α-sinensal.

[0330] Example 5: We tested whether the M1-alkB module could oxidize other sesquiterpenes to aldehydes. First, synthetic constructs expressing β-bisabolene synthase (ZoBBS) (BAI67934.1) from ginger (Zingiber officinale) and santalene synthase (CiCaSSy) (QNV69588.1) from camphor tree (Cinnamomum camphora) were synthesized by a standard service provider and delivered in the plasmid pUC57.

[0331] The nucleic acid sequences of SEQ ID NO:15 and SEQ ID NO:16 correspond to ginger (Zingiber officinale)-(S)-β-bisabolene synthase (ZoBBS) and a plant terpene synthase from camphor tree (Cinnamomum camphora) (CiCaSSy).

[0332] Plasmids pUC57-ZoBBS and pUC57-CiCaSSy were then digested using restriction enzymes EcoRI and HindIII. Plasmids pUC57-M1-alkB-rr-rrr and pUC57-D000 were digested with BamHI and HindIII, respectively, and combined with BamHI-EcoRI digested pm-LPppa-CiCaSSy-mpmii alt in a ligation reaction following standard procedures. The ligation was transformed into E. coli S17-1 and selected on LB containing 100 μg / ml neomycin.

[0333] The resulting E. coli S17-1 colonies harbored the plasmids pBBR-MVA-ZoBBS-M1-alkB-rr-rrr, or pBBR-MVA-ZoBBS-D000, and BBR-MVA-CiCaSSy-M1-alkB-rr-rrr, or pBBR-MVA-CiCaSSy-D000. These plasmids were conjugated into Rhodobacter sphaeroides strain Rs265-9c using standard methods, and trans-formed conjugates were selected using plates containing Ra medium and 100 μg / ml neomycin. Rhodobacter colonies carrying the plasmids pBBR-MVA-ZoBBS-M1-alkB-rr-rrr, or pBBR-MVA-ZoBBS-D000, and BBR-MVA-CiCaSSy-M1-alkB-rr-rrr, or pBBR-MVA-CiCaSSy-D000 were selected. The resulting strains were named after the plasmids.

[0334] Strains (IS4) Rs265-9c-pBBR-MVA-ZoBBS-M1-alkB-rr-rrr and (CS4) Rs265-9c-pBBR-MVA-ZoBBS-D000 as controls, (IS5) Rs265-9c-pBBR-MVA-CiCaSSy-M1-alkB-rr-rrr and (CS5) Rs265-9c-pBBR-MVA-CiCaSSy-D000 as controls were cultivated in 20 ml of RS102 medium using 2 ml of n-dodecane as an overlay essentially as described in WO2018160066A1. After 72 hours of cultivation, the n-dodecane layer was harvested and analyzed by GC-MS essentially as described in WO2018160066A1.

[0335] Referring to Figure 6, n-dodecane from culture Rs265-9c-pBBR-MVA-CiCaSSy-D000 contained β-α-santalene, bergamotene and β-santalene as predominant peaks at retention times of 17.23 and 18.23 min, respectively. Dodecane from Rs265-9c-pBBR-MVA-CiCaSSy-M1-alkB-rr-rrr showed additional peaks at 23.75 and 24.66 min, which were absent in (CS5) Rs265-9c-pBBR-MVA-CiCaSSy-M1-D000. Comparison of the mass spectrum and retention times of the peaks eluting at 23.75 and 24.66 min with those from the NIST library and sandalwood standard revealed that they corresponded to trans-α-santalol and trans-β-santalol.

[0336] Referring to FIG. 5, the n-dodecane of culture (CS4) Rs265-9c-pBBR-MVA-ZoBBS-D000 contained β-bisabolene as the predominant peak eluting at 19.37 min. The n-dodecane of (IS4) Rs265-9c-pBBR-MVA-ZoBBS-M1-alkB-rr-rrr showed an additional peak at 25.16 min that was not present in (CS4) Rs265-9c-pBBR-MVA-ZoBBS-M1-D000. GC MS analysis revealed that this peak represented lantheol-aldehyde, an allylic oxidation product derived from β-bisabolene.

[0337] Example 6: Effects of M1-alkB mutations Two mutants of the M1-alkB protein were designed to generate the M1-alkB-F93S mutant and the M1-alkB-W189S mutant, and the expression constructs of these two mutants were synthesized by a standard service provider.

[0338] Plasmid pUC57-AaBFS was then digested using restriction enzymes EcoRI and HindIII. Plasmids pUC57-M1-alkB-F93S-rr-rrr (having the sequence of SEQ ID NO: 17) and pUC57-M1-alkB-W189S-rr-rrr (having the sequence of SEQ ID NO: 18) were digested with BamHI and HindIII, respectively, and combined in a ligation reaction with BamHI-EcoRI digested pm-LPppa-CiCaSSy-mpmii alt (described in US Patent Publication No. 2020 / 0010822) according to standard procedures. The ligation was transformed into E. coli S17-1 and selected on LB containing 100 μg / ml neomycin.

[0339] The resulting E. coli S17-1 colonies harbored the plasmids pBBR-AaBFS-M1-alkB-F93S-rr-rrr or pBBR-MVA-AaBFS-M1-alkB-W189S-rr-rrr. These plasmids were conjugated to Rhodobacter sphaeroides strain Rs265-9c using standard methods, and trans-form conjugates were selected using plates containing Ra medium and 100 μg / ml neomycin. Rhodobacter colonies harboring the plasmids pBBR-MVA-AaBFS-M1-alkB-F93S-rr-rrr or pBBR-MVA-AaBFS-M1-alkB-W189S-rr-rrr were selected. The resulting strains were named after the plasmids.

[0340] Strains (IS6a) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93S-rr-rrr and (IS6b) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-W189S-rr-rrr were cultivated in 20 ml of RS102 medium with 2 ml of n-dodecane as an overlay as described in Example 3. After 72 hours of cultivation, the n-dodecane layer was collected and analyzed by GC-MS.

[0341] Quantitative analysis revealed that both strains (IS6a) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93S-rr-rrr and (IS6b) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-W189S-rr-rrr produced 16.0 g of farnesene per kg of dodecane. Strain (IS6a) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93S-rr-rrr produced 0.81 g of sinensal per kg of dodecane, while (IS6b) Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-W189S-rr-rrr produced 0.51 g of sinensal per kg of dodecane. Therefore, both mutations in M1-alkB appear to contribute to higher sinensal production.

[0342] The nucleic acid sequences of SEQ ID NO:17 and SEQ ID NO:18 correspond to alkane oxidation system components a and c having a point mutation of F93S, i.e., M1-alkB-F93S-rr-rrr, and alkane oxidation system components a and c having a point mutation of W189S, i.e., M1-alkB-W189S-rr-rrr.

[0343] [Table 1-1]

[0344] [Table 1-2]

[0345] [Table 1-3]

[0346] Example 7: Point Mutations Position 93 of the alkB protein codes for phenylalanine. Position F93 was changed to serine, and this M1-alkB was introduced in combination with β-farnesene synthase, rubredoxin, and optionally rubredoxin reductase. This point mutation was associated with improved productivity of β-sinensal. This point mutation was also associated with the oxidation of terpene end products as well as farnesol as a by-product.

[0347] Point mutations observed at position 93 include F93V, F93I, F93A, F93R, F93W, F93T, and F93G. Of these, F93A produced the highest amount of β-sinensal when combined with β-farnesene synthase. No formation of farnesol was observed in the F93A mutant.

[0348] [Table 2]

[0349] Table 2 above shows the amounts of β-farnesene and β-sinensal over a 72-hour period obtained by point-mutating the 93rd residue of the alkB protein, which is normally an "F". The point mutations F93S, F93V, F93G, and F93A associated with strains (IS7a), i.e., Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93S-rr-rrr-2, (IS7b), i.e., Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93V-rr-rrr, (IS7c), i.e., Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93G-rr-rrr, and (IS7d), i.e., Rs265-9c-pBBR-MVA-AaBFS-M1-alkB-F93A-rr-rrr, were associated with improved production of β-farnesene and β-sinensal compared to the native alkB strain with an F at position 93.

[0350] However, the point mutations F93R, F93I, F93W, and F93T were not associated with improved production of oxygenated terpene products, and this improvement was with respect to the original 93rd residue, i.e., "F."

[0351] Example 8: Production of sinensal in the absence of component d without MVA Similar to Example 3 above, strain IS8 carrying construct Rs265-9c-pBBR-AaBFS-M1-alkB-rr-rrr was tested. The difference from Example 3 was that strain IS8 did not contain rubredoxin reductase or another foreign component d, and did not contain a heterologous MVA pathway. The resulting amount of sinensal produced was comparable to that of IS2, demonstrating that components a, b, and c alone were sufficient to produce the oxidized terpene product sinensal.

[0352] Example 9 The construct of SEQ ID NO: 44 was examined in a similar manner to the examination of the alkB construct in Example 3 above. The results showed that the CMR5c protein is also suitable as an oxidase enzyme in an alkane oxidation system.

[0353] Example 10 Based on the method for preparing mutant polypeptides described above, mutant polypeptides of SEQ ID NOs: 25 to 42 were obtained. Figures 7a and 7b show alignments of the polypeptides of the invention with conserved amino acids at the positions indicated. The polypeptide variants of the invention contain conserved amino acids at the positions shown in Figures 7a and 7b. In the figures, the positions of the conserved amino acids are shown in white font letters on a black background.

[0354] Example 11 Based on the method disclosed above, further experimental analysis was performed using the above-mentioned strains of Rhodobacter sp. One strain was developed to possess all of the components a, b, c, and d of the alkane oxidation system. The other strain was developed to possess only component a (oxidase enzyme) and component b (terpene synthase), but not component c (rubredoxin) and component d (rubredoxin reductase). Both strains were shown to oxidize β-farnesene to β-sinensal.

[0355] Table 3 below shows the amounts of β-farnesene and β-sinensal for the strains after 72 hours.

[0356] [Table 3]

Claims

1. 1. An artificial alkane oxidation system comprising: a. an oxidase enzyme, and b. one or more enzymes to provide one or more alkanes; and c. optionally, an electron transfer compound suitable for transferring at least one electron to said oxidase enzyme; and d. optionally, an electron transfer compound regenerating enzyme suitable for reducing said electron transfer compound from an oxidized state; An artificial alkane oxidation system, wherein the oxidase enzyme is an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment thereof.

2. the alkane has at least 5 carbon atoms or at least one 5-carbon unit; or 2. The artificial alkane oxidation system of claim 1, wherein component b is at least one terpene synthase protein and the at least one alkane is a terpene.

3. 2. The artificial alkane oxidation system according to claim 1, wherein the electron transfer compound is an electron transfer protein and / or the electron transfer compound regenerating enzyme is an electron transfer protein reductase.

4. 10. A synthetic nucleic acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 4, 12, 17, 18, 19, or 44, comprising a nucleic acid sequence encoding the oxidase enzyme of claim 1.

5. The artificial alkane oxidation system kit comprises: a first isolated nucleic acid sequence encoding the oxidase enzyme of claim 1; a second isolated nucleic acid sequence encoding an electron transfer protein, preferably not physically associated with said first nucleic acid; Optionally, a third nucleic acid sequence encoding an electron transfer protein reductase, which may be fused to the first and / or second nucleic acid sequence; and Kit including:

6. An expression cassette comprising: A synthetic nucleic acid sequence according to claim 4, or The nucleic acid sequence of the alkane oxidation system, a. has at least 70% sequence identity with SEQ ID NO: 20, or a nucleic acid sequence of an oxidase enzyme encoding an oxidase enzyme having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43; b. has at least 70% sequence identity with SEQ ID NO: 21; or a nucleic acid sequence of a rubredoxin peptide encoding rubredoxin of SEQ ID NO: 2 or SEQ ID NO: 45, and optionally c. has at least 70% sequence identity with SEQ ID NO: 22; or the nucleic acid sequence of the alkane oxidation system comprising a rubredoxin reductase peptide encoding the rubredoxin reductase peptide of SEQ ID NO: 3; an expression cassette comprising:

7. 1. A method for the oxidation of at least one alkane substrate, comprising: a. for the oxidation of said at least one alkane substrate, i. an artificial alkane oxidase system according to claim 1, and / or iii. Expression of a nucleic acid sequence encoding said oxidase enzyme comprising an amino acid sequence having at least 70% sequence identity to any one of SEQ ID NOs: 1, 10, 11, 23-43, or a fragment or variant thereof; and v. Optionally, providing one or more alkane substrates; b. allylic oxidizing the alkane substrate to produce at least one oxidized alkane product; c. optionally extracting said at least one oxidized alkane product; A method comprising:

8. 8. The method of claim 7, wherein the at least one alkane substrate is selected from a diterpene, monoterpene, or sesquiterpene, including α-farnesene, β-farnesene, α-bisabolene, β-bisabolene, α-bergamotene, β-bergamotene, α-santalene, β-santalene, valencene, α-guayene, the monoterpene geraniol, nerol monoterpene, geranyl lactone, linalyl acetate, limonene, β-pinene, or a combination thereof.

9. 9. A method for producing at least one oxidized alkane product by oxidation of an alkane substrate, comprising steps a to c of claim 7, wherein preferably the alkane substrate is a terpene as defined in claim 8.

10. An artificial alkane oxidation system according to claim 1, or 8. The oxidation of the alkane substrate by the method of claim 7. A non-human host cell comprising:

11. 8. A composition produced by the method of claim 7, comprising: The oxidase enzyme of claim 1; at least one oxidized terpene product selected from myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol, trans-β-santalol, lantheol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderalasin, bicyclo-octanediol, α-guaiene oxide, or β-guaiene oxide, or a combination thereof; Optionally, one or more terpene substrates A composition comprising:

12. A non-human host cell according to claim 10 cultured in a culture medium; and said at least one oxidized alkane product produced from said non-human host cell as a major compound, the fermentation composition comprises the alkane substrate, and optionally one or more by-products are trace compounds; Fermented composition.

13. 1. A fermentative method for the production of at least one oxidized alkane product, comprising: providing a non-human host cell with an oxidase enzyme system or expression cassette according to claim 1; Optionally, providing said host cell with at least one alkane synthase for the production of an alkane substrate; Cultivating the non-human host cells in a culture medium under conditions suitable for the host cells to produce the encoded polypeptide in an active form; Optionally, generating and / or providing one or more alkane substrates to said host cell; producing at least one oxidized alkane product; optionally purifying said at least one oxidized alkane product; A method comprising:

14. 12. The method of claim 7 or the composition of claim 11, wherein the alkane oxidation product is selected from an alcohol or an aldehyde, or both, including myrcene aldehyde, α-sinensal, β-sinensal, trans-α-santalol, trans-β-santalol, lantheol-aldehyde, nootkatone, vetivone, rotundone, rebaudioside, 8-hydroxygeraniol, 8-hydroxynerol, 9,10-epoxygeranylacetone, hexadecenal, farnesol, denderalasin, bicyclo-octanediol, α-guaiene oxide, β-guaiene oxide, or a combination thereof.

15. 1. A method for preparing a mutant polypeptide having oxidase enzyme activity, comprising: a) selecting a nucleic acid encoding a synthetic nucleic acid according to claim 4 or an amino acid sequence having at least 70% sequence identity with any one of SEQ ID NOs: 1, 10, 11, 23 to 43, or a fragment thereof; b) modifying the selected nucleic acids to obtain at least one mutant nucleic acid; c) transforming the non-human host cell with the mutant nucleic acid sequence to express the oxidase enzyme encoded by the mutant nucleic acid sequence; d) screening the oxidase enzyme for at least one altered property in addition to the function of the oxidase enzyme encoded by the selected nucleic acid of a); e) optionally repeating process steps (a) to (d) to obtain an oxidase enzyme with the desired mutant function; f) optionally isolating said mutant nucleic acid sequence from said transformed non-human host cell obtained in step (c); A method comprising:

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