Biotechnological production of phytol

Abstract

The present invention relates to a bacterium suitable for producing phytol, characterized in that it contains chlorophyll and expresses a heterologous and / or overexpressed chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll. The invention further refers to methods for preparing such bacterium and for preparing phytol using such bacterium. Moreover, the invention relates to a composition comprising phytol obtainable from a method of the invention and uses of such composition and phytol obtainable from a method of the invention such as use as fragrance or aroma or for preparing vitamin E and / or K, isophytol, pristane, and / or phytane.

Description

[0001] Biotechnological Production of Phytol

[0002] The present invention relates to a bacterium suitable for producing phytol, characterized in that it contains chlorophyll and expresses a heterologous and / or overexpressed chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll. The invention further refers to methods for preparing such bacterium and for preparing phytol using such bacterium. Moreover, the invention relates to a composition comprising phytol obtainable from a method of the invention and uses of such composition and phytol obtainable from a method of the invention such as use as fragrance or aroma or for preparing vitamin E and / or K, isophytol, pristane, and / or phytane.

[0003] Phytol is a compound of notable interest. It is known as a valuable fragrance molecule (McGinty et al., Fragrance material review on phytol, Food and Chemical Toxicology, 2010, 48: S59-S63) and can serve as intermediate in the chemical synthesis of vitamin E and vitamin K. Therefore, considerable amounts of phytol are required. It is thus of general interest to prepare phytol by a cost- and energy-efficient route.

[0004] Phytol has two stereochemical centers. Furthermore, there are cis- / trans-isomers. Biopotency of the different possible isomers is known to differ. As an example, the RRR-o-tocopherol isomer often shows highest potency among the eight possible isomers. Thus, there is a particular desire for RR-phytol, in particular phytol with the formula (2E,7R,11 R)-3,7,11 ,15-tetramethylhexadec-2-en-1-ol that is regularly found in nature. Due to the comparably complex structure and stereochemical requirements, chemical synthesis is challenging and often not as stereoselective as desired. Furthermore, synthetic routes are often undesirably energy- and resources-consuming.

[0005] Thus, it has been considered to obtain phytol from natural sources, in particular plants. Indeed, phytol is considered as widely abundant acyclic isoprenoid (Rontani and Volkman, Phytol degradation products as biogeochemical tracers in aquatic environments, Organic Geochemistry, 2003,34(1): 1-35). A moiety of phytol is, as the phytyl chain, forming part of chlorophyll an essential component of the light harvesting machinery of plants, algae, cyanobacteria, and pho-totrophic bacteria. Here, it may serve as a hydrophobic anchor for the integration of chlorophyll into the hydrophobic core of membranes. In a biochemical pathway, chlorophyll may be biosynthesized from two main building blocks: chlorophyllide, and geranyl geranyl diphosphate (GGPP) (Gutbrod et al., Phytol metabolism in plants and algae, Progress in Lipid Research, 2019, 74:1-17). The chlorin skeleton of chlorophyllide may be assembled from glutamic acid via the tetrapyrrole pathway. GGPP is built up from the universal isoprenoid precursors isopentenyl diphosphate (IPP) and dimethylallyl diphosphate (DMAPP) which may be condensed over several steps catalyzed by enzymes such as the farnesyl diphosphate (FPP) synthase and GGPP synthase. Chlorophyllide is decorated with a geranyl geranyl tail from GGPP by the chlorophyll synthase. In a subsequent step, the three terminal double bonds of the geranyl geranyl tail are typically enantioselecti vly hydrogenated to the RR-configured phytyl chain by a geranyl geranyl reductase (GGR) under consumption of three equivalents of NADPH yielding the functional chlorophyll. In plants and algae, minor amounts of free phytol may occur in the course of the degradation of chlorophyll. Here, the ester hydrolysis liberating free phytol can proceed via two potential routes. First, with a preceding extraction of the central magnesium ion of chlorophyll which may be catalyzed by pheophytin pheophorbide hydrolase (PPH). Second, without the magnesium extraction and hydrolysis catalyzed by chlorophyll dephytylase (CLD). These processes are typical for plants and algae.

[0006] Phytol and chlorophyll are only present at low concentrations in plants, algae and bacteria (Brown and Lascelles, Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides. Plant Physiol, 1972, 50(6)747-749; Zavfel et al., Phenotypic characterization of Synechocystis sp. PCC 6803 substrains reveals differences in sensitivity to abiotic stress, PLOS ONE, 2017, 12(12):e0189130). Low amounts of phytol have also been obtained from photo-trophic bacteria for experimental reasons (Brown and Lascelles, Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomonas spheroides. Plant Physiol, 1972, 50(6)747-749).

[0007] To obtain higher yields of chlorophyll, plant material containing chlorophyll can be subjected to chemical saponification. Saponification is typically conducted under rather harsh conditions such as basic buffers. This often leads to undesired side products.

[0008] Other processes for extracting phytol such as chromatographic methods (Thakor et al., Extraction and purification of phytol from Abutilon indicum: cyto-toxic and apoptotic activity. RSC Advances, 2016, 6 (54): 48336-48345) appear even less efficient and seem to be suitable for analytical purposes only. These processes are not as economical as desired.

[0009] Using cell cultures and of plant and algae cells require comparingly big efforts. Furthermore, there are no sufficient industrial fermentation capabilities to obtain desirably high yields of phytol from plants, algae of cell cultures thereof. Most bacteria such as E. coli do neither generate chlorophyll nor phytol. Phototrophic bacteria produce bacteriochlorophyll. However, there are no means for efficiently preparing phytol from such phototrophic bacteria. Experiments were performed on a laboratory scale to isolate an enzyme that can catalyzes cleavage of phytol from chlorophyll in E. coli and using such enzyme in a cell-free chlorophyll-containing medium in vitro (Lin et al., Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis, The Plant Cell, 2016.28(12): 2974-2990). This does, however, not allow feasible production of phytol. It is required to express it and isolate the enzyme and, subsequently, adding it to a chlorophyll-containing medium. This is rather laborious and inefficient.

[0010] In view of the above, there is still an unmet need for efficient means for preparing phytol, in particular with desired stereochemical properties. It is particularly preferred to obtain a method that allows producing higher amounts of phytol.

[0011] It has surprisingly been found that bacteria containing chlorophyll that expresses a heterologous and / or overexpressed chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll enable to obtain a desired phytol by a particularly efficient route using biologically derived raw material and having a low carbon footprint. The residual chlorophyll may also be understood and designated as chlorophyllide. Such bacteria allow biotechnological production of phytol. This process can be scaled up for producing higher amounts of phytol.

[0012] A first aspect of the present invention relates to a bacterium suitable for producing phytol, in particular (2E,7R, 11 R)-3,7,11 , 15-tetramethylhexadec-2-en-1 -ol, wherein the bacterium is characterized in that it:

[0013] (a) contains chlorophyll, in particular bacteriochlorophyll; and

[0014] (b) expresses a heterologous and / or overexpressed chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll.

[0015] A chlorophyll-hydrolyzing enzyme may also be designated as "chlorophyllase”, "chlase”, or "chlorophyll chlorophyl-lidohydrolase”. A chlorophyll-hydrolyzing enzyme may catalyze the reaction from [chlorophyll + H2O] to [phytol + chlo-rophyllide] . As a non-binding example, the reaction as catalyzed by the chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, exemplified as a chlorophyll dephytylase, the following is depicted for illustrative purposes:

[0016]

[0017] It will be understood that each other type of chlorophyll may also be used.

[0018] Chlorophyll may be any chlorophyll known in the art. In other words, the chlorophyll may be any type of chlorophyll known in the art. It may, for instance be chlorophyll a, chlorophyll b, chlorophyll d, chlorophyll c2, chlorophyll d, chlorophyll f, or a combination of two or more thereof. Chlorophyll may be such typically found in plants, algae, cyanobacteria, and phototrophic bacteria (Brown and Lascelles, Phytol and Bacteriochlorophyll Synthesis in Rhodopseudomo-nas spheroides. Plant Physiol, 1972, 50(6):747-749; Sadukha et al., Sequential Downstream Process for Concurrent Extraction of Lutein, Phytol, and Biochemicals from Marine Microalgal Biomass as a Sustainable Bio-refinery, ACS Sustainable Chemistry & Engineering, 2023, 11 (2):547-558; Thakor et al., Extraction and purification of phytol from Abutilon indicum: cyto-toxic and apoptotic activity. RSC Advances, 2016, 6 (54): 48336-48345). In a preferred embodiment, the chlorophyll is bacteriochlorophyll. In a preferred embodiment, the chlorophyll is bacteriochlorophyll inherently present in the bacterium used in the context of the present invention.

[0019] The bacterium may be any bacterium suitable to contain chlorophyll, in particular bacteriochlorophyll and expressing the chlorophyll-hydrolyzing enzyme.

[0020] In a preferred embodiment, the bacterium is of a phototrophic bacterial species inherently containing bacteriochlorophyll. As used herein, a phototrophic bacterium may be photoheterotrophic or photoautotrophic. In a preferred embodiment, the bacterium is of a photoheterotrophic bacterial species. In a preferred embodiment, the bacterium is of a photoheterotrophic bacterial species inherently containing bacteriochlorophyll.

[0021] The bacterium may be of any phylum. In a preferred embodiment, the bacterium is of the phylum Pseudomonadota. In a preferred embodiment, the bacterium is of the class Alphaproteobacteiial. In a preferred embodiment, the bacterium is of the order Rhodobacterales. In a preferred embodiment, the bacterium is Rhodobacteraceae. In a preferred embodiment, the bacterium is of the genus Rhodobacter, in other words is a Rhodobacter bacterium. Such Rhodo-bacter bacterium may be of any Rhodobacter spoecies and of any strain thereof. In a preferred embodiment, the bacterium is Rhodobacter sphaeroides or Rhodobacter capsulatus. In a preferred embodiment, the bacterium is Rhodobacter sphaeroides. The bacterium may be of any strain, such as may be Rhodobacter sphaeroides strain Rs265.

[0022] Surprisingly, it has been found that phytol can be directly produced using bacteriochlorophyll producing bacteria Rhodobacter sphaeroides and equipping them with a chlorophyll-hydrolyzing enzyme such as heterologous chlorophyll dephytylase gene.

[0023] The chlorophyll-hydrolyzing enzyme may be any enzyme suitable for catalyzing cleavage of phytol off the residual chlorophyll. This may be an enzyme that catalyzes cleaving phytol off chlorophyll containing the complexed magnesium and / or an enzyme that catalyzes cleaving chlorophyll not containing the complexed magnesium. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme cleaved off catalyzes cleaving phytol off chlorophyll containing the complexed magnesium and / or an enzyme that catalyzes cleaving chlorophyll not containing the complexed magnesium inside (i.e., intracellularly in, i.e. , in the cyctoplasm of) the bacterium suitable for producing phytol. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is a chlorophyll dephytylase or a pheo-phytin pheophorbide hydrolase. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is a chlorophyll dephytylase.

[0024] The expressed chlorophyll-hydrolyzing enzyme may be derived from any species. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant or algae species. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant. Surprisingly, it has been found that enzymes that cleave off phytol from plant-derived chlorophyll in plants are also active to cleave off phytol from bacteriochlorophyll. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant further characterized in that it is of the clade of Tracheophytes, the clade of Angiosperms, the clade of Eudicots, and / or the clade of Rosids. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant of the order of Brassicales, in particular of the family of Brassicaceae. In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme is derived from a plant of the genus Arabidopsis, in particular of the species Arabidopsis thaliana. Arabidopsis thali-ana is a species that is commonly used in science including The Netherlands and Germany for a long time.

[0025] In a preferred embodiment, the chlorophyll-hydrolyzing enzyme is Arabidopsis thaliana chlorophyllase I chlorophyll dephytylase (Benedetti and Arruda, Altering the expression of the chlorophyllase gene ATHC0R1 in transgenic Arabidopsis caused changes in the chlorophyll-to-chlorophyllide ratio, Plant Physiology, 2002 128:1255-1263; Schelbert et al., Pheophytin pheophorbide hydrolase (pheophytinase) is involved in chlorophyll breakdown during leaf senescence in Arabidopsis, Plant Cell, 2009, 21:767-785; Lin et al., Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis, The Plant Cell, 2016.28(12): 2974-2990).

[0026] In further embodiments of the present invention, the chlorophyll-hydrolyzing enzyme is selected from the group consisting of:

[0027] citrus chlorophyllase (Jacob-Wilk et al., Chlorophyll breakdown by chlorophyllase: isolation and functional expression of the Chlasel gene from ethylene-treated Citrus fruit and its regulation during development, 1999, The Plant Journal 20:653-661);

[0028] Chenopodium chlorophyllase (Tsuchiyaz et al., Cloning of chlorophyllase, the key enzyme in chlorophyll degradation: finding of a lipase motif and the induction by methyl jasmonate, Proc Natl Acad Sci U S A, 1999, 96:15362-15367);

[0029] In a preferred embodiment, expressed chlorophyll-hydrolyzing enzyme comprises or consists of a polypeptide sequence having sequence identity of at least 80% of SEQ ID NO: 1 :

[0030] ATASSSATVSGGGWEAVELAEIGERSKKWKWKGEYSVNYFVKDSPEEVTPASQTVLLVHGFGASIPHWRRNINALS KNHTVYAIDLLGFGASDKPPGFSYTMESWAELILNFLEEWQKPTILIGNSVGSLACVIAASGTKFLIYLEKKTESRGDL VKGLVLLNCADGMNNKAVFDDWRIKLLMPLLLLIDFLLKQRGIASALFNRVKDRENLKNILTNVYGNKDNVDDTLVEIIA GPANTEGALDAFVSILTGPPGPNPIKLIPEITKPVLVLWGDQDGLTPLDGPVGKYFTSLPDQLPNFNLYVLQGVGHCP QDDRPDLVHERLLPWLAQLSST

[0031] This is a G193D (which is underlined in the above) mutated form of the chlorophyll dephytylase CDL1 from Arabidopsis thaliana (AtCLDI). AtCLDI is encoded by chlorophyll dephytylase CLD1 gene from the classical lab organism Arabidopsis thaliana. The enzyme AtCLDI is located in the thylakoid of the chloroplast.

[0032] The mutated enzyme of SEQ ID NO: 1 may also be designated as "AtCLDI (G193D)” or “AtCLDI”. This is described by Lin et al. (Identification of a Chlorophyll Dephytylase Involved in Chlorophyll Turnover in Arabidopsis, The Plant Cell, 2016.28(12): 2974-2990). It was found that the mutated form AtCLDI bears higher activity than the wild-type form CLD1. Lin et al. described that the primary expressed polypeptide product and mature polypeptide (lacking the transit peptide) can be functionally expressed in E. coli. Cell-free protein extracts of containing such enzyme were incubated in vitro with chlorophyll a, chlorophyll b and Phein, and produced substantial amounts of Chlide a, Chlide b, and Pheide a, respectively. This process was however not suitable for preparing phytol efficiently as the enzyme has to be isolated and plant material has to be provided in rather pure form.

[0033] In a preferred embodiment, the expressed chlorophyll-hydrolyzing enzyme comprises or consists of a polypeptide sequence having sequence identity of at least 90%, of at least 95%, of at least 98%, of at least 99%, of at least 99.5%, of at least 99.8%, or 100% (i.e., identity) of SEQ ID NO: 1.

[0034] It will be understood that throughout the present invention the term "polypeptide” and "protein” may be used interchangeably. An enzyme as used herein may also be designated as a polypeptide.

[0035] It will be further understood that throughout the present invention, a polypeptide (including an enzyme) as described by a (partial) identity to a given polypeptide sequence may also include any salt, any complex, and any hydrate thereof. Furthermore, it may embrace any posttranslational modification thereof such as, e.g., glycosylation, oxidation, reduction, phosphorylation, sulfatation, amidation, acylation (e.g., acetylation or amino acid conjugation), cyclization, etc. at of one or more amino acid side chains and / or at the N-terminus and / or the C-terminus. It will be understood that formation of salts, complexes and / or hydrates, and / or one or more posttranslational modifications may inherently occur in the bacterium suitable for producing phytol.

[0036] It will be understood that also a respective enzyme wherein the G193D mutation is not occurring may be used. Thus, in a preferred embodiment, expressed chlorophyll-hydrolyzing enzyme comprises or consists of a polypeptide sequence having sequence identity of at least 80%, at least 90%, of at least 95%, of at least 98%, of at least 99%, of at least 99.5%, of at least 99.8%, or 100% (i.e., identity) of SEQ ID NO: 3.

[0037] ATASSSATVSGGGWEAVELAEIGERSKKWKWKGEYSVNYFVKDSPEEVTPASQTVLLVHGFGASIPHWRRNINALS KNHTVYAIDLLGFGASDKPPGFSYTMESWAELILNFLEEWQKPTILIGNSVGSLACVIAASGTKFLIYLEKKTESRGDL VKGLVLLNCAGGMNNKAVFDDWRIKLLMPLLLLIDFLLKQRGIASALFNRVKDRENLKNILTNVYGNKDNVDDTLVEIIA GPANTEGALDAFVSILTGPPGPNPIKLIPEITKPVLVLWGDQDGLTPLDGPVGKYFTSLPDQLPNFNLYVLQGVGHCP QDDRPDLVHERLLPWLAQLSST

[0038] This sequence may be derived from a sequence of UniProt ID No. F4KBJ3, which further comprises an N-terminal transit peptide of 38 amino acid moieties targeting it to the chloroplast thylakoid. This is not required in the bacteria as used in the context of the present invention. This polypeptide derived from an open reading frame of Arabidopsis thaliana has the sequence SEQ ID NO: 4: MRALTWTAMSPPVMSRTATSTVNLRRISLRRDRVCVRATASSSATVSGGGWEAVELAEIGERSKKWKWKGEYSVN YFVKDSPEEVTPASQTVLLVHGFGASIPHWRRNINALSKNHTVYAIDLLGFGASDKPPGFSYTMESWAELILNFLEEW QKPTILIGNSVGSLACVIAASGTKFLIYLEKKTESRGDLVKGLVLLNCAGGMNNKAVFDDWRIKLLMPLLLLIDFLLKQR GIASALFNRVKDRENLKNILTNVYGNKDNVDDTLVEIIAGPANTEGALDAFVSILTGPPGPNPIKLIPEITKPVLVLWGDQ DGLTPLDGPVGKYFTSLPDQLPNFNLYVLQGVGHCPQDDRPDLVHERLLPWLAQLSST

[0039] Homologous sequences of AtCLDI were found in other green organisms from cyanobacteria, algae, moss, gymnosperms, and angiosperms. Homologous chlorophyll-hydrolyzing enzymes such as chlorophyll dephytylases may in particular be found in other photosynthetic purple bacteria, such as, e.g., Rhodobacter capsulatus. Such homologous enzymes may also be suitable for producing phytol. Therefore, it is scientifically plausible and justified that any chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll located in the bacterium may be used in the context of the present invention, in particular any such chlorophyll-hydrolyzing enzyme that catalyzes cleavage of phytol off chlorophyll without the need of removal of the complexed magnesium ion, in particular any chlorophyll dephytylase.

[0040] The chlorophyll-hydrolyzing enzyme may be expressed in the bacterium suitable for producing phytol as a polypeptide of its own or may comprise one or more extensions at the N- and / or C-terminus thereof. This may, modify the performance of the enzyme in the bacterium such as, e.g., the localization thereof in the bacterium, the lifetime in the bacterium, and / or the binding properties and / or catalytic activity of the enzyme. Such extension may also be a fused other polypeptide, forming a fusion protein.

[0041] In a preferred embodiment, the chlorophyll-hydrolyzing enzyme forms part of a fusion polypeptide with one or more other polypeptides. Such one or more other polypeptides fused to the chlorophyll-hydrolyzing enzyme may be any polypeptide.

[0042] In a preferred embodiment, the chlorophyll-hydrolyzing enzyme forms part of a fusion polypeptide with one or more other polypeptides. Such one or more other polypeptides may be any polypeptide. In a preferred embodiment, such other polypeptide enhances expression of the chlorophyll-hydrolyzing enzyme and / or its stability. Thus, in a preferred embodiment, the chlorophyll-hydrolyzing enzyme forms part of a fusion polypeptide with one or more other polypeptides enhancing the expression of the chlorophyll-hydrolyzing enzyme and / or its stability.

[0043] Additionally or alternatively, the one or more other polypeptides used to the chlorophyll-hydrolyzing enzyme may enhance the solubility of the chlorophyll-hydrolyzing enzyme and / or influence the localization in the bacterium expressing the polypeptide including the chlorophyll-hydrolyzing enzyme.

[0044] The person skilled in the art knows a considerable number of such polypeptides that can be used for this purpose. It will be understood that the present invention can, in general, also be carried out without any of such fused polypeptides. Thus, it will be understood that the present invention can, in general, be carried out with any such polypeptides. For instance, such one or more other polypeptides may be selected from the group consisting of maltose binding protein (MBP), glutathione S-transferase (GST), thioredoxin (TRX), a fluorescent polypeptide (e.g., green fluorescent protein (GFP), MYC tag, FLAG tag, His tag, MISTIC tag, P17 tag, small ubiquitin-like Modifier (SUMO) tag, calcium binding proteins (CaBP), Z-basic tag, Fh8 tag, Skp tag, NusA tag, mysB tag, Tsf tag, RpoA tag, Ecotin tag, etc., or polypeptides endogenous to the host organisms, or polypeptides of heterologous enzymes required in the involved metabolic pathways, or a combination of two or more polypeptides or fragments thereof. Exemplarily, the one or more polypeptide may comprise or may consist of a maltose binding protein (MBP). Such maltose binding protein (MBP) may be any maltose binding protein known in the art. In a preferred embodiment, the chlorophyll-hydrolyzing enzyme forms part of a fusion polypeptide with a maltose binding protein comprising or consisting of a polypeptide sequence having sequence identity of at least 80%, of at least 90%, of at least 95%, of at least 98%, of at least 99%, of at least 99.5%, of at least 99.8%, or 100% (i.e., identity) of SEQ ID NO: 5.

[0045] MKIEEGKLVIWINGDKGYNGLAEVGKKFEKDTGIKVTVEHPDKLEEKFPQVAATGDGPDIIFWAHDRFGGYAQSGLLA EITPDKAFQDKLYPFTWDAVRYNGKLIAYPIAVEALSLIYNKDLLPNPPKTWEEIPALDKELKAKGKSALMFNLQEPYFT WPLIAADGGYAFKYENGKYDIKDVGVDNAGAKAGLTFLVDLIKNKHMNADTDYSIAEAAFNKGETAMTINGPWAWSNI DTSKVNYGVTVLPTFKGQPSKPFVGVLSAGINAASPNKELAKEFLENYLLTDEGLEAVNKDKPLGAVALKSYEEELVK DPRIAATMENAQKGEIMPNIPQMSAFWYAVRTAVINAASGRQTVDEALKDAQTGDDDDKLI

[0046] The fusion polypeptide may be encoded by any polynucleotide sequence suitable for such purpose. In a preferred embodiment, the fusion peptide is encoded by a polynucleotide sequence comprising or consisting of a polynucleotide (DNA) sequence having sequence identity of at least 80%, of at least 90%, of at least 95%, of at least 98%, of at least 99%, of at least 99.5%, of at least 99.8%, or 100% (i.e., identity) of SEQ ID NO: 2.

[0047] ATGAAGATCGAGGAGGGCAAGCTGGTCATCTGGATCAACGGCGACAAGGGCTACAACGGCCTGGCCGAGGTC GGCAAGAAGTTCGAGAAGGACACCGGCATCAAGGTGACGGTGGAGCACCCGGACAAGCTCGAGGAGAAGTTC CCGCAGGTGGCGGCCACCGGCGACGGCCCGGACATCATCTTCTGGGCCCATGACCGCTTCGGCGGCTACGCC CAGTCGGGCCTGCTCGCGGAGATCACGCCGGACAAGGCGTTCCAGGACAAGCTGTATCCCTTCACCTGGGAC GCCGTGCGCTACAACGGCAAGCTCATCGCGTATCCCATCGCGGTGGAGGCCCTGTCGCTCATCTACAACAAGG ACCTGCTCCCGAACCCGCCCAAGACCTGGGAGGAGATCCCCGCCCTCGACAAGGAGCTGAAGGCCAAGGGCA AGTCGGCGCTCATGTTCAACCTGCAGGAGCCGTACTTCACGTGGCCCCTGATCGCGGCCGACGGCGGCTACG CGTTCAAGTATGAGAACGGCAAGTATGACATCAAGGACGTGGGCGTGGACAACGCGGGCGCCAAGGCCGGCC TGACGTTCCTCGTGGACCTGATCAAGAACAAGCACATGAACGCCGACACCGACTACTCGATCGCGGAGGCCGC GTTCAACAAGGGCGAGACGGCCATGACGATCAACGGCCCGTGGGCGTGGTCGAACATCGACACGTCGAAGGT GAACTATGGCGTGACCGTGCTGCCCACGTTCAAGGGCCAGCCCTCGAAGCCCTTCGTGGGCGTGCTCTCGGC GGGCATCAACGCCGCGTCGCCGAACAAGGAGCTGGCGAAGGAGTTCCTCGAGAACTACCTGCTCACGGACGA GGGCCTCGAGGCCGTGAACAAGGACAAGCCCCTGGGCGCCGTGGCCCTCAAGTCGTATGAGGAAGAGCTGGT GAAGGACCCGCGCATCGCGGCCACCATGGAGAACGCGCAGAAGGGCGAGATCATGCCGAACATCCCCCAGAT GTCGGCCTTCTGGTATGCGGTGCGCACGGCCGTGATCAACGCGGCGTCGGGCCGCCAGACCGTGGACGAGG CCCTCAAGGACGCCCAGACGGGCGACGACGACGACAAGCTTATCGCCACCGCCTCGTCGTCGGCCACCGTGT CGGGCGGCGGCGTGGTGGAGGCCGTGGAGCTCGCCGAGATCGGCGAGCGCTCGAAGAAGTGGAAGTGGAAG GGCGAGTACTCGGTGAACTACTTCGTGAAGGACTCGCCGGAGGAGGTGACCCCGGCCTCGCAGACCGTGCTC CTCGTGCACGGCTTCGGCGCCTCGATCCCGCACTGGCGCCGCAACATCAACGCCCTCTCGAAGAACCACACCG TGTACGCCATCGACCTCCTCGGCTTCGGCGCCTCGGACAAGCCGCCGGGCTTCTCGTACACCATGGAGTCGTG GGCCGAGCTCATCCTCAACTTCCTCGAGGAGGTGGTGCAGAAGCCGACCATCCTCATCGGCAACTCGGTGGGC TCGCTCGCCTGCGTGATCGCCGCCTCGGGCACCAAGTTCCTCATCTACCTCGAGAAGAAGACCGAGTCGCGCG GCGACCTCGTGAAGGGCCTCGTGCTCCTCAACTGCGCCGGCGGCATGAACAACAAGGCCGTGTTCGACGACT GGCGCATCAAGCTCCTCATGCCGCTCCTCCTCCTCATCGACTTCCTCCTCAAGCAGCGCGGCATCGCCTCGGC CCTCTTCAACCGCGTGAAGGACCGCGAGAACCTCAAGAACATCCTCACCAACGTGTACGGCAACAAGGACAAC GTGGACGACACCCTCGTGGAGATCATCGCCGGCCCGGCCAACACCGAGGGCGCCCTCGACGCCTTCGTGTCG ATCCTCACCGGCCCGCCGGGCCCGAACCCGATCAAGCTCATCCCGGAGATCACCAAGCCGGTGCTCGTGCTCT GGGGCGACCAGGACGGCCTCACCCCGCTCGACGGCCCGGTGGGCAAGTACTTCACCTCGCTCCCGGACCAGC TCCCGAACTTCAACCTCTACGTGCTCCAGGGCGTGGGCCACTGCCCGCAGGACGACCGCCCGGACCTCGTGC ACGAGCGCCTCCTCCCGTGGCTCGCCCAGCTCTCGTCGACCTGA

[0048] In a preferred embodiment, the bacterium is characterized in that:

[0049] the bacterium is a phototrophic bacterium inherently containing bacteriochlorophyll, in particular of the genus Rhodo-bacter, and

[0050] the expressed chlorophyll-hydrolyzing enzyme is a chlorophyll dephytylase, preferably comprising (or consisting of) a polypeptide sequence that has a sequence identity of at least 80% of SEQ ID NO: 1,

[0051] in particular wherein the bacterium comprises a polynucleotide having a sequence identity of at least 80% of SEQ ID NO: 2.

[0052] The bacterium suitable for producing phytol may be optionally further modified to generate mor chlorophyll, which may lead to an increased yield of phytol (as each compared with a not further modified bacterium). This may be achieved by any means such as by increasing production of chlorophyll and / or by decreasing one or more non-phy-tol-releasi ng degradation routes of chlorophyll and / or by decreasing one or more phytol- degradation routes.

[0053] In a preferred embodiment, the bacterium is further characterized in that:

[0054] (I) geranylgeranyl pyrophosphate synthase (GGPP synthase) is overexpressed; and / or

[0055] (II) one or more enzymes of the mevalonate pathway are overexpressed; and / or

[0056] (III) one or more genes for the 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP / DOXP) pathway are expressed, and / or

[0057] (IV) one or more genes of the tetrapyrrole pathway are overexpressed, and / or

[0058] (V) geranyl geranyl reductase (GGR) is overexpressed, and / or

[0059] (VI) phytoene synthase is downregulated or deleted. Thus, phytol production may be enhanced by overexpression of a geranylgeranyl pyrophosphate synthase (GGPP synthase). Also, overexpression of a mevalonate pathway may contribute to a higher phytol production. Furthermore, using a bacterium which is deleted in phytoene synthase may enhance phytol production. Also a bacterium deleted in the regulators of the photosynthesis machinery, such as ppsR or regA, may increase phytol yields.

[0060] a

[0061] As indicated above, the fusion polypeptide as suitable for cleaving off phytol from chlorophyll is also novel.

[0062] Thus, the present invention further relates to a fusion polypeptide comprising (or (essentially) consisting of):

[0063] (b1) chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll; fused to

[0064] (b2) another polypeptide having at least 20 consecutive amino acid moieties that improves cellular localization, in particular wherein said other polypeptide is maltose binding protein.

[0065] A further aspect of the present invention relates to a fusion polypeptide comprising (or (essentially) consisting of): (b1) a polypeptide sequence having a sequence identity of at least 80% of SEQ ID NO: 1; fused to

[0066] (b2) another polypeptide having at least 20 consecutive amino acid moieties that improves cellular localization, in particular wherein said other polypeptide is maltose binding protein.

[0067] It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol of the present invention mutatis mutandis apply to the fusion polypeptide of the present invention.

[0068] The genetic information may be inserted into the bacterium by any means. Preferably, it is inserted into the bacterium by a vector (e.g., by transfection).

[0069] A still further aspect of the present invention relates to a vector comprising (or (essentially) consisting of):

[0070] (v1) at least one promoter suitable for promoting expression of expression in a bacterium, in particular a phototrophic bacterium containing chlorophyll, preferably located upstream in the vector; and

[0071] (v2) a polynucleotide sequence encoding for a chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, in particular having a sequence identity of at least 80% of SEQ ID NO: 1; and (v3) one or more polynucleotide sequences different from (v2) encoding for one or more polypeptide sequences forming a fusion polypeptide with the chlorophyll-hydrolyzing enzyme when expressed; and

[0072] (v4) optionally an expression-modifying sequence, in particular an expression enhancer or a sequence that enables controlling and / or silencing expression; and

[0073] (v5) optionally one or more polynucleotide sequences facilitating an antibiotic resistance suitable for selection of a transfected bacterium; and

[0074] (v6) optionally an origin of replication,

[0075] preferably wherein the vector is a circular plasmid,

[0076] in particular wherein said vector comprises a polynucleotide sequence having a sequence identity of at least 80% of SEQ ID NO: 2. It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol and the fusion polypeptide of the present invention mutatis mutandis apply to the vector of the present invention.

[0077] Such vector may be any type of vector known in the art. It may be a viral vector, plain DNA or RNA or an analogue thereof, formulated DNA and / or RNA (e.g., embedded or attached to in a liposome, a micelle, a cell-penetrating peptide, fatty acids, and / or an artificial polymer), or a conjugate of DNA and / or RNA to one or more other structures (e.g., a liposome, a micelle, a cell-penetrating peptide, fatty acids, and / or an artificial polymer). Such vector may be a linear or circular vector. In a preferred embodiment, the vector is a circular vector, in particular a plasmid. The generation of a vector may optionally be conducted as described in WO2011 / 074954.

[0078] A bacterium suitable for producing phytol may be prepared by any means. A further aspect of the present invention relates to a method for preparing a bacterium suitable for producing phytol according to the present invention, comprising (or (essentially) consisting of) the step of transfecting a bacterium, preferably a bacterium containing bacteriochlorophyll, with a gene, in particular a gene forming part of a vector according to the present invention, encoding for a chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, and

[0079] optionally selecting the bacterium suitable for producing phytol, and optionally propagating the bacterium.

[0080] It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol, the fusion polypeptide, and the vector of the present invention mutatis mutandis apply to the method for preparing a bacterium suitable for producing phytol of the present invention.

[0081] In a preferred embodiment, the bacterium is transfected with a plasmid encoding for the chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, optionally fused to one or more other polypeptides.

[0082] Transfecting may be performed by any means such as, e.g., by using electroporation, a gene gun, a chariot system, a cell-penetrating peptide and / or another transfection reagent. Several routine techniques are known.

[0083] As indicated above, it is of particular interest in the context of the present invention to prepare phytol.

[0084] A further aspect of the present invention relates to a method for preparing phytol, comprising (or (essentially) consisting of) the steps:

[0085] (i) providing bacteria suitable for producing phytol according to the present invention and / or obtainable (or obtained) from a method of the present invention, and optionally propagating the bacteria;

[0086] (ii) cultivating the bacteria of step (i) under conditions that allow expression of the chlorophyll-hydrolyzing enzyme and enzymatically catalyzed cleavage of phytol off the residual chlorophyll and optionally propagating the bacteria; and

[0087] (iii) optionally isolating phytol from the bacteria. It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol, a method for preparing such, the fusion polypeptide, and the vector of the present invention mutatis mutandis apply to the method for preparing phytol of the present invention.

[0088] In a preferred embodiment, in step (ii) of cultivating the bacteria, the conditions further allow the generation of chlorophyll in the bacterium. Then, the bacterium may generate chlorophyll and, concomitantly, cleave the of phytol off the residual chlorophyll. In other words, in step (ii), phytol is continuously produced. In a preferred embodiment, in step (ii) of cultivating the bacteria, the conditions further allow the inherent generation of bacteriochlorophyll in the bacterium. Then, the bacterium may generate (inherent) bacteriochlorophyll and, concomitantly, cleave the of phytol off the residual bacteriochlorophyll. In other words, in step (ii), phytol is continuously produced.

[0089] Step (ii) of cultivating the bacteria may be conducted for any time period. In a preferred embodiment, step (ii) of cultivating the bacteria is conducted for at least 30 min, for at least 1 hour, for at least 2 hours, for at least 3 hours, for at least 5 hours, for at least 10 hours, for at least a day, for at least two days, for at least three days, or for at least a week o two or more weeks.

[0090] Step (ii) of cultivating the bacteria may be conducted at any temperature. In a preferred embodiment, step (ii) of cultivating the bacteria is conducted at a temperature in the range of 0 to 50°C, at a temperature in the range of 4 to 45°C, at a temperature in the range of 10 to 42°C, at a temperature in the range of 18 to 40°C, at a temperature in the range of 25 to 39°C, at a temperature in the range of 30 to 38°C, at a temperature in the range of 22 to 32°C, at a temperature in the range of 25 to 35°C, at a temperature in the range of 25 to 30°C, at a temperature in the range of 28 to 32°C, or at a temperature in the range of 32 to 37°C, such as at (approximately) 25°C, at (approximately) 26°C, at (approximately) 27°C, at (approximately) 28°C, at (approximately) 29°C, at (approximately) 30°C, at (approximately) 31°C, at (approximately) 32°C, at (approximately) 33°C, at (approximately) 34°C, at (approximately) 35°C, at (approximately) 36°C, at (approximately) 37°C, at (approximately) 38°C, at (approximately) 39°C, at (approximately) 40°C, at (approximately) 41 °C, or at (approximately) 42°C. It will be understood that the temperature will be adapted to the bacterium and / or the expression optimum of the chlorophy l-hydrolyzing enzyme and may also be different from the above temperature ranges.

[0091] Optionally, the bacteria may be stirred during conducting step (ii). Optionally, feed and / or gasses may be added and / or used medium and or gasses may be removed during step (ii).

[0092] Step (ii) of cultivating the bacteria may be conducted in any culture medium as suitable for the chosen bacterium. In a preferred embodiment, the medium used in step (ii) is a liquid medium. In a preferred embodiment, the medium used in step (ii) is a liquid medium and the bacteria are cultivated in suspension. In a preferred embodiment, the method comprises a step (iii) of isolating phytol from the bacteria. In a preferred embodiment, the method comprises step (iii) of isolating phytol from the bacteria comprising (or (essentially) consisting of):

[0093] (iii-1) contacting a hydrophobic solvent, in particular a Ce-Ci6 alkane, with the bacteria or lysate thereof and allowing the phytol to at least partly dissolve into the hydrophobic solvent;

[0094] (iii-2) separating the hydrophobic solvent containing phytol from the hydrophilic phase; and

[0095] (iii-3) optionally further isolating phytol from the hydrophobic solvent of step (iii-2), preferably by distillation, by a chromatographic method, or a combination thereof,

[0096] wherein said step (iii-1) may be optionally conducted concomitantly with step (ii) and / or subsequently to step (ii).

[0097] As used herein, the hydrophobic solvent may be any hydrophobic solvent. In a preferred embodiment, the hydrophobic solvent is acceptable for the bacterium and does not prevent its viability. In a preferred embodiment, the hydrophobic solvent is liquid at a temperature in the range of between 20 and 40°C, in particular at a temperature range at which the bacterium is cultivated in step (ii).

[0098] In a preferred embodiment, the hydrophobic solvent has a (decadic) logarithmic octanol / water partition coefficient logP (at 25°C) of at least 2, of at least 3, of at least 4, of at least 5, of at least 6, of at least 7, of at least 8, of at least 9, or of at least 10.

[0099] In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of compounds bearing 6 to 16 carbon atoms, more preferably 7 to 15 carbon atoms, even more preferably 8 to 14 carbon atoms, in particular 10 to 12 carbon atoms. In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of an Ce-Ci6 alkane. In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of an C7-C15 alkane, C8-C14 alkane, C10-C12 alkane. In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of compounds bearing 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 carbon atoms, in particular wherein the hydrophobic solvent is liquid. In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of a Ce-alkane, aCz-alkane, aCs-alkane, aCg-alkane, Cw-alkane, aCn-alkane, aCi2-alkane, aC -alkane, aCn-alkane, a Cis-alkane, or a C -alkane. An alkane may be a linear or branched alkane and may, optionally, be a cycloalkane or may comprise a cycloalkyl residue. In a preferred embodiment, an alkane is a linear alkane (n-alkane). In a preferred embodiment, the hydrophobic solvent comprises or (essentially) consists of n-decane, n-undecane, n-dodecane, n-tridecane, n-tetradecane, white oil, sunflower oil, safflower oil, rape seed oil, isopropyl myristate, or a combination of two or more thereof, in particular is n-dodecane.

[0100] It will be understood that the hydrophobic solvent may also be a combination of two or more types of compounds.

[0101] The step (iii-1) of contacting a hydrophobic solvent with the bacteria or lysate thereof may be conducted at any temperature. In a preferred embodiment, step (iii-1) 0 is conducted at a temperature in the range of 0 to 50°C, at a temperature in the range of 4 to 45°C, at a temperature in the range of 10 to 42°C, at a temperature in the range of 18 to 40°C, at a temperature in the range of 25 to 39°C, at a temperature in the range of 30 to 38°C, at a temperature in the range of 22 to 32°C, at a temperature in the range of 25 to 35°C, at a temperature in the range of 25 to 30°C, at a temperature in the range of 28 to 32°C, or at a temperature in the range of 32 to 37°C, such as at (approximately) 25°C, at (approximately) 26°C, at (approximately) 27°C, at (approximately) 28°C, at (approximately) 29°C, at (approximately) 30°C, at (approximately) 31°C, at (approximately) 32°C, at (approximately) 33°C, at (approximately) 34°C, at (approximately) 35°C, at (approximately) 36°C, at (approximately) 37°C, at (approximately) 38°C, at (approximately) 39°C, at (approximately) 40°C, at (approximately) 41°C, or at (approximately) 42°C.

[0102] The step (iii-1) of contacting a hydrophobic solvent with the bacteria or lysate thereof may be conducted with any volume ratio of bacterial suspension to hydrophobic solvent. In a preferred embodiment, the volume ratio of bacterial suspension : hydrophobic solvent is in the range of between 1000 : 1 and 1 : 1000, more preferably in the range of between 500 : 1 and 1 : 100, even more preferably in the range of between 100 : 1 and 1 : 10, in particular in the range of 100 : 1 and 1 : 1, in the range of 50 : 1 and 2 : 1, or in the range of 20 : 1 and 5 : 1, such as in the range of approximately 10 : 1.

[0103] In a preferred embodiment, the step (iii-1) of contacting a hydrophobic solvent with the bacteria or lysate thereof is contacting the hydrophobic solvent with the bacteria. In a preferred embodiment, the step (iii-1) comprises contacting the hydrophobic solvent with the bacteria suspension as used in step (ii) as a whole.

[0104] In a preferred embodiment, however, the step (iii-1) is conducted concomitantly with step (ii). In this case, the preferred conditions are as described for step (ii) above. The step (iii-1) may optionally be conducted as described in WO2018 / 160066. In a preferred embodiment, the hydrophobic solvent is used as an overlay during conducting step (ii).

[0105] In a preferred embodiment, the method for preparing phytol, comprising (or (essentially) consisting of) the steps: (I) providing bacteria suitable for producing phytol according to the present invention and / or obtainable (or obtained) from a method of the present invention, and optionally propagating the bacteria;

[0106] (ii) cultivating the bacteria of step (I) under conditions that allow expression of the chlorophyll-hydrolyzing enzyme and enzymatically catalyzed cleavage of phytol off the residual chlorophyll and optionally propagating the bacteria and optionally generating chlorophyll; and

[0107] (ill) isolating phytol from the bacteria, including the following steps:

[0108] (iii-1) contacting a hydrophobic solvent, in particular a Ce-Ci6 alkane, with the bacteria during conducting step (ii) and allowing the phytol to at least partly dissolve into the hydrophobic solvent during conducting step (ii);

[0109] (iii-2) separating the hydrophobic solvent containing phytol from the hydrophilic phase; and

[0110] (iii-3) optionally further isolating phytol from the hydrophobic solvent of step (iii-2), preferably by distillation, by a chromatographic method, or a combination thereof. The step (iii-2) of separating the hydrophobic solvent containing phytol from the hydrophilic phase may be conducted by any means such as, e.g., by withdrawing the overlay (e.g., by suction), bay centrifugation, or by other means of phase separation. It will be understood that the step (iii-2) may also be conducted continuously or batchwise in the process while step (ii) is conducted. Optionally steps (iii-1) and (iii-2) may also be conducted continuously or batchwise in the process while step (II) is conducted. Then, the hydrophobic solvent is added during conducting step (ii) and withdrawn during conducting step (ii). Adding and withdrawing may be conducted concomitantly or sequentially and may be conducted continuously or discontinuously. In one embodiment, step (iii-3) including removal of phytol and optional other components from the hydrophobic solvent may optionally also conducted in the withdrawn stream. The remaining hydrophobic solvent may be led back to the bacteria. This may be conducted continuously or discontinuously.

[0111] The step (iii-3) of further isolating phytol from the hydrophobic solvent of step (iii-2) may be conducted by any means. In a preferred embodiment, it is conducted by distillation, by a chromatographic method, or a combination thereof.

[0112] In an alternative embodiment, the step (iii-1) of contacting a hydrophobic solvent with the bacteria or lysate thereof may be conducted subsequent to step (ii). Then, the hydrophobic solvent may be contacted with the bacteria suspension as used in step (ii) as a whole or may further comprise a previous step of separating the bacteria from the medium and optionally lysing the bacteria. Separating the bacteria from the medium may be conducted by any means such as, e.g., by centrifugation or filtration (dead end and / or cross-flow filtration). It will be understood that such step of contacting the hydrophobic solvent with the bacteria separated from the medium or a lysate of the bacteria may also be combined with a previous step of contacting the hydrophobic solvent with the bacteria in the medium optionally during conducting step (ii).

[0113] A further aspect of the present invention relates to phytol obtainable (or obtained) from a method of the present invention or a composition comprising such.

[0114] The obtained phytol may be isolated as pure compound or may be isolated as forming part of a composition further comprising residual contents of other material obtainable (or obtained) by the bacterium and / or its cultivation. In other words, the composition comprising phytol obtainable (or obtained) from a method of the present invention may comprise one or more composition that are typical for the route of preparation. Thus, a composition bearing special characteristics is obtained.

[0115] Accordingly, a further aspect of the present invention relates to a composition comprising phytol obtainable (or obtained) from a method of the present invention, comprising:

[0116] (A) phytol, in particular (2E,7R,11R)-3,7,11, 15-tetramethylhexadec-2-en-1-ol; and

[0117] (B) at least 0.1 ppm, based on the composition as a whole, of one or more other metabolic products of the bacterium in which the phytol is produced, preferably wherein the one or more other metabolic products are selected from the group consisting of farnesol, geranyl geraniol, 14,15-dihydrogeranylgeraniol, 10,11-dihydrogeranylgera- niol , 6,7-dihy d rogerany Igeraniol , 10, 11 , 14, 15-tetrahydrogerany Igeraniol , 6,7, 10, 11 -tetrahydrogeranylgeraniol, 6,7, 14,15-tetrahydrogeranylgeraniol, (bacterio)chlorophyllide, (bacterio)chlorophyll, phytenic acid, phytanol, phytadiene, phytene, phytane, pristene, and pristane; and

[0118] (C) optionally hydrophobic solvent.

[0119] It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol, a method for preparing such, the fusion polypeptide, the vector, and the method for preparing phytol of the present invention mutatis mutandis apply to the composition comprising phytol of the present invention.

[0120] The other metabolic products of the bacterium in which the phytol is produced may be any products obtained from the bacterium as used. For instance, the composition may comprise at least 0.1 ppm, or at least 0.5 ppm, or at least 1 ppm, or at least 5 ppm, or at least 10 ppm, or at least 100 ppm, based on the composition as a whole, of one or more other metabolic products of the bacterium. For instance, the composition may comprise between 0.1 ppm and 10% by weight, between 0.5 ppm and 5% by weight, between 1 ppm and 0.1% by weight, between 5 ppm and 0.05% by weight, or between 10 ppm and 100 ppm of one or more other metabolic products of the bacterium.

[0121] Furthermore, the composition may comprise DNA or fragments thereof and / or RNA or fragments thereof stemming from the bacterium in which phytol is prepared. This may be genomic DNA and / or may be plasmid DNA.

[0122] The hydrophobic solvent may be the hydrophobic solvent as used in step (ii).

[0123] The composition of the present invention may be used for any purpose and may form part of any product in combination with one or more further ingredients.

[0124] Accordingly, a further aspect of the present invention relates to a product comprising the composition of the present invention and one or more further ingredients

[0125] It will be understood that the definitions and embodiments as laid out in the context of the bacterium suitable for producing phytol, a method for preparing such, the fusion polypeptide, the vector, and the method for preparing phytol and the composition comprising phytol of the present invention mutatis mutandis apply to the product comprising the composition of the present invention.

[0126] The product may be any product. In a preferred embodiment, the is selected from the group consisting of a fragrance such as, e.g., as fine fragrance, perfume, Eau de Cologne, Eau de Toilette, an aftershave, or an antiperspirant; another cosmetic and / or body care product such as, e.g., a shower gel, a soap (e.g., bar soap, liquid soap), a shampoo, a conditioner, a hair gel, a hair spray, a cream (e.g., a body cream, a face cream, a day cream, a night cream, an after sun cream, etc.), a cosmetic oil, a lotion (e.g., a body lotion, a face lotion, a hand lotion, optionally sprayable, etc.), a sun protectant (a sun blocker, a sun protecting lotion / cream / spray, etc.), a make-up, a lipstick, a shaving gel / foam / cream, toothpaste, mouthwash, hairspray, etc.),

[0127] a household chemical such as, e.g., general cleaner, toilet cleaner, an acid cleaner, a laundry agent, a dishwashing agent, another detergent (e.g., detergent powder or liquid detergent) or a softener; and

[0128] a scented product such as, e.g., a candle; and

[0129] a consumable good such as, e.g., candy, flavored chocolate, bakery goods.

[0130] The product mixture may contain ingredients at any content ranges of the composition of the present invention. In a preferred embodiment, the product comprises:

[0131] (A) 0.0001 to 99.9 wt.%, preferably 0.001 to 50 wt.%, more preferably 0.01 to 20 wt.%, in particular 0.01 to 10 wt.%, 0.05 to 5 wt.%, or 0.1 to 5 wt.%, as referred to the total mass of the product mixture as a whole, of the composition of the present invention; and

[0132] (B) 0.1 to 99.9999 wt.%, preferably 50 to 99.999 wt.%, more preferably 80 to 99.99 wt.%, in particular 90 to 99.99 wt.%, 95 to 99.95 wt.%, or 95 to 99.9 wt.%, as referred to the total mass of the product mixture as a whole, of one or more other ingredients different from phytol and the other compounds of the composition of the present invention, preferably wherein the one or more other ingredients are pharmaceutically acceptable in the used content ranges.

[0133] The obtained phytol or composition of the present invention may be used for any purpose. For instance, the phytol may be used as a fragrance and / or aroma compound and / or may be used as an educt for preparing vitamin E and / or vitamin K.

[0134] Thus, a further aspect of the present invention relates to the use of phytol, in particular (2E,7R,11 R)-3,7, 11 ,15-tetra-methylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention as a fragrance and / or aroma compound.

[0135] It will be understood that the definitions and embodiments as laid out in the context of the above aspects, in particular the bacterium suitable for producing phytol, a method for preparing phytol and the composition comprising phytol of the present invention, mutatis mutandis apply to the use of such phytol or composition as a fragrance and / or aroma compound.

[0136] In other words, the present invention relates to a method for preparing a fragrance and / or aroma composition, comprising a step of adding phytol, in particular (2E,7R,11 R)-3,7, 11 ,15-tetramethylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention to said composition.

[0137] In a preferred embodiment, when used as a fragrance or aroma ingredient, the phytol, in particular (2E,7R,11R)-3,7,11,15-tetramethylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention is used in combination with one or more other ingredients different from phytol selected from the group consisting of one or more solvents (e.g., water, aqueous solvents, aqueous buffers, organic solvents, or mixtures of two or more thereof), one or more surfactants / detergents, one or more colorants or other staining agents, one or more thickening agents, one or more filling agents, one or more other fragrances than phytol, one or more pharmaceutically active agents, one or more cosmetically active agents (e.g., conditioning agents), one or more chelating agents, one or more cleaning agents other than surfactants / detergents, one or more acid / base regulators, one or more buffers, one or more preserving agents, and mixtures of two or more thereof.

[0138] In a preferred embodiment, when used as a fragrance and / or aroma compound, the phytol, in particular (2E,7R, 11R)-3,7,11,15-tetramethylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention forms part of a product. Preferably such product is selected from the group consisting of:

[0139] a fragrance such as, e.g., as fine fragrance, perfume, Eau de Cologne, Eau de Toilette, an aftershave, or an antiperspirant;

[0140] another cosmetic and / or body care product such as, e.g., a shower gel, a soap (e.g., bar soap, liquid soap), a shampoo, a conditioner, a hair gel, a hair spray, a cream (e.g., a body cream, a face cream, a day cream, a night cream, an after sun cream, etc.), a cosmetic oil, a lotion (e.g., a body lotion, a face lotion, a hand lotion, optionally sprayable, etc.), a sun protectant (a sun blocker, a sun protecting lotion / cream / spray, etc.), a make-up, a lipstick, a shaving gel / foam / cream, toothpaste, mouthwash, hairspray, etc.),

[0141] a household chemical such as, e.g., general cleaner, toilet cleaner, an acid cleaner, a laundry agent, a dishwashing agent, another detergent (e.g., detergent powder or liquid detergent) or a softener; and

[0142] a scented product such as, e.g., a candle; and

[0143] a consumable good such as, e.g., candy, flavored chocolate, bakery goods.

[0144] As indicated above, the phytol, in particular (2E,7R, 11R)-3,7,11,15-tetramethylhexadec-2-en-1-ol, may be used as educt in chemical syntheses. In particular, the phytol, in particular (2E,7R, 11 R)-3,7, 11, 15-tetramethylhexadec-2-en-1-ol, may be used for preparing vitamin E and / or vitamin K and / or pristane and / or phytane.

[0145] Therefore, a further aspect of the present invention relates to the use of phytol, in particular (2E,7R, 11 R)-3,7,11 ,15-tetramethylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention as an educt for preparing vitamin E (also: (alpha-)tocopherol) and / or vitamin K and / or pristane and / or phytane.

[0146] It will be understood that the definitions and embodiments as laid out in the context of the above aspects, in particular the bacterium suitable for producing phytol, a method for preparing phytol and the composition comprising phytol of the present invention, mutatis mutandis apply to the use of such phytol or composition as an educt for preparing vitamin E and / or vitamin K and / or pristane and / or phytane. The person skilled in the art will understand that each product may be in an uncharged or charged form (i.e., as salt) wherever salts technically exist and / or may optionally also comprise conjugates thereof (such as one or more acylated forms, in particular acetylated forms thereof). Accordingly, for instance, vitamin E may optionally also be a vitamin E salt and / or vitamin E acetate or salt thereof. A salt may comprise any counterion. In particular, a counterion may be a pharmaceutically and / or cosmetically acceptable ion. For instance, an ion may be selected from the group consisting of sodium ion, potassium ion, ammonium ion, sulfate ion, phosphate ion, hydrogen phosphate ion, dihydrogen phosphate ion, acetyl ion, a fatty acid, an amino acid ion, and a combination of two or more thereof.

[0147] In other words, the present invention relates to a method for preparing vitamin E and / or vitamin K, comprising a step of conjugating a phytol-derived moiety to a precursor of vitamin E and / or vitamin K, wherein the phytol, in particular (2E,7R, 11 R)-3,7, 11 ,15-tetramethylhexadec-2-en-1-ol, is obtainable (or obtained) from a method of the present invention or forms part of a composition of the present invention to said composition.

[0148] Vitamin E and / or vitamin K may be prepared by any means. There is a variety of possible synthetic routes well known in the art. In a preferred embodiment, vitamin E and / or K may be prepared as described in the review article Bonrath and Netscher (Applied Catalysis A: General, 2005, 280:55-73.). In a preferred embodiment, vitamin E (and analogously vitamin K) may be prepared as described in the review article Kundu and Sarkar (Journal of Heterocyclic Chemistry, 2023, 60:345-368). For instance, these routes involve methods starting from phytol as such, via isophytol and / or via (iso)phytol halide and / or via acetates thereof.

[0149] In another preferred embodiment, vitamin E may be prepared via phytol and / or isophytol and / or phytyl halide and / or isophytyl halide according to US2411967, WC2000 / 001686, WC2004 / 046126, EP0100471, and / or EP06775201.

[0150] For instance, vitamin E or an acetate thereof may be obtained by reacting (2,3,5-)trimethylhydroquinone with phytol (and / or isophytol, which may be obtained from phytol) in the presence of a catalyst (e.g., a Lewis acid catalyst, in particular a zinc halogenide) as obtainable (or obtained) from the present invention as taught in GB1568559A or EP0850937B1. Furthermore, synthetic routes of preparing vitamin E from isophytol is described in Ye et al. ("Revolution of vitamin E production by starting from microbial fermented farnesene to isophytol”, The Innovation, 2022, 3(3): 100228) and in WC2024 / 180228.

[0151] In a preferred embodiment, vitamin E may be prepared according to the method (via isophytol) as described in Raila et al. (J Anim Physiol Anim Nutr., 2024, 108:646-663), in particular the chapter "2.3 Industrial syntheses” thereof. In another preferred embodiment, vitamin E and / or vitamin K (in particular vitamin K1, K2 and / or K3) may be prepared (via isophytol and / or methylnaphtochinon) according to the method as described in Rahman et a. (Global Drugs and Therapeutics, 2017, 2(4):1-6), in particular pages 2 and 3, Schemes 2-4 thereof. In another preferred embodiment, vitamin K (in particular vitamin K1 and / or K2) may be may be prepared (via phytol halogenide and / or isophytol halogenide) according to the method as described in US4229356A and / or US4853156A. A further aspect of the present invention relates to the use of phytol, in particular (2E,7R, 11R)-3,7,11,15-tetra-methylhexadec-2-en-1-ol, obtainable (or obtained) from a method of the present invention or a composition of the present invention as an educt for preparing pristane and / or phytane. This may be achieved by hydrogenation of the phytol (phytane) and decarboxyl aton (pristane). Hydrogenation and decarboxyl aton may be conducted by any means. For example, hydrogenation may be performed by using a hydrogen stream contacted with the phytol in the presence of a Pd / C catalyst. Decarboxylation may be performed by means of tosylation and subsequent hydration with aluminium or boro hydrides in analogy to the route described by Denmark et al. ("Total Synthesis of (+)-Papulacandin D”, Tetrahedron, 2010, 66(26): 4745-4759). In a preferred embodiment, pristane is available from phytol by oxidation, decarboxylation of the resulting phytenic acid, leading to pristene, which finally may be hydrogenated. In a preferred embodiment, phytane is available either by dehydration and hydrogenation or by hydrogenation, dehydration and again hydrogenation. It will be understood that the method of the present invention may also be a method for preparing one or more of the intermediate products such as phytenic acid, pristene, phytanol, phytadiene, and / or phytene are also embraced by the scope of protection.

[0152] Phytol may also be used for preparing isophytol. Thus, a further aspect of the present invention refers to the isomerization of phytol to isophytol. The present invention thus also refers to isophytol as obtainable (or obtained) according to the present invention. Phytol may be isomerized into isophytol by any means known in the art. In one embodiment, the phytol of the present invention is subjected to epoxidation of the double bond, followed by mesylation of the OH group, and subsequent reductive elimination. An example of a feasible synthetic route is described in Wu et al. ("An Efficient Procedure for the 1 ,3-Transposition of Allylic Alcohols Based on Lithium Naphthalenide Induced Reductive Elimination of Epoxy Mesylates”, Synlett, 2008, 4:0621-0623).

[0153] A further aspect of the present invention relates to isophytol thereof obtainable (or obtained) from a method of the present invention.

[0154] A further aspect of the present invention relates to vitamin E or a salt thereof obtainable (or obtained) from a method of the present invention.

[0155] A further aspect of the present invention relates to vitamin K or a salt thereof obtainable (or obtained) from a method of the present invention.

[0156] A further aspect of the present invention relates to phytane obtainable (or obtained) from a method of the present invention.

[0157] A further aspect of the present invention relates to pristane obtainable (or obtained) from a method of the present invention. A further aspect of the present invention relates to phytenic acid obtainable (or obtained) from a method of the present invention.

[0158] A further aspect of the present invention relates to phytene obtainable (or obtained) from a method of the present invention.

[0159] A further aspect of the present invention relates to pristene obtainable (or obtained) from a method of the present invention.

[0160] A further aspect of the present invention relates to phytanol obtainable (or obtained) from a method of the present invention.

[0161] A further aspect of the present invention relates to phytadiene obtainable (or obtained) from a method of the present invention.

[0162] It will be understood that the definitions and preferred embodiments as defined in the context of the methods above mutatis mutandis apply to the products or salts thereof obtainable (or obtained) from a method of the present invention. Brief description of the Figures

[0163] Figure 1 shows the experimentally exemplified plasmid vector (pBBR MCS AtCLDI) expressing a chlorophyll-hydro-lyzing enzyme (chlorophyll dephytylase, AtCLDI) as a fusion polypeptide fused with a fusion polypeptide (experimentally exemplified as maltose binding protein (MBP)). The vector comprises an origin of replication (rep) and a kanamy-cin-expressing site (kan) usable for selecting transfected bacterial cells.

[0164] Figure 2 shows a gas chromatography (GC) chromatogram of n-dodecane hydrophobic phases obtained from Rhodo-bacterspaeroides bacteria transfected with (A) an empty vector (pBBR MCS) and (B) a vector expressing a chlorophyllhydrolyzing enzyme (chlorophyll dephytylase, pBBR MCS AtCLDI) as a fusion polypeptide fused with another polypeptide (experimentally exemplified as maltose binding protein (MBP)). It can be seen that the expression of chlorophyll-hydrolyzing enzyme significantly increases the content of obtained phytol.

[0165] Example

[0166] Phototrophic bacterium transfected with a chlorophyll-hydrolyzing enzyme

[0167] Preparation of bacteria expressing a chlorophyll-hydrolyzing enzyme:

[0168] The phototrophic bacterium Rhodobacter sphaeroides , which inherently comprises bacteriochlorophyll, was transfected with a plasmid pBBR-MBP-AtCLD1, which is according to the scheme as depicted in Figure 1. As depicted in Figure 1, the plasmid further comprises a promoter sequence active in Rhodobacter (including Rhodobacter sphaeroides). This plasmid is obtained from inserting the DNA sequence of SEQ ID NO: 2 into a commercially available plasmid pBBR-MCS. The pBBR-MBP-AtCLD1 plasmid and an empty pBBR-MCS plasmid were introduced in E. coli strain S17-1 by standard E coli transformation methodology and selected on LB with 100 pig / ml Neomycin. The phototrophic bacterium Rhodobacter sphaeroides was transfected with the plasmid as obtained in the previous step. The transfection using methods disclosed in the international patent application WO2011 / 074954 (see pages 64 to 67 thereof). Thus, transformation of E. coli S17-1 with plasmids and subsequent transfer of plasmids from S17-1 to Rhodobacter sphaeroides by conjugation were performed using standard procedures (Nishimura et al., A rapid and highly efficient method for preparation of competent Escherichia coli cells, Nucl. Acids Res., 1990, 18:6169). Rhodobacter sphaeroides recipient strain was grown in RA-medium. The composition and preparation of medium RA is summarized in WO2011 / 074954, Table 3. thereof. In parallel, E. coli S17-1 donor strain that carries the plasmid to be transferred was grown in LB-broth containing the appropriate antibiotic. For the conjugation, 450 l culture aliquots of the Rhodobacter sphaeroides recipient strain and of the E. coli S17-1 donor strain were mixed together, and then pelleted by centrifugation. The supernatant was discarded. Cells were washed twice with fresh RA-medium to remove the antibiotics, and then resuspended in 0.05 mL fresh RA-medium and spotted onto a PY-plate. The composition and preparation of medium PY is summarized in WO2011 / 074954, Table 4. thereof. After 4 to 5 hours incubation at 30°C, the cells were harvested with an inoculating loop and resuspended in 0.3 mL of RA-medium. Dilutions of this suspension were spread onto RA-plates containing the appropriate antibiotic and incubated at 30°C for 2 to 3 days. Colonies were picked from the plates, streaked onto RS102-plates (i.e., plates containing RS102 medium as described in WC2018 / 160066) containing the appropriate antibiotic, and incubated at 30°C for 2 to 3 days to obtain single colonies. One single colony from each clone (putatively transformed cells of Rhodobacter sphaeroides) was again grown in liquid RS102 medium (i.e., as described in WC2018 / 160066) containing the appropriate antibiotic and the presence of the expected plasmid was confirmed by PCR using appropriate primers. The final transformants were partly preserved by adding glycerol to the culture (15% v / v) and freezing at -80°C.The transformed bacteria were selected using plates with Ra medium and 100 pig / ml Neomycin. Rhodobacter sphaeroides colonies were selected which harbored the plasmid pBBR-MBP-AtCLD1, or pBBR-MCS. Resulting strains were named after their plasmids. In summary, the phototrophic bacterium Rhodobacter sphaeroides, when transfected with the plasmid expresses a fusion polypeptide of a mutant form of chlorophyll dephytylase CDL1 (AtCLDI) of SEQ ID NO: 1 fused with another polypeptide (exemplified as maltose binding protein of SEQ ID NO: 5 (MBP-AtCLD1 )). The obtained bacteria strain was designated as Rs-pBBR-MBP-AtCLD1.

[0169] Preparation of phytol:

[0170] Bacteria of strain Rs-pBBR-MBP-AtCLD1 as expressing the AtCLDI and control Rs-pBBR-MCS were cultivated in 20 ml RS102 medium, using a hydrophobic solvent (2 ml n-dodecane) as an overlay, basically as described in WQ2018 / 160066. The n-dodecane layer was harvested after 72 hours of cultivation and was analyzed by gas chro-matography-mass spectrometry (GC-MS) basically as described in WQ2018 / 160066. The GC-MS analysis was performed on an Agilent Technologies system, comprising a 7980A GC system, a 597C inert MSD detector (70 eV), a 7683 auto-sampler and injector and a Phenomenex Zebron ZB-5ms column of 30 m length x 0.25 mm internal diameter and 0.25 m stationary phase, with a Guardian precolumn (5 m). In this system, 1 pl of the sample of n-dodec-ane containing hydrophobic compounds such as phytol was injected. The injection chamber was at 250°C, the injection was splitless, and the ZB5 column was maintained at 45°C for 2 min after which a gradient of 10°C per minute was started, until 300°C. Peaks were detected in chromatograms of the total ion count. Compounds were identified by their retention index and by their mass spectrum in combination with comparison of the mass spectrum to libraries (NIST8 and in-house).

[0171] Results:

[0172] In gas chromatograph (GC), the n-dodecane hydrophobic phase of negative control of culture Rs-pBBR-MCS produced a minor peak at 14.7 min (see Figure 2A). The n-dodecane hydrophobic phase obtained from culture Rs-pBBR-MBP-AtCLD1 (expressing the chlorophyll-hydrolyzing enzyme AtCLDI) displayed a much higher peak at 14.7 min (see Figure 2B). Comparison of the mass spectrum and retention time of the peak eluting at 14.7 min and the mass spectrum and retention time of a standard of phytol (Sigma), revealed that this peak is representing phytol. A quantitative analysis revealed that 700 mg phytol per kg n-dodecane was produced by strain Rs265-pBBR-MBP-AtCLDI, while the amount of phytol in strain Rs pBBR-MCS was lower than 50 mg / kg.

[0173] In summary, it was surprisingly found that expressing a chlorophyll-hydrolyzing enzyme (exemplified chlorophyll dephytylase, AtCLDI) in a bacterium containing chlorophyll (exemplified as Rhodobacter sphaeroides) is suitable for produce significant amounts of phytol.

Claims

CLAIMS1. A bacterium suitable for producing phytol, in particular (2E,7R,11 R)-3,7,11 ,15-tetramethylhexadec-2-en-1-ol, wherein the bacterium is characterized in that it:(a) contains chlorophyll, in particular bacteriochlorophyll; and(b) expresses a heterologous and / or overexpressed chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll.

2. The bacterium of any of claim 1 , wherein the bacterium is of a phototrophic bacterial species inherently containing bacteriochlorophyll, preferably wherein the bacterium is of the phylum Pseudomonadota, more preferably of the class Alphaproteobacterial, even more preferably of the order Rhodobacterales, even more preferably of the family Rhodobacteraceae, in particular of the genus Rhodobacter.

3. The bacterium of any of claims 1 or 2, wherein the bacterium is Rhodobacter sphaeroides or Rhodobacter capsulatus.

4. The bacterium of any of claims 1 to 3, wherein the expressed chlorophyll-hydrolyzing enzyme is a chlorophyll dephytylase or a pheophytin pheophorbide hydrolase,preferably is a chlorophyll dephytylase,in particular comprises or consists of a polypeptide sequence having sequence identity of at least 80% of SEQ ID NO: 1.

5. The bacterium of any of claims 1 to 4, wherein the chlorophyll-hydrolyzing enzyme forms part of a fusion polypeptide with one or more other polypeptides, preferably with one or more other polypeptides enhancing the expression of the chlorophyll-hydrolyzing enzyme and / or its stability.

6. The bacterium of any of claims 1 to 5, wherein:the bacterium is a phototrophic bacterium inherently containing bacteriochlorophyll, in particular of the genus Rhodobacter, andthe expressed chlorophyll-hydrolyzing enzyme is a chlorophyll dephytylase, preferably comprising a polypeptide sequence that has a sequence identity of at least 80% of SEQ ID NO: 1.

7. The bacterium of any of claims 1 to 6, wherein it is further characterized in that:(I) geranylgeranyl pyrophosphate synthase is overexpressed; and / or(II) one or more enzymes of the mevalonate pathway are overexpressed; and / or(III) one or more genes for the 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate pathway are expressed, and / or(IV) one or more genes of the tetrapyrrole pathway are overexpressed, and / or(V) geranyl geranyl reductase (GGR) is overexpressed, and / or(VI) phytoene synthase is downregulated or deleted.

8. A fusion polypeptide comprising:(b1) a polypeptide sequence having a sequence identity of at least 80% of SEQ ID NO: 1; fused to(b2) another polypeptide having at least 20 consecutive amino acid moieties that improves cellular localization.

9. A vector comprising:(v1) at least one promoter suitable for promoting expression of expression in a bacterium, in particular a phototrophic bacterium containing chlorophyll, preferably located upstream in the vector; and(v2) a polynucleotide sequence encoding for a chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, in particular having a sequence identity of at least 80% of SEQ ID NO: 1; and (v3) one or more polynucleotide sequences different from (v2) encoding for one or more polypeptide sequences forming a fusion polypeptide with the chlorophyll-hydrolyzing enzyme when expressed; and(v4) optionally an expression-modifying sequence, in particular an expression enhancer or a sequence that enables controlling and / or silencing expression; and(v5) optionally one or more polynucleotide sequences facilitating an antibiotic resistance suitable for selection of a transfected bacterium; and(v6) optionally an origin of replication,preferably wherein the vector is a circular plasmid,in particular wherein said vector comprises a polynucleotide sequence having a sequence identity of at least 80% of SEQ ID NO: 2.

10. A method for preparing a bacterium suitable for producing phytol according to any of claims 1 to 7, comprising the step of transfecting a bacterium, preferably a bacterium containing bacteriochlorophyll, with a gene, in particular a gene forming part of a vector according to claim 9, encoding for a chlorophyll-hydrolyzing enzyme that catalyzes cleaving phytol off the residual chlorophyll, andoptionally selecting the bacterium suitable for producing phytol, and optionally propagating the bacterium.

11. A method for preparing phytol, comprising the steps:(i) providing bacteria suitable for producing phytol according to any of claims 1 to 7 and / or obtainable from a method of claim 10, and optionally propagating the bacteria;(ii) cultivating the bacteria of step (i) under conditions that allow expression of the chlorophyll-hydrolyzing enzyme and enzymatically catalyzed cleavage of phytol off the residual chlorophyll and optionally propagating the bacteria; and(iii) optionally isolating phytol from the bacteria.

12. The method of claim 11 , wherein the method comprises step (iii) of isolating phytol from the bacteria comprising:(iii-1) contacting a hydrophobic solvent, in particular a Ce-Ci6 alkane, with the bacteria or lysate thereof and allowing the phytol to at least partly dissolve into the hydrophobic solvent;(iii-2) separating the hydrophobic solvent containing phytol from the hydrophilic phase; and(iii-3) optionally further isolating phytol from the hydrophobic solvent of step (iii-2), preferably by distillation, by a chromatographic method, or a combination thereof,wherein said step (iii-1) may be optionally conducted concomitantly with step (II) and / or subsequently to step (II).

13. A composition comprising phytol obtainable from a method of any of claims 11 or 12, comprising:(A) phytol, in particular (2E,7R,11R)-3,7,11, 15-tetramethylhexadec-2-en-1-ol; and(B) at least 0.1 ppm, based on the composition as a whole, of one or more other metabolic products of the bacterium in which the phytol is produced, preferably wherein the one or more other metabolic products are selected from the group consisting of farnesol, geranyl geraniol, 14, 15-dihydrogeranylgeraniol, 10,11 -dihydrogeranylgeraniol, 6, 7-di hydrogerany Igeraniol , 10, 11 , 14, 15-tetrahydrogerany Igeraniol, 6,7, 10, 11 -tetrahydrogeranylgeraniol, 6,7,14, 15-tetrahydrogeranylgeraniol, (bacterio)chlorophyllide, (bacterio)chlorophyll, phytenic acid, phytanol, phytadiene, phytene, phytane, pristene, and pristane; and(C) optionally hydrophobic solvent.

14. Use of phytol, in particular (2E,7R,11 R)-3,7,11 ,15-tetramethylhexadec-2-en-1-ol, obtainable from a method of any of claims 11 or 12 or a composition of claim 13 as a fragrance and / or aroma compound.

15. Use of phytol, in particular (2E,7R,11 R)-3,7,11 ,15-tetramethylhexadec-2-en-1-ol, obtainable from a method of any of claims 11 or 12 or a composition of claim 13 as an educt for preparing vitamin E and / or vitamin K and / or isophytol and / or pristane and / or phytane.

Images