Recombinant host cells producing iridone and uses thereof

Abstract

The present invention relates to recombinant host cells genetically modified to produce irisone compounds. The invention also relates to a method for producing an irisone compound by using the recombinant host cell.

Description

Technical Field

[0001] The present invention relates to the field of production of bio-based compounds, in particular the production of irone compounds using recombinant host cells. Background Art

[0002] Plant natural products (PNPs) are non-nutritive, bioactive compounds that are widely used as pharmaceuticals, chemicals, flavoring agents, and fragrances. These compounds are extracted from plant biomass, and their supply requires a large investment in terms of land, water, and time, and is often limited. In addition, PNPs are often present in plants at low abundance, which makes them inadequate for research and market needs. Some PNPs can be chemically synthesized, however, obtaining structurally complex, enantiomerically pure, bioactive PNPs by large-scale synthesis is often a challenge.

[0003] Recent developments in synthetic biology and metabolic engineering have made it possible to produce PNPs in fast-growing and fermentable microorganisms. This strategy is one of the most promising alternatives for obtaining high-value molecules, bypassing the problem of limited and unstable supply of plant biomass. To this end, all or part of the plant biosynthetic pathway is transferred to an engineered microbial chassis such as Saccharomyces cerevisiae or Escherichia coli, enabling heterologous production of the final PNP or its biosynthetic intermediates for downstream chemical synthesis. Successful examples using this approach include the production of semi-synthetic artemisinin (Paddon et al., Nat. Rev. Microbiol. 12 (2014) 355–367), rose oil (Kutyna et al., Genes 2018, 9(7), 326) and resveratrol (Li et al., Scientific Reports, 2016, volume 6, article number: 36827). However, in many cases, the natural biosynthetic pathways in plants remain completely or partially unknown. Therefore, heterologous production requires the de novo design of new pathways or the use of alternative enzymes to compensate for the missing steps.

[0004] Iris oil is one of the most expensive natural products used in perfumery, costing more than 140,000 euros / kg. Despite the high price, global production of iris oil is declining due to long production cycles, low yields and insufficient incentives for farmers. Irisone is the active compound of iris oil, responsible for its violet-like smell and powdery notes. Five stereoisomers / enantiomers of iris oil occur naturally in fresh iris extracts in different proportions. Cis-α-, β- and γ-irone are present in most cases, while trans-α- and γ-irone are rare. The smell and price of iris oil are determined by the ratios between the different irises.

[0005] Due to the presence of two chiral centers, the chemical synthesis of single enantiomers of irone is challenging and has been rarely reported. ® It is mainly composed of diastereomers of α-irone, with a small amount of β-irone. The synthesis involves the acid-catalyzed cyclization of methyl-3-pseudoionone. ® Initially, a biocatalyst-assisted procedure was developed that afforded all ten isomers of irone (Brenna et al., Helvetica Chimica Acta, 2001, 84(12):3650-3666). However, this process is lengthy and requires lipase-mediated resolution of several racemic mixtures and separation of diastereomeric products, making it unsuitable for industrial production. A recent study reported a challenging de novo enantioselective synthesis of (-)-(2S,6R)-cis-α- and (-)-(2S,6R)-cis-γ-irone (Bugoni et al., Chemistry, 2015 Jan 7;21(2):791-9). The difficulties in chemical synthesis have highlighted the interest in developing procedures for the production of irone through synthetic biology. Summary of the invention

[0006] The inventors herein demonstrate that the irone biosynthetic pathway can be expressed in recombinant microbial host cells to produce α-irone and / or β-irone.

[0007] Thus, in a first aspect, the present invention relates to a method for producing an irone compound, the method comprising culturing a recombinant microbial host cell comprising a heterologous nucleic acid encoding a lycopene cyclase that catalyzes the β- or ɛ-cyclization of one or both termini of 2-methyl-lycopene and / or the β- or ɛ-cyclization of one or both termini of 2,2'-dimethyl-lycopene under conditions suitable for the production of the irone compound, and optionally recovering the irone compound.

[0008] The lycopene cyclase may be selected from wild-type cyanobacterial CrtL-type lycopene ɛ-cyclase, wild-type bacterial CrtY-type lycopene β-cyclase and wild-type heterodimeric lycopene cyclase of Gram-positive bacteria and variants thereof, said variant exhibiting lycopene cyclase activity and having at least 70% sequence identity with any of these cyclases. Preferably, the lycopene cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 10, 8, 9, 11, 1, 2 and 3 and variants thereof, said variant exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of SEQ ID NOs: 10, 8, 9, 11, 1, 2 and 3.

[0009] In particular, the lycopene cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8 to 11. Preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 10.

[0010] Alternatively, the lycopene cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 3 and variants thereof, which exhibit lycopene β-cyclase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3.

[0011] Preferably, the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase. Specifically, the GPP C6-methyltransferase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 17 to 31 and variants thereof, wherein the variant exhibits GPP C6-methyltransferase activity and has at least 70% sequence identity to SEQ ID NOs: 17 to 31.

[0012] The recombinant microbial host cell may further comprise a heterologous nucleic acid encoding a farnesyl diphosphate (FPP) synthase and / or a heterologous nucleic acid encoding a geranylgeranyl diphosphate (GGPP) synthase and / or a heterologous nucleic acid encoding a polypeptide exhibiting FPP synthase and GGPP synthase activity; and / or a heterologous nucleic acid encoding a phytoene synthase; and / or a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase; and / or a heterologous nucleic acid encoding a carotenoid-cleaving dioxygenase.

[0013] The recombinant microbial host may have been genetically modified to reduce the activity of an endogenous PgpB phosphatase, an endogenous CpdB phosphatase, and / or an endogenous MetJ repressor protein compared to an unmodified microbial host cell.

[0014] Preferably, the recombinant microbial host is a bacterium or a yeast.

[0015] In a second aspect, the present invention also relates to a recombinant microbial host as defined above.

[0016] In another aspect, the present invention also relates to use of the recombinant microbial host cell of the present invention for producing an irone compound. BRIEF DESCRIPTION OF THE DRAWINGS

[0017] Figure 1 : Design pathway for isomeric irones. For clarity, only carotenoids with all-E configuration are shown.

[0018] Figure 2 :Production C 31 -Engineering pathways for carotenoids.

[0019] Figure 3 : Production of methylated C by expressing bezA in Escherichia coli 30 Carotenoids. A) Schematic representation of the genetic constructs used. B) Shown is 4,4'-diapo-lycopene (1, M + ° m / z at 400.3121), 4,4'-diapoprotin (2, M + ° m / z at 402.277), 2- or 2'-methyl-4,4'-diapoprotin (3, M + ° m / z at 416.3435) and 2- or 2'-methyl-4,4'-diapto- -Carotene (4, M + ° Extracted ion chromatogram (EIC) of the molecular ion of the molecule with m / z 418.3591. C) The generated C 30 and C 31 The structure of carotenoids.

[0020] Figure 4 : Production of methylated C by expressing bezA in Escherichia coli 40 Carotenoids. A) Schematic representation of the genetic constructs used. B) Lycopene (5, M + ° m / z at 536.4377), 2- or 2'-methyl-lycopene (6, M + ° m / z at 550.4533) and 2,2'-dimethyl-lycopene (7, M + ° EIC of the molecular ion with m / z at 564.4690).

[0021] Figure 5: Production of carotene with irone using CrtY-β and CrtL-ɛ cyclases in Escherichia coli with a methyl-lycopene pathway. A) Schematic representation of the genetic constructs used. The corresponding cyclase genes were cloned together with bezA to generate a series of plasmids pL2k-Bsn2-CXyz (X and yz describe the cyclase family and strain, respectively). For the cyclase CrtY PA -β, the plasmid pAC-BETA (Cunningham et al., Plant Cell. 8 (1996) 1613–1626) was used directly. B) shows M + ° EICs of molecular ions at m / z 536.4377, 550.4533 and 564.4690. They correspond to lycopene (5, C40H56) and carotene (8 or 11, C40H56), 2- / 2'-methyl lycopene (6, C41H58) and 2- / 2'-methyl-carotene (9 or 12, C41H58) and 2,2'-dimethyl-lycopene (7, C42H56) and 2,2'-dimethyl-carotene (10 or 13, C42H56). The annotations of the peaks were inferred from the tandem MS analysis. C) Expected carotenoid structures. Only structures with the E configuration are shown.

[0022] Figure 6 : Through CCD1 OF Cleavage produces irone in vitro. The EIC of the [M+H]+ ion at m / z 207.1743 (corresponding to irone) is shown. The irone standard from Sigma is a racemic mixture of trans- and cis-α-irone. BezAsn and CXyz describe the recombinant strain from which the extracts used were derived. The corresponding genetic constructs are shown in Figure 5A middle.

[0023] Figure 7: A proposed biosynthetic pathway for cycloirials in Iris plants, yielding various irones via unknown oxidation processes (Belcour et al., Phytochemistry. 34 (1993) 1313–1315; Ritzdorf et al., Phytochemistry. 50 (1999) 995–1003).

[0024] Figure 8 : Chemical structures of (+)-cis-α-irone, (-)-cis-α-irone, (+)-trans-α-irone, (-)-trans-α-irone, (+)-β-irone and (-)-β-irone. DETAILED DESCRIPTION

[0025] The pathway for irone biogenesis in Iris is only partially known ( Figure 7 ) (Marner et al., Helv. Chim. Acta. 71 (1988) 1331–1338). In fact, irones are absent in fresh rhizomes. During the senescence of rhizomes, they are converted from a C 31 Terpene precursors are slowly generated via an elusive oxidation mechanism. Cycloiral biosynthesis originates from the squalene pathway, although most of the enzymes involved remain unknown (Marner et al., supra).

[0026] As shown in the experimental section of this application, the inventors herein demonstrate that the irone biosynthetic pathway can be expressed in microbial host cells (e.g., Escherichia coli) leading to the production of α-irone and β-irone. Specifically, they demonstrate that lycopene β-cyclase or lycopene ɛ-cyclase can be used to cyclize monomethyl and dimethyl lycopene and produce the irone motif. They also show that microbial host cells such as Escherichia coli expressing 6-GPP methyltransferase can produce non-canonical C 11 The building block 6meGPP, which can be accepted by downstream carotenoid enzymes (including CrtE, CrtB and CrtI enzymes) to produce monomethyl and dimethyl lycopene. After the irone motif is generated using lycopene β-cyclase or lycopene ɛ-cyclase, regardless of the configuration of the irone motif, the last step of the designed irone pathway requires cleavage of the double bond between C9-C10 and C9'-C10' of the demethylated carotene. Therefore, the inventors demonstrated the applicability of carotenoid cleavage enzymes (CCDs) in cleaving non-natural methylated carotenes to produce irones, thereby producing α-irone and / or β-irone.

[0027] definition

[0028] In the context of the present invention, the term "recombinant host cell" refers to a cell not found in nature, which contains a genome modified by the deletion, insertion or modification of one or more genetic elements. The term "host cell" also encompasses any offspring of a parent host cell that differs from the parent host cell due to mutations that occur during replication. Preferably, the host cell is a microbial host cell. The term "microbial host cell" as used herein refers to bacteria, filamentous fungi or yeast, preferably bacteria or yeast.

[0029] "Recombinant nucleic acid" or "recombinant nucleic acid molecule" refers to a nucleic acid (e.g., a DNA, cDNA, or RNA molecule) that has been engineered and does not originally occur in nature. Typically, the term refers to a nucleic acid molecule that contains segments that have been generated and / or joined together using recombinant DNA techniques (e.g., molecular cloning and nucleic acid amplification). A recombinant nucleic acid molecule contains one or more non-naturally occurring sequences and / or contains joined nucleic acid molecules that are from different original sources and are not naturally attached together.

[0030] The term "gene" refers to any nucleic acid encoding a protein. The term encompasses DNA such as cDNA or gDNA and RNA. A gene can be first prepared, for example, by recombinant, enzymatic and / or chemical techniques and then replicated in a host cell or in vitro system. A gene typically comprises an open reading frame encoding a desired protein. A gene may contain additional sequences, such as a transcription terminator or a signal peptide.

[0031] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence such that the control sequence directs the expression of the coding sequence.

[0032] The term "control sequence" means a nucleic acid sequence necessary for gene expression. The control sequence may be native or heterologous. Control sequences known to those skilled in the art and currently used are preferred. Such control sequences include, but are not limited to, leader sequences, polyadenylation sequences, propeptide sequences, promoters, signal peptide sequences, ribosome binding sites, and transcription terminators. Preferably, the control sequence includes a promoter and a transcription terminator.

[0033] The term "expression cassette" refers to a nucleic acid construct comprising an operably linked coding region (i.e., one or several genes) and a regulatory region (i.e., comprising one or more control sequences). Optionally, an expression cassette may comprise several coding regions operably linked to several regulatory regions. Specifically, an expression cassette may comprise several coding sequences, each of which is operably linked to the same promoter. Alternatively, an expression cassette may comprise one or several coding sequences, each of which is operably linked to different promoters. An expression cassette may also comprise one or several coding sequences, each of which is operably linked to different promoters, and one or several other coding sequences operably linked to a common promoter.

[0034] The term "expression vector" as used herein means a DNA or RNA molecule comprising an expression cassette. Preferably, the expression vector is a linear or circular double-stranded DNA molecule. The vector may also contain a replication origin, a selection marker, etc.

[0035] As used herein, the term "native" or "endogenous" with respect to a host cell refers to a genetic element or protein that is naturally present in the host cell.

[0036] The term "heterologous" refers to a genetic element or protein that is not naturally present in the host cell for a host cell. The origin of the genetic element or protein may be different from the cell in which it is introduced. However, the genetic element or protein may also be derived from the same species as the cell in which it is introduced, but due to its non-natural environment, it is considered to be heterologous. For example, a genetic element such as a gene is heterologous because it is under the control of a promoter other than its natural promoter, and it is introduced into a position different from its natural position. Before the introduction of a heterologous genetic element or protein, the host cell may contain a copy of an endogenous genetic element or protein, or it may not contain an endogenous copy. In addition, the genetic element or protein may be heterologous in the sense that the coding sequence has been optimized for expression in the host cell. Codon optimization can be performed by conventional methods known in the art (see, for example, Welch, M. et al., (2011), Methods in Enzymology 498: 43-66). Preferably, in this document, a heterologous nucleic acid sequence encodes a protein that is heterologous to the host cell, i.e., is not naturally present in the host cell. In particular, the term may refer to a genetic element or protein provided from a cell of a different species or a different genus than the host cell, more preferably a different genus than the host cell.

[0037] The terms "peptide," "oligopeptide," "polypeptide," and "protein" are used interchangeably and refer to a chain of amino acids linked by peptide bonds, regardless of the number of amino acids forming the chain.

[0038] As used herein, the term "wild-type protein" refers to the non-mutated version of a polypeptide as it occurs naturally in a species.

[0039] The term "variant" as used herein refers to a polypeptide derived from a wild-type protein and comprising changes (i.e., substitutions, insertions and / or deletions) at one or more (e.g., several) positions. The term "deletion" when used in association with a position or an amino acid means that the amino acid in the particular position has been deleted or does not exist. The term "insertion" when used in association with a position or an amino acid means that one or more amino acids have been inserted or exist adjacent to and immediately following the amino acid occupying the particular position. The term "replacement" as used herein means that the amino acid in a particular position has been replaced by another amino acid, or there are amino acids different from those of the wild-type protein. The variant can be obtained by various techniques known in the art. Specifically, examples of techniques for changing the DNA sequence encoding the wild-type protein include, but are not limited to, site-directed mutagenesis, random mutagenesis, and synthetic oligonucleotide construction.

[0040] The terms "sequence identity" or "identity" as used herein refer to the number (%) of position matches (same amino acid residues) from an alignment of two polypeptide sequences. Sequence identity is determined by comparing the sequences while aligning them to maximize overlap and identity while minimizing sequence gaps. Specifically, sequence identity can be determined using any of a variety of mathematical global or local alignment algorithms depending on the length of the two sequences. Sequences of similar length are preferably aligned using a global alignment algorithm (e.g., the Needleman and Wunsch algorithm; Needleman and Wunsch, 1970, J. Mol. Biol 48:443) that optimally aligns the sequences over their entire length, while sequences of significantly different lengths are preferably aligned using a local alignment algorithm (e.g., the Smith and Waterman algorithm (Smith and Waterman, Adv. Appl. Math. 2:482, 1981) or the Altschul algorithm (Altschul et al., 1997, Nucleic Acids Res. 25:3389-3402; Altschul et al., 2005, FEBS J. 272:5101-5109). Alignment for determining percent amino acid sequence identity can be accomplished in various ways within the skill of the art, such as using publicly available computer software on Internet sites such as http: / / blast.ncbi.nlm.nih.gov / or http: / / www.ebi.ac.uk / Tools / emboss / . One skilled in the art can determine suitable parameters for measuring alignment, including any algorithm required to achieve maximum alignment over the full length of the compared sequences. Preferably, for purposes herein, % values ​​for amino acid sequence identity refer to the percentage of amino acid sequence identity calculated using BLAST (Basic Local Alignment Search Tool). The value generated by the method, wherein all search parameters are set to default values. In some specific embodiments, all sequence identities (particularly variant sequence identities) are the same and are set to at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99%. In some more specific embodiments, all sequence identities (particularly variant sequence identities) are the same and are set to at least 80% sequence identity. In some other specific embodiments, all sequence identities (particularly variant sequence identities) are the same and are set to at least 90% or at least 95% sequence identity. In some embodiments, relative to the wild-type sequence or the sequence described in the SEQ ID NO, the variant may include 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additions, substitutions or deletions. Specifically, these additions, substitutions or deletions can be introduced at the N-terminal end, the C-terminal end or both ends.The variant may optionally take the form of a fusion protein.

[0041] As used herein, the terms "overexpression" and "increased expression" are used interchangeably and mean that the expression of a gene or enzyme is increased relative to an unmodified microorganism, such as a wild-type microorganism or a microorganism that does not contain a genetic modification described herein. The term "wild type" refers to an unmodified microorganism present in nature. Increased expression of an enzyme is generally obtained by increasing the expression of a gene encoding the enzyme. In embodiments where the gene or enzyme does not naturally exist in the host cell of the present invention (i.e., a heterologous gene or enzyme), the terms "overexpression" and "expression" are used interchangeably. In order to increase the expression of a gene, those skilled in the art may use any known technique, such as increasing the number of copies of the gene in the microorganism, using a promoter (i.e., a strong promoter) that induces high-level expression of the gene, using an element that stabilizes the corresponding messenger RNA, or a sequence that isolates a ribosome binding site (RBS) and its surrounding sequences. Specifically, overexpression can be obtained by increasing the number of copies of the gene in the microorganism. One or more copies of the gene may be introduced into the genome by recombination methods known to those skilled in the art, including replacement or multi-copy integration of the gene. Preferably, an expression cassette comprising a gene preferably placed under the control of a strong promoter is integrated into the genome. As an alternative, the gene can be carried by an expression vector, preferably a plasmid, which comprises an expression cassette, wherein the gene of interest is preferably placed under the control of a strong promoter. The expression vector can be present in the microorganism with one or more copies, depending on the nature of the replication origin. The overexpression of the gene can also be obtained by using a promoter that induces the high-level expression of the gene. For example, the promoter of the endogenous gene can be replaced with a stronger promoter, i.e., a promoter that induces a higher level of expression. The endogenous gene under the control of the promoter as a non-natural promoter is referred to as a heterologous nucleic acid. Promoters suitable for use in the present invention are well known to those skilled in the art, can be constitutive or inducible, and can be endogenous or heterologous.

[0042] As used herein, the term "irone" or "irone compound" refers to a compound selected from α-irone, β-irone and γ-irone. Preferably, the term refers to a compound selected from α-irone and β-irone. As used herein, the term "α-irone" refers to a compound selected from (+)-cis-α-irone, (-)-cis-α-irone, (+)-trans-α-irone, (-)-trans-α-irone, and mixtures thereof. As used herein, the term "β-irone" refers to a compound selected from (+)-β-irone and (-)-β-irone, and mixtures thereof. These compounds are shown in Figure 8 middle.

[0043] Recombinant microbial host cells

[0044] The present inventors have demonstrated herein that lycopene β-cyclase or lycopene ɛ-cyclase can be used to cyclize monomethyl- and dimethyl-lycopene and produce irone motifs in recombinant microbial host cells, particularly Escherichia coli, thereby paving the way for the production of bio-based irone compounds.

[0045] Thus, in a first aspect, the present invention relates to a recombinant microbial host cell comprising a heterologous nucleic acid encoding a lycopene cyclase, which catalyzes the production of 2-methyl-carotene from 2-methyl-lycopene and / or the production of 2,2'-dimethyl-carotene from 2,2'-dimethyl-lycopene.

[0046] The microbial host cell of the present invention may be a eukaryotic microorganism selected from yeast and filamentous fungi, or may be a prokaryotic microorganism.

[0047] In one embodiment, the microbial host cell is a yeast, preferably selected from yeasts of the order Saccharomyces, the order Saccharomyces and the order Schizosaccharomyces. Specifically, the yeast can be selected from yeasts of the genus Pichia, the genus Kluyveromyces, the genus Saccharomyces, the genus Schizosaccharomyces, the genus Candida, the genus Lipomyces, the genus Rhodotorula, the genus Rhodosporidium, the genus Yarrowia or the genus Debaryomyces. Preferably, the yeast is selected from Pichia pastoris, Kluyveromyces lactis, Kluyveromyces marxianus, Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis, Saccharomyces oviformis, Schizosaccharomyces pombe, Candida albicans, Candida tropicalis, Rhodotorula glutinis, Rhodosporidium More preferably, the yeast is a yeast of the genus Saccharomyces, preferably Saccharomyces cerevisiae.

[0048] In another embodiment, the microbial host cell is a filamentous fungus, i.e. a fungus grown in the form of mycelium of mycelium. The feature of filamentous fungi is generally the mycelial wall composed of chitin, cellulose, glucan, chitosan, mannan and other complex polysaccharides. The filamentous fungus is preferably selected from the fungi of Acremonium, Aspergillus, Short-stalked Mould, Tomopidium, Pseudomonas, Chrysosporium, Coprinus, Versicolor, Cryptococcus, Endocytosis, Filibasidium, Fusarium, Humicola, Giant Seat Shell, Mucor, Myceliophthora, Neobeta, Neurospora, Penicillium, Penicillium, Original Hairy Phanerochaete, Radius, Pyriphage, Pyriphage, Pyriphage, Pleurotus, Podospora, Pyriphage, Schizophyllum, Talaromyces, Thermoascus, Thielavia, Toxoplasma, Trametes and Trichoderma. Preferably, the filamentous fungus is selected from the genus Aspergillus, Neurospora or Trichoderma. Specifically, the filamentous fungus can be selected from Aspergillus nidulans, Aspergillus niger, Aspergillus awomari, Aspergillus oryzae, Aspergillus terreus, Neurosporacrassa, Trichoderma reesei and Trichoderma viride.

[0049] In another embodiment, the microbial host cell is a prokaryotic organism, preferably a bacterium. Specifically, the bacterium can be selected from the group consisting of Acidobacteria, Actinobacteria, Aquabacteria, Bacteroidetes, Chlamydia, Chlorobacteria, Chloroflexi, Aureobacteria, Cyanobacteria, Deferrobacillaceae, Deinococcus-Thermus, Digitococcus, Fibrobacter, Firmicutes, Fusobacteria, Gemmatimonads, Nitrospira, Planctomycetes, Proteobacteria, Spirochaetes, Thermodesulfobacteria, Thermomicrobacteria, Thermococcidiales, or Verrucomicrobia. Preferably, the bacteria belongs to Acaryochloris, Acetobacter, Actinobacillus, Agrobacterium, Alicyclobacillus, Anabaena, Anastrozole, Anaerospira, Liquid-producing Bacteria, Arthrobacter, Arthrospira, Azotobacter, Bacillus, Brevibacterium, Burkholderia, Chlorobacter, Chromobacterium, Chlorobacter, Clostridium, Corynebacterium, Cupricobacter, Cyanobacterium, Enterobacter, Deinococcus, Erwinia, Escherichia, Geobacter, Gloeobacter, Gluconobacter, Hydrogenobacter, Klebsiella, Lactobacillus, Lactococcus, Mannheimia, Mesorhizobium, Methylobacterium, Microbacterium, Microcystis, Nitrobacter, Nitrosomonas, Nitrospinosus, Nitrospira, Nostoc, Pseudomonas, Ralstonia, Rhizobium, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus, Streptomyces, Synechococcus, Synechocystis, Thermosynechococcus, Trichoderma or Zymomonas. More preferably, the bacterium is selected from Agrobacterium tumefaciens, Anaerobiospirillumsucciniciproducens, Actinobacillus succinogenes, Aquifex aeolicus, Aquifex pyrophilus, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium acetobutylicum, Clostridium beijerinckii, Clostridium tumefaciens ...beigerinckii、Corynebacterium glutamicum、Cupriavidus necator、Cupriavidus metallidurans、Enterobacter sakazakii、Escherichia coli、Gluconobacter oxydans、Hydrogenobacter thermophilus、Klebsiella oxytoca、Lactococcus lactis、Lactobacillus plantarum、Mannheimia succiniciproducens、Mesorhizobium loti、Pseudomonas aeruginosa、Pseudomonas mevalonii、Pseudomonas pudica, Pseudomonas putida, Pseudomonas fluorescens, Rhizobium etli, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Streptomyces coelicolor, Zymomonas mobilis, Acaryochloris marina, Anabaenavariabilis, Arthrospira platensis, Arthrospira maxima, Chlorobiumtepidum, Chlorobaculum sp., Cyanothecesp., Gloeobacter violaceus, Microcystis aeruginosa, Nostoc punctiforme, Prochlorococcus marinus, Synechococcus elongatus, Synechocystis sp., Thermosynechococcuselongatus, Trichodesmium erythraeum and Rhodopseudomonas palustris. In a specific embodiment, the microorganism is a bacterium that does not belong to the genus Streptomyces. In a preferred embodiment, the microorganism is an Escherichia coli bacterium.

[0050] In a specific embodiment, the microbial host cell is selected from yeast, filamentous fungi and bacteria not belonging to the genus Streptomyces. Preferably, the microbial host cell is a yeast, a filamentous fungus or an Escherichia coli bacterium.

[0051] Lycopene cyclase activity

[0052] The recombinant microbial host cell of the present invention comprises a heterologous nucleic acid encoding a lycopene cyclase, which catalyzes the β- or ɛ-cyclization of one or both ends of 2-methyl-lycopene and / or the β- or ɛ-cyclization of one or both ends of 2,2'-dimethyl-lycopene.

[0053] The term "lycopene cyclase" as used herein refers to an enzyme exhibiting lycopene β-cyclase activity (EC 5.5.1.19) and / or lycopene ɛ-cyclase activity (EC 5.5.1.18), i.e., an enzyme that catalyzes the β-cyclization of one terminus of lycopene to form γ-carotene, the β-cyclization of both termini of lycopene to produce β-carotene, the ɛ-cyclization of one terminus of lycopene to form δ-carotene and / or the ɛ-cyclization of both termini of lycopene to produce ɛ-carotene. In the context of the present invention, the term "lycopene cyclase activity" refers to the β- or ɛ-cyclization of one or both ends of 2-methyl-lycopene, preferably of both ends of 2-methyl-lycopene, to give 2-methyl-carotene, or the β- or ɛ-cyclization of one or both ends of 2,2'-dimethyl-lycopene, preferably of both ends of 2,2'-dimethyl-lycopene, to give 2,2'-dimethyl-carotene. The lycopene cyclase activity can be assessed by any method known to the skilled person. For example, this activity can be assessed by co-expressing the lycopene cyclase gene together with the crtE, crtB and crtI genes in a host cell such as Escherichia coli and coupling subsequent pigment analysis. The pigment is extracted and analyzed by LC and / or MS analysis coupled with UV / Vis spectroscopic detection. Detection of the β- or ɛ-cyclization product of 2-methyl-lycopene (preferably 2-methyl-carotene) and / or the β- or ɛ-cyclization product of 2,2'-dimethyl-lycopene (preferably 2,2'-dimethyl-carotene) indicates that the tested enzyme exhibits lycopene cyclase activity.

[0054] Lycopene cyclases can be divided into four categories: bacterial CrtY-type lycopene β-cyclases, which are specifically found in many carotenoid-producing Proteobacteria (e.g., Misawa et al., J Bacteriol, 172, 6704–6712, 1990; Matsumura et al., Gene 189, 169–174, 1997), Streptomyces (Krugel et al., Biochim Biophys Acta 1439, 57–64, 1999), and Chloroflexi species; CrtL-type lycopene cyclases, which include β- and ε-cyclases in certain cyanobacteria, algae, and plants (Cunningham et al., Plant Cell 6, 1107–1121, 1994; Stickforth et al., Arch Microbiol 179, 409–415, 1998); 2003); heterodimeric cyclases (CrtYc / CrtYd) from some Gram-positive bacteria (Krubasik et al., Mol Gen Genet 263, 423–432, 2000; Viveiros et al., FEMS Microbiol Lett 187, 95–101, 2000), monomeric cyclases CrtYc-Yd from archaea, and fused bifunctional cyclases CrtYB from fungi (Hemmi et al., 2003. Biochem. Biophys. Res. Commun., 35, 586–591; Takaichi et al., 2011. Mar. Drugs, 9, 1101–1118); and the CruA / CruP family of lycopene cyclases, which are found in green sulfur bacteria and cyanobacteria that do not have CrtY or CrtL cyclases (Takaichi et al., 2013, “Tetraterpenes: carotenoids”, in Natural Products, edited by Merillon, JM and Ramawat, KG, Springer, Berlin pp. 3251–3283).

[0055] Examples of lycopene cyclases include, but are not limited to, those from Brevibacterium linens (CrtY BL -β; heterodimer CrtYc / CrtYd, Uniprot accession number of CrtYc: Q9KK78, SEQ ID NO: 1, Uniprot accession number of CrtYd: Q9KK79, SEQ ID NO: 2), Pantoea agglomerans (CrtYPA -β, Uniprot accession number: K7WHX6, SEQ ID NO: 3), Porphyra umbilicalis (GenBank accession number: QHA79699.1, SEQ ID NO: 4), Synechococcus elongatus (Uniprot accession number: Q55276, SEQ ID NO: 5), and yeast Phaffia rhodozyma (Uniprot accession number: Q7Z859, SEQ ID NO: 6), Vulcanococcus limneticus (CrtL VL -ɛ, NCBI reference sequence: WP_094588021, SEQ ID NO: 8), Synechococcus sp. BS55DK (CrtL SB -ɛ, NCBI reference sequence: WP_131594774, SEQ ID NO: 9), Cyanobium sp. CACIAM 14 (CrtL CC -ɛ, NCBI GenBank accession number: KEF41217, SEQ ID NO: 10), Prochlorococcus sp. HOT208 (CrtL PH -ɛ, NCBI reference sequence: WP_079293938, SEQ ID NO: 11), Porphyra umbilicalis (GenBank accession number: QHA79700.1, SEQ ID NO: 12), Oryza sativa (NCBI reference sequence: XP_015622198.1, SEQ ID NO: 13), Zea mays (Uniprot accession number: B7S825, SEQ ID NO: 14), Chromochloris zofingiensis (Uniprot accession number: J7Q2X9, SEQ ID NO: 15), and lycopene cyclase from Haematococcus lacustris (GenBank accession number: AKT95178.1, SEQ ID NO: 16). Other lycopene cyclases can be readily identified using known databases or any sequence alignment software applied to the lycopene cyclases listed above.

[0056] In particular, the lycopene cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 6 and 8 to 16 and variants thereof, said variant exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6 and 8 to 16. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 1 to 6 and 8 to 16 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0057] Preferably, the lycopene cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variant exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3 and 8 to 11. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 1 to 3 and 8 to 11 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0058] In one embodiment, the recombinant microbial host cell of the present invention comprises a heterologous nucleic acid encoding a lycopene β-cyclase.

[0059] As used herein, the term "lycopene β-cyclase" refers to an enzyme that catalyzes the β-cyclization of one end of lycopene to form γ-carotene or the β-cyclization of both ends of lycopene to produce β-carotene (EC 5.5.1.19). In the context of the present invention, the term "lycopene β-cyclase activity" refers to the β-cyclization of one or both ends of 2-methyl-lycopene, preferably both ends of 2-methyl-lycopene, to give 2-methyl-β-carotene, or the β-cyclization of one or both ends of 2,2'-dimethyl-lycopene, preferably both ends of 2,2'-dimethyl-lycopene, to give 2,2'-dimethyl-β-carotene. Lycopene β-cyclase activity can be assessed by any method known to the skilled person. For example, such activity can be assessed by co-expressing a lycopene cyclase gene together with crtE, crtB and crtI genes in a host cell such as Escherichia coli and coupling it to a subsequent pigment analysis. The pigment is extracted and analyzed by LC and / or MS analysis coupled with UV / Vis spectroscopic detection. Detection of β-cyclization products of 2-methyl-lycopene (preferably 2-methyl-β-carotene) and / or β-cyclization products of 2,2'-dimethyl-lycopene (preferably 2,2'-dimethyl-β-carotene) indicates that the tested enzyme exhibits lycopene β-cyclase activity.

[0060] Examples of lycopene β-cyclases include, but are not limited to, those from Brevibacterium linens (CrtY BL -β; heterodimer CrtYc / CrtYd, SEQ ID NO: 1 and CrtYd SEQ ID NO: 2), Pantoea agglomerans (CrtY PA -β, SEQ ID NO: 3), Porphyra umbilicalis (SEQ ID NO: 4), Synechococcus elongatus (SEQ ID NO: 5), and the yeast Phaffia rhodozyma (SEQ ID NO: 6). Other lycopene β-cyclases can be easily identified using known databases or any sequence alignment software applied to the lycopene β-cyclases listed above.

[0061] In particular, the lycopene β-cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 1 to 6 and variants thereof, said variant exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 1 to 6 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0062] In a preferred embodiment, the lycopene cyclase is a lycopene β-cyclase selected from the group consisting of a wild-type bacterial CrtY-type lycopene β-cyclase and a wild-type heterodimeric lycopene β-cyclase of a Gram-positive bacterium, and variants thereof, said variant exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases.

[0063] Examples of bacterial CrtY-type lycopene β-cyclases include, but are not limited to, Pantoea agglomerans (CrtY PA -β, SEQ ID NO: 3).

[0064] Examples of heterodimeric lycopene cyclases of Gram-positive bacteria include, but are not limited to, lycopene cyclases from Brevibacterium linens (SEQ ID NOs: 1 and 2).

[0065] In a specific embodiment, the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 1 to 3 and variants thereof, said variant exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 1 to 3 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0066] In a more specific embodiment, the lycopene cyclase comprises or consists of the following amino acid sequences: (i) an amino acid sequence selected from SEQ ID NO: 3 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 3, and (ii) a heterodimeric lycopene cyclase comprising a first subunit and a second subunit, said first subunit comprising or consisting of an amino acid sequence selected from SEQ ID NO: 1 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 1, and a second subunit comprising or consisting of an amino acid sequence selected from SEQ ID NO: 2 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: ID NO: 2 has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity.

[0067] In another embodiment, the recombinant microbial host cell of the present invention comprises a heterologous nucleic acid encoding a lycopene ɛ-cyclase.

[0068] As used herein, the term "lycopene ɛ-cyclase" refers to an enzyme that catalyzes the ɛ-cyclization of one end of lycopene to form δ-carotene or the ɛ-cyclization of both ends of lycopene to produce ɛ-carotene (EC 5.5.1.18). In the context of the present invention, the term "lycopene ɛ-cyclase activity" refers to the ɛ-cyclization of one or both ends of 2-methyl-lycopene, preferably both ends of 2-methyl-lycopene, to give 2-methyl-α-carotene, or the ɛ-cyclization of one or both ends of 2,2'-dimethyl-lycopene, preferably both ends of 2,2'-dimethyl-lycopene, to give 2,2'-dimethyl-α-carotene. The lycopene ɛ-cyclase activity can be assessed by any method known to the skilled person. For example, this activity can be assessed by co-expressing the lycopene cyclase gene together with the crtE, crtB and crtI genes in a host cell such as Escherichia coli and coupling it to a subsequent pigment analysis. The pigment is extracted and analyzed by LC and / or MS analysis coupled to UV / Vis spectroscopic detection. The detection of ɛ-cyclization products of 2-methyl-lycopene (preferably 2-methyl-α-carotene) and / or ɛ-cyclization products of 2,2'-dimethyl-lycopene (preferably 2,2'-dimethyl-α-carotene) indicates that the enzyme tested exhibits lycopene ɛ-cyclase activity. Some lycopene ɛ-cyclases may further exhibit lycopene β-cyclase activity as defined above, and in particular may produce 2-methyl-β-carotene and / or 2,2'-dimethyl-β-carotene, preferably 2,2'-dimethyl-β-carotene.

[0069] Examples of lycopene ɛ-cyclases include, but are not limited to, those from Vulcanococcus limneticus (CrtL VL -ɛ, SEQ ID NO: 8), Synechococcus sp. BS55DK (CrtL SB -ɛ, SEQ ID NO: 9), Cyanobium sp. CACIAM 14 (CrtL CC -ɛ, NCBI SEQ ID NO: 10), Prochlorococcus sp. HOT208 (CrtL PH-ɛ, SEQ ID NO: 11), Porphyraumbilicalis (SEQ ID NO: 12), Oryza sativa (SEQ ID NO: 13), Zea mays (SEQ ID NO: 14), Chromochloris zofingiensis (SEQ ID NO: 15) and Haematococcus lacustris (SEQ ID NO: 16). Other lycopene ɛ-cyclases can be easily identified using known databases or any sequence alignment software applied to the lycopene ɛ-cyclases listed above.

[0070] In particular, the lycopene ɛ-cyclase may comprise or consist of an amino acid sequence selected from SEQ ID NOs: 8 to 16 and variants thereof, said variant exhibiting lycopene cyclase activity, preferably lycopene ɛ-cyclase activity and optionally lycopene β-cyclase activity, and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8 to 16. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 8 to 16 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0071] In a preferred embodiment, the lycopene cyclase is a lycopene ɛ-cyclase selected from the wild-type cyanobacterial CrtL-type ɛ-cyclase and variants thereof, said variants exhibiting lycopene cyclase activity, preferably lycopene ɛ-cyclase activity and optionally lycopene β-cyclase activity, and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases.

[0072] Examples of cyanobacterial CrtL-type lycopene ɛ-cyclases include, but are not limited to, those from Vulcanococcus limneticus (CrtL VL -ɛ, SEQ ID NO: 8), Synechococcus sp. BS55DK (CrtL SB -ɛ, SEQ ID NO: 9), Cyanobium sp. CACIAM 14 (CrtL CC-ɛ, NCBI SEQ ID NO: 10) and Prochlorococcus sp. HOT208 (CrtL PH -ɛ, SEQ ID NO: 11).

[0073] In a specific embodiment, the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 8 to 11 and variants thereof, said variant exhibiting lycopene cyclase activity, preferably lycopene ɛ-cyclase activity and optionally lycopene β-cyclase activity, and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8 to 11. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 8 to 11 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0074] In a specific embodiment, the irone compound is β-irone, and the lycopene cyclase is selected from the group consisting of lycopene β-cyclase and lycopene ɛ-cyclase exhibiting lycopene β-cyclase activity, preferably selected from the group consisting of wild-type bacterial CrtY-type lycopene β-cyclase, wild-type heterodimeric lycopene β-cyclase of Gram-positive bacteria and wild-type cyanobacterial CrtL-type lycopene ɛ-cyclase exhibiting lycopene β-cyclase activity, and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases. Preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, 8 and 9 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6, 8 and 9. More preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 8 and 9 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3, 8 and 9. In particular, the variant may comprise a sequence that differs from the sequence set forth in any one of SEQ ID NOs: 1 to 6, 8 and 9, preferably the sequence set forth in any one of SEQ ID NOs: 1 to 3, 8 and 9 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions, or consists of said sequence.

[0075] In another specific embodiment, the irone compound is α-irone, and the lycopene cyclase is selected from the group consisting of lycopene ɛ-cyclase, preferably selected from the group consisting of wild-type cyanobacterial CrtL-type lycopene ɛ-cyclase and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases. Preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 16 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of SEQ ID NOs: 8 to 16. More preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8 to 11. Even more preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 10 and 11. In particular, the variant may comprise a sequence that differs from a sequence set forth in any one of SEQ ID NOs: 8 to 16, preferably a sequence set forth in any one of SEQ ID NOs: 8 to 11, more preferably a sequence set forth in any one of SEQ ID NOs: 10 and 11 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions, or consists of said sequence.

[0076] In another specific embodiment, the irone compound is cis-α-irone, and the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, the variant exhibits lycopene ɛ-cyclase activity and has at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 10. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in SEQ ID NO: 10 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0077] The recombinant microbial host cell of the present invention may naturally express the lycopene cyclase. In this case, the recombinant host cell may be genetically modified to overexpress this protein or further express a heterologous lycopene cyclase. In embodiments where the recombinant microbial host cell does not naturally express the lycopene cyclase, the host cell is genetically modified to express the heterologous lycopene cyclase. In a preferred embodiment, the host cell is genetically modified to express a heterologous lycopene cyclase.

[0078] Production of non-standard components

[0079] The recombinant microbial host cells of the present invention are capable of producing the non-canonical building block 14-methylgeranylgeranyl diphosphate (14-meGGPP), which can be accepted by downstream carotenoid enzymes, including CrtB and CrtI enzymes, to produce monomethyl- and dimethyl-lycopene.

[0080] In a preferred embodiment, the recombinant microbial host cell of the present invention exhibits geranyl pyrophosphate (GPP) C6-methyltransferase activity. The geranyl pyrophosphate (GPP) C6-methyltransferase catalyzes the methylation of GPP to obtain 6-methylgeranyl diphosphate (6-meGPP); 6-meGPP is then converted by farnesyl diphosphate synthase and CrtE enzymes to produce 14-meGGPP.

[0081] As used herein, the term "geranyl pyrophosphate C6-methyltransferase", "geranyl pyrophosphate (GPP) C6-methyltransferase" or "GPP C6-methyltransferase" refers to an enzyme that catalyzes the S-adenosylmethionine (SAM)-dependent methylation of geranyl diphosphate (GPP) to give 6-methylgeranyl diphosphate (6-meGPP). GPP C6-methyltransferase activity can be assessed by any method known to the skilled person. For example, such activity can be assessed by incubating a putative GPP C6-methyltransferase with GPP, SAM and Mg. 2+ Incubate and assess the production of 6-meGPP using any suitable method, such as liquid chromatography coupled to electrospray ionization mass spectrometry (LC-ESI-MS) in negative mode (see, e.g., Lee et al., 2011. Mass Spectrometry Letters, 2(4), 92-95). The position of the methyl group can be confirmed by derivatization of me-GPP (pyrophosphorylation followed by epoxidation of the double bond) and coupled use of LC-tandem MS analysis (see, e.g., Tsutsumi et al., Angew Chem Int Ed Engl. 2022 Jan 3;61(1):e202111217).

[0082] The GPP C6-methyltransferase may be selected from known GPP C6-methyltransferases and variants thereof, which exhibit GPP C6-methyltransferase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the known GPP C6-methyltransferases.

[0083] Examples of GPP C6-methyltransferases include, but are not limited to, the BezA enzyme from the benzastatin biosynthetic pathway of Streptomyces niveus (SEQ ID NO: 17), BezA from Streptomyces sp. RI18 (SEQ ID NO: 18, Tsutsumi et al., Angew Chem Int Ed Engl. 2022 Jan 3;61(1):e202111217, J. Am. Chem. Soc. 2018, 140, 21, 6631–6639), from Streptomyces sp. 4R-3d (SEQ ID NO: 19), Streptomyces ipomoeae (SEQ ID NO: 20), Streptomyces malaysiensis (SEQ ID NO: 21), Streptomyces sp. (SEQ ID NO: 22), Streptomyces antimycoticus (SEQ ID NO: 23), Streptomyces antioxidans (SEQ ID NO: 24), Streptomyces sp. 150FB (SEQ ID NO: 25), Streptomyces caeruleatus (SEQ ID NO: 26), Streptomyces adelaidensis (SEQ ID NO: 27), Streptomyces kasugaensis (SEQ ID NO: 28), Streptomyces acidiscabies (SEQ ID NO: 29), Lentzea indica (SEQ ID NO: 30), and homologs of BezA of Saccharothrix espanaensis (SEQ ID NO: 31).

[0084] Preferably, the GPP C6-methyltransferase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 17 to 31 and variants thereof, which exhibit GPP C6-methyltransferase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 17 to 31. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 1 to 15 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0085] More preferably, the GPP C6-methyltransferase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variant exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17. In particular, the variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 17 or 18, preferably SEQ ID NO: 1 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0086] In a preferred embodiment, the substitutions, insertions and / or deletions of the variant of the GPP C6-methyltransferase do not affect the residues of the enzyme known to be important for catalysis, phosphate binding and substrate specificity, in particular the residues of the enzyme corresponding to Y28, K31, K38, Y47, E170, Y174, W210, Y213 F214, S277 or N281 of SEQ ID NO: 18. The residues in the sequence of the GPPC6-methyltransferase corresponding to Y28, K31, K38, Y47, E170, Y174, W210, Y213 F214, S277 or N281 of SEQ ID NO: 18 can be easily identified by conventional sequence alignment techniques.

[0087] The recombinant microbial host cell of the present invention may naturally express GPP C6-methyltransferase, for example when the host cell is a Streptomyces bacterium. In this case, the recombinant host cell may be genetically modified to overexpress endogenous GPP C6-methyltransferase or further express heterologous GPP C6-methyltransferase. In a preferred embodiment, in particular when the recombinant microbial host cell does not naturally express GPP C6-methyltransferase, the host cell is genetically modified to express heterologous GPP C6-methyltransferase.

[0088] In certain embodiments, the recombinant microbial host cell comprises

[0089] - a heterologous nucleic acid encoding a lycopene cyclase, preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and 8 to 16 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6 and 8 to 16, more preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3 and 8 to 11;

[0090] - A heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase, preferably a GPP C6-methyltransferase comprising or consisting of SEQ ID NOs: 17 to 31 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17 to 31, more preferably a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17.

[0091] In a preferred embodiment, the recombinant microbial host cell comprises

[0092] - a heterologous nucleic acid encoding a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 1 to 3 and 8 to 11; and

[0093] - A heterologous nucleic acid encoding a GPP C6-methyltransferase comprising or consisting of an amino acid sequence of SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17.

[0094] In embodiments wherein the recombinant microbial host cell exhibits geranyl pyrophosphate (GPP) C6-methyltransferase activity, the host cell preferably further exhibits farnesyl diphosphate synthase activity and geranylgeranyl diphosphate synthase activity, allowing for the production of 14-methylgeranylgeranyl diphosphate (14-meGGPP) from 6-meGPP.

[0095] As used herein, the term "farnesyl diphosphate synthase," "FPP synthase," or "IspA" refers to an enzyme (EC 2.5.1.10, EC 2.5.1.1) that catalyzes the sequential condensation of isopentenyl pyrophosphate (IPP) with allyl pyrophosphate, dimethylallyl pyrophosphate, and then the resulting geranyl pyrophosphate (GPP) to form the final product, farnesyl pyrophosphate (FPP). The term also refers to an enzyme that catalyzes the condensation of IPP and 6-meGPP to produce 10-methylfarnesyl pyrophosphate (10-meFPP). FPP synthase activity can be assessed by any method known to those skilled in the art. For example, such activity can be assessed by incubating a purified FPP synthase with IPP and GPP in the presence of Mg 2+ The presence of β-lactamase can be assessed by incubation, and the generated FPP can be analyzed by LC-ESI-MS analysis in negative mode (see, e.g., Lee et al., Mass Spectrometry Letters, 2011, 2(4), 92-95).

[0096] The recombinant microbial host cells of the present invention may naturally express a polypeptide that exhibits FPP synthase activity. In this case, the recombinant host cell may optionally be genetically modified to overexpress such a polypeptide or further express a heterologous polypeptide that exhibits FPP synthase activity. In embodiments where the recombinant microbial host cell does not naturally express a polypeptide that exhibits FPP synthase activity, the host cell is genetically modified to express a heterologous polypeptide that exhibits FPP synthase activity.

[0097] As used herein, the term "geranylgeranyl diphosphate synthase," "GGPP synthase," or "CrtE" refers to an enzyme (EC 2.5.1.29) that catalyzes the condensation of FPP and IPP to produce geranylgeranyl diphosphate (GGPP). The term also refers to an enzyme that catalyzes the condensation of IPP and 10-meFPP to produce 14-meGGPP. GGPP synthase activity can be assessed by any method known to those skilled in the art. For example, such activity can be assessed by incubating a purified GGPP synthase with IPP and FPP in the presence of Mg 2+ The presence of β-catenin was evaluated by incubation, and the generated GGPP can be detected by LC-ESI-MS analysis in negative mode (see, e.g., Lee et al., Mass Spectrometry Letters, 2011, 2(4), 92-95).

[0098] The recombinant microbial host cells of the present invention may naturally express a polypeptide that exhibits GGPP synthase activity. In this case, the recombinant host cell may optionally be genetically modified to express such a polypeptide or further express a heterologous polypeptide that exhibits GGPP synthase activity. In embodiments where the recombinant microbial host cell does not naturally express a polypeptide that exhibits GGPP synthase activity, the host cell is genetically modified to express a heterologous polypeptide that exhibits GGPP synthase activity.

[0099] The FPP synthase activity and GGPP synthase activity of the recombinant microbial host cell of the present invention can be provided by two different enzymes (i.e., FPP synthase and GGPP synthase), or by only one enzyme that exhibits these two activities. Therefore, the recombinant microbial host cell of the present invention may contain (i) a nucleic acid encoding an FPP synthase and / or a nucleic acid encoding a GGPP synthase, and / or (ii) a nucleic acid encoding a polypeptide exhibiting FPP synthase and GGPP synthase activities.

[0100] In a preferred embodiment, the recombinant microbial host cell comprises a heterologous nucleic acid encoding a GGPP synthase and, optionally, a heterologous nucleic acid encoding a FPP synthase.

[0101] The FPP synthase can be selected from known FPP synthases and variants thereof, which exhibit FPP synthase activity and have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% with any of the known FPP synthases.

[0102] Examples of FPP synthases include, but are not limited to, those from Escherichia coli (gene: ispA, Uniprot Accession No.: P22939, SEQ ID NO: 32), Bacillus subtilis (Uniprot Accession No.: P54383, SEQ ID NO: 33), Pantoea agglomerans (GenBank Accession No.: UJQ23241.1, SEQ ID NO: 34), Saccharomyces cerevisiae (Uniprot Accession No.: P08524, SEQ ID NO: 35), Geobacillus stearothermophilus (Uniprot Accession No.: Q08291, SEQ ID NO: 36), Gallus gallus (Uniprot Accession No.: P08836, SEQ ID NO: 37), and Artemisia tridentata (Uniprot Accession No.: Q7XYS9, SEQ ID NO: 38). Other FPP synthases can be readily identified using known databases or any sequence alignment software applied to the FPP synthases listed above.

[0103] Preferably, the FPP synthase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 32 to 38 and variants thereof, which exhibit FPP synthase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 32 to 38. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 32 to 38 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0104] More preferably, the FPP synthase comprises or consists of an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, the variant exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 32. In particular, the variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 32 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0105] The GGPP synthase can be selected from known GGPP synthases and variants thereof, which exhibit GGPP synthase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the known GGPP synthases.

[0106] Examples of GGPP synthases include, but are not limited to, those from Pantoea agglomerans (Uniprot Accession No.: K7WMD5, SEQ ID NO: 39), Saccharomyces cerevisiae (Uniprot Accession No.: Q12051, SEQ ID NO: 40), Deinococcus radiodurans (NCBI Accession No.: WP_010888034.1, SEQ ID NO: 41), Synechococcus sp. (Unitprot Accession No.: B1XJV9, SEQ ID NO: 42), and Rhodobacter capsulatus (Unitprot Accession No.: P17060, SEQ ID NO: 43). Other GGPP synthases can be readily identified using known databases or any sequence alignment software applied to the GGPP synthases listed above.

[0107] Preferably, the GGPP synthase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 39 to 43 and variants thereof, which exhibit GGPP synthase activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 39 to 43. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 39 to 43 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0108] More preferably, the GGPP synthase comprises or consists of an amino acid sequence selected from SEQ ID NO: 39 and variants thereof, the variant exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 39. In particular, the variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 39 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0109] Examples of polypeptides exhibiting FPP synthase and GGPP synthase activity include, but are not limited to, IdsA from Methanobacterium thermoautotrophicum (Uniprot Accession No.: O26156, SEQ ID NO: 44) and FPP / GGPP synthase from Pyrococcus furiosus (Uniprot Accession No.: Q8U1V3, SEQ ID NO: 45). Other polypeptides exhibiting FPP synthase and GGPP synthase activity can be easily identified using known databases or any sequence alignment software.

[0110] Preferably, the polypeptide exhibiting FPP synthase and GGPP synthase activity comprises or consists of an amino acid sequence selected from SEQ ID NOs: 44 to 45 and variants thereof, said variant exhibiting FPP synthase and GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 44 to 45. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 44 to 45 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0111] In certain embodiments, the recombinant microbial host cell comprises

[0112] - a heterologous nucleic acid encoding a lycopene cyclase, preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and 8 to 16 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6 and 8 to 16, more preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3 and 8 to 11;

[0113] - a heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase, preferably a GPP C6-methyltransferase comprising or consisting of SEQ ID NOs: 17 to 31 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17 to 31, more preferably a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17;

[0114] - A heterologous nucleic acid encoding a GGPP synthase, preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 43 and variants thereof, said variant exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 39 to 43, more preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 and variants thereof, said variant exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 39.

[0115] Optionally, the recombinant microbial host cell may further comprise a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 38 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 32 to 38, more preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32.

[0116] In a preferred embodiment, the recombinant microbial host cell comprises

[0117] - a heterologous nucleic acid encoding a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 1 to 3 and 8 to 11; and

[0118] - a heterologous nucleic acid encoding a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17; and

[0119] - A heterologous nucleic acid encoding a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 39 and variants thereof, said variants exhibiting GGPP synthase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 39.

[0120] Optionally, the recombinant microbial host cell may further comprise a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, wherein the variants exhibit FPP synthase activity and have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 32.

[0121] Phytoene synthase activity and phytoene desaturase activity

[0122] The recombinant microbial host cell of the present invention preferably further exhibits phytoene synthase activity and phytoene desaturase activity, allowing for the production of 2-methyl-lycopene and / or 2,2'-dimethyl-lycopene, preferably 2-methyl-lycopene and 2,2'-dimethyl-lycopene, from 14-meGGPP.

[0123] As used herein, the term "phytoene synthase" or "CrtB" refers to an enzyme that catalyzes the condensation of two geranylgeranyl diphosphate (GGPP) molecules to produce phytoene (EC 2.5.1.32). This term also refers to an enzyme that catalyzes the condensation of two 14-meGGPP molecules or one 14-meGGPP and one GGPP molecule to produce 2,2'-dimethyl-phytoene or 2-methyl-phytoene. Phytoene synthase activity can be assessed by any method known to the skilled person. For example, such activity can be assessed by a bacterial complementation assay in Escherichia coli, in which a putative crtB is co-expressed with a plasmid encoding all enzymes except phytoene synthase to produce carotene (see, e.g., Cunningham et al., Photosynth Res, 2007, 92, 245–259). The function of CrtB can be detected by a color change of the Escherichia coli colonies to yellow. The production of carotene can then be detected by LC-MS and / or UV / Vis spectroscopy.

[0124] The recombinant microbial host cells of the present invention may naturally express phytoene synthase. In this case, the recombinant host cell may optionally be genetically modified to overexpress this protein or further express a heterologous phytoene synthase. In embodiments where the recombinant microbial host cell does not naturally express phytoene synthase, the host cell is genetically modified to express a heterologous phytoene synthase.

[0125] As used herein, the term "phytoene desaturase", "CrtI" refers to an enzyme that catalyzes four desaturation steps to convert phytoene to lycopene (EC 1.3.99.31). This term also refers to an enzyme that catalyzes two desaturation steps to convert 2-methyl-phytoene and 2, 2'-dimethyl-phytoene to 2-methyl-lycopene and 2,2'-dimethyl-lycopene, respectively. Phytoene desaturase activity can be assessed by any method known to the skilled person. For example, such activity can be assessed by a bacterial complementation assay in Escherichia coli, wherein a putative crtI is co-expressed with a plasmid encoding all enzymes except the phytoene desaturase to produce carotene (see, e.g., Cunningham et al., Photosynth Res, 2007, 92, 245-259). The function of CrtI can be detected by the color change of the Escherichia coli colonies to light yellow. The production of carotene can then be detected by LC-MS and / or UV / Vis spectroscopy.

[0126] The recombinant microbial host cells of the present invention may naturally express a phytoene desaturase. In this case, the recombinant host cell may optionally be genetically modified to overexpress this protein or further express a heterologous phytoene desaturase. In embodiments where the recombinant microbial host cell does not naturally express a phytoene desaturase, the host cell is genetically modified to express a heterologous phytoene desaturase.

[0127] In one embodiment, a recombinant microbial host cell of the invention comprises a heterologous nucleic acid encoding a phytoene synthase.

[0128] In another embodiment, the recombinant microbial host cell of the present invention comprises a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase.

[0129] In a preferred embodiment, the recombinant microbial host cell of the present invention comprises a heterologous nucleic acid encoding a phytoene synthase and a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase.

[0130] The phytoene synthase may be selected from known phytoene synthases and variants thereof, said variants exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of said known phytoene synthases.

[0131] Examples of phytoene synthases include, but are not limited to, phytoene synthases from Pantoea agglomerans (Uniprot Accession No.: D5KXJ0, SEQ ID NO: 46), Rhodobacter capsulatus (Uniprot Accession No.: P17056, SEQ ID NO: 47). Other phytoene synthases can be easily identified using known databases or any sequence alignment software applied to the phytoene synthases listed above.

[0132] Preferably, the phytoene synthase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 46 to 47 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 46 to 47. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 46 to 47 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0133] More preferably, the phytoene synthase comprises or consists of an amino acid sequence selected from SEQ ID NO: 46 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 46. In particular, said variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 46 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0134] The lycopene forming phytoene desaturase may be selected from known lycopene forming phytoene desaturases and variants thereof, said variants exhibiting lycopene forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of said known lycopene forming phytoene desaturases.

[0135] Examples of lycopene-forming phytoene desaturases include, but are not limited to, lycopene-forming phytoene desaturases from Pantoea agglomerans (Uniprot Accession No.: L0BGV3, SEQ ID NO: 48), Rhodobacter capsulatus (Uniprot Accession No.: A0A4U1JQP1, SEQ ID NO: 49). Other lycopene-forming phytoene desaturases can be easily identified using known databases or any sequence alignment software applied to the phytoene desaturases listed above.

[0136] Preferably, the lycopene-forming phytoene desaturase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 49 and variants thereof, said variants exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 48 to 49. In particular, said variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 48 to 49 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0137] More preferably, the lycopene-forming phytoene desaturase comprises or consists of an amino acid sequence selected from SEQ ID NO: 48 and variants thereof, said variants exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 48. In particular, said variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 48 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably by 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0138] In certain embodiments, the recombinant microbial host cell comprises

[0139] - a heterologous nucleic acid encoding a lycopene cyclase, preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and 8 to 16 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6 and 8 to 16, more preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3 and 8 to 11;

[0140] - a heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase, preferably a GPP C6-methyltransferase comprising or consisting of SEQ ID NOs: 17 to 31 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17 to 31, more preferably a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17;

[0141] - a heterologous nucleic acid encoding a GGPP synthase, preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 43 and variants thereof, said variants exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 39 to 43, more preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 and variants thereof, said variants exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 39; and

[0142] - a heterologous nucleic acid encoding a phytoene synthase, preferably a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 47 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 46 to 47, more preferably a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46; and

[0143] - a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase, preferably a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 49 and variants thereof, said variant exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 48 to 49, more preferably a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and variants thereof, said variant exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 48.

[0144] Optionally, the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 38 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 32 to 38, more preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32.

[0145] In a preferred embodiment, the recombinant microbial host cell comprises

[0146] - a heterologous nucleic acid encoding a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 1 to 3 and 8 to 11; and

[0147] - a heterologous nucleic acid encoding a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17; and

[0148] - a heterologous nucleic acid encoding a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 39 and variants thereof, said variants exhibiting GGPP synthase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 39; and

[0149] - a heterologous nucleic acid encoding a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and variants thereof, said variants exhibiting phytoene synthase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 46; and

[0150] - a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 48 and variants thereof, said variants exhibiting lycopene-forming phytoene desaturase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 48.

[0151] Optionally, the recombinant microbial host cell may further comprise a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, wherein the variants exhibit FPP synthase activity and have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 32.

[0152] Dioxygenase activity that cleaves carotenoids

[0153] The recombinant microbial host cell of the present invention preferably further exhibits a carotenoid-cleaving dioxygenase activity, which allows the production of irone compounds, in particular β- and / or α-irone from 2-methyl-carotene and / or 2,2'-dimethyl-carotene.

[0154] As used herein, the term "carotenoid cleaving dioxygenase" or "CCD" refers to an enzyme that catalyzes the formation of apocarotenoids by cleavage of double bonds of cyclic carotenoids. Preferably, this term refers to CCD class 1 enzymes (CCD1), i.e. enzymes that symmetrically cleave various carotenoids at both the 9-10 and 9'-10' double bonds. In particular, the CDD enzymes used in the present invention catalyze the cleavage of double bonds between C9-C10 and C9'-C10' of 2-methyl-carotene and / or 2,2'-dimethyl-carotene. CCD activity, in particular CCD1 activity, can be assessed by any method known to the skilled person. For example, this activity can be assessed by incubating an acetone extract of cells producing a carotene substrate and purified CCD1 in the presence of β-octylglucoside and then analyzing the cleavage products by LC-ESI-MS.

[0155] The recombinant microbial host cells of the present invention may naturally express CCD. In this case, the recombinant host cells may optionally be genetically modified to overexpress this protein or further express a heterologous CCD. In embodiments where the recombinant microbial host cells do not naturally express CCD, the host cells are genetically modified to express a heterologous CCD.

[0156] In a preferred embodiment, the recombinant microbial host cell of the invention comprises a heterologous nucleic acid encoding a CCD.

[0157] The CCD may be selected from known CCDs and variants thereof, which exhibit CCD activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of the known CCDs.

[0158] Examples of CCDs include, but are not limited to, those from Osmanthus fragrans (CCD1 OF, NCBI GenBank accession number: BAJ05401.1, SEQ ID NO: 50), Zea mays (GenBank accession number: ABF85668.1, SEQ ID NO: 51), Arabidopsis thaliana (Uniprot accession number: O65572, SEQ ID NO: 52), Solanum lycopersicum (Uniprot accession number: Q6E4P5, SEQ ID NO: 53) and Chrysanthemum morifolium (Uniprot accession number: A0JBX5, SEQ ID NO: 54) CCD. Other CCDs can be easily identified using known databases or any sequence alignment software applied to the CCDs listed above.

[0159] Preferably, the CCD comprises or consists of an amino acid sequence selected from SEQ ID NOs: 50 to 54 and variants thereof, which exhibit CCD activity and have at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 50 to 54. In particular, the variant may comprise or consist of a sequence that differs from the sequence set forth in any one of SEQ ID Nos. 50 to 54 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0160] More preferably, the CCD comprises or consists of an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, the variant exhibiting CCD activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 50. Specifically, the variant may comprise or consist of a sequence that differs from the sequence of SEQ ID NO. 50 by 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 substitutions, insertions and / or deletions, preferably 1, 2, 3, 4 or 5 substitutions, insertions and / or deletions.

[0161] In certain embodiments, the recombinant microbial host cell comprises

[0162] - a heterologous nucleic acid encoding a lycopene cyclase, preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6 and 8 to 16 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6 and 8 to 16, more preferably a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3 and 8 to 11;

[0163] - a heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase, preferably a GPP C6-methyltransferase comprising or consisting of SEQ ID NOs: 17 to 31 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 17 to 31, more preferably a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NOs: 17 or 18, preferably SEQ ID NO: 17;

[0164] - a heterologous nucleic acid encoding a GGPP synthase, preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 to 43 and variants thereof, said variants exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 39 to 43, more preferably a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 39 and variants thereof, said variants exhibiting GGPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 39; and

[0165] - a heterologous nucleic acid encoding a phytoene synthase, preferably a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 46 to 47 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 46 to 47, more preferably a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and variants thereof, said variant exhibiting phytoene synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 46; and

[0166] - a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase, preferably a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 to 49 and variants thereof, said variant exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 48 to 49, more preferably a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 48 and variants thereof, said variant exhibiting lycopene-forming phytoene desaturase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 48;

[0167] - A heterologous nucleic acid encoding a CCD, preferably a CCD comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 50 to 54 and variants thereof, said variants exhibiting CCD activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 50 to 54, more preferably a CCD comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 50 and variants thereof, said variants exhibiting CCD activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with SEQ ID NO: 50.

[0168] Optionally, the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 to 38 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 32 to 38, more preferably a FPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 32 and variants thereof, said variants exhibiting FPP synthase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 32.

[0169] In a preferred embodiment, the recombinant microbial host cell comprises

[0170] - a heterologous nucleic acid encoding a lycopene cyclase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3 and 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 1 to 3 and 8 to 11; and

[0171] - a heterologous nucleic acid encoding a GPP C6-methyltransferase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17 and variants thereof, said variants exhibiting GPP C6-methyltransferase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 17 or 18, preferably SEQ ID NO: 17; and

[0172] - a heterologous nucleic acid encoding a GGPP synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 39 and variants thereof, said variants exhibiting GGPP synthase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 39; and

[0173] - a heterologous nucleic acid encoding a phytoene synthase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 46 and variants thereof, said variants exhibiting phytoene synthase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 46; and

[0174] - a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase comprising or consisting of an amino acid sequence selected from the group consisting of SEQ ID NO: 48 and variants thereof, said variants exhibiting lycopene-forming phytoene desaturase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 48; and

[0175] - a heterologous nucleic acid encoding a CCD comprising or consisting of an amino acid sequence selected from SEQ ID NO: 50 and variants thereof, said variants exhibiting CCD activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 50.

[0176] Optionally, the recombinant microbial host cell may further comprise a heterologous nucleic acid encoding a FPP synthase, preferably a FPP synthase comprising or consisting of an amino acid sequence selected from SEQ ID NO: 32 and variants thereof, wherein the variants exhibit FPP synthase activity and have a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO: 32.

[0177] In a preferred embodiment, the recombinant microbial host cell of the present invention exhibits (i) lycopene β-cyclase and / or lycopene ɛ-cyclase activity, and (ii) GPP C6-methyltransferase, FPP synthase, GGPP synthase, phytoene synthase and lycopene-forming phytoene desaturase activity. Optionally, the recombinant microbial host cell of the present invention may further exhibit CCD activity. Each of these activities may be provided by a heterologous or endogenous enzyme.

[0178] Inhibition of phosphatases that degrade phosphorylated precursors

[0179] It has been previously shown that inhibition of PgpB and CpdB, which encode phosphatases that degrade phosphorylated isoprene precursors, leads to enhanced carotenoid accumulation in Escherichia coli due to increased availability of terpene precursors such as GPP and FPP (Wang et al., Appl. Microbiol. Biotechnol. 102 (2018) 9771–9780). These phosphatases are also able to degrade 13-meFPP to produce 13-methylfarnesyl.

[0180] Therefore, the recombinant microbial host cell can be further genetically modified to reduce the activity of endogenous PgpB phosphatase and / or the activity of endogenous CpdB phosphatase compared to the unmodified microbial host cell. Preferably, the recombinant microbial host cell is genetically modified to reduce the activity of endogenous PgpB phosphatase and the activity of endogenous CpdB phosphatase. In a preferred embodiment, the activity of endogenous PgpB phosphatase and / or the activity of endogenous CpdB phosphatase is inhibited.

[0181] As used herein, the term "PgpB", "PgpB phosphatase" or "phosphatase encoding PgpB" refers to a phosphatidylglycerol phosphatase B that catalyzes the dephosphorylation of diacylglycerol diphosphate (DGPP) to phosphatidic acid (PA) and the subsequent dephosphorylation of PA to diacylglycerol (DAG). In Escherichia coli, this enzyme is encoded by the gene pgpB.

[0182] As used herein, the term "CpdB", "CpdB phosphatase" or "phosphatase encoding CpdB" refers to a 2',3'-cyclic nucleotide 2'-phosphodiesterase / 3'-nucleotidase that converts a 2',3'-cyclic nucleotide into a 3'-nucleotide, which is then converted into the corresponding nucleoside and phosphate. In Escherichia coli, this enzyme is encoded by the gene cpdB.

[0183] The nomenclature of these enzymes and encoding genes may vary depending on the organism. However, for the sake of clarity, in this specification, these terms are used independently of the origin of the enzyme or gene.

[0184] The activity of PgpB phosphatase can be assessed using any method known to the skilled person. For example, the activity can be assessed by incubating a suitable lipid phosphate substrate (e.g., lysophosphatidic acid) with purified PgpB protein in the presence of n-dodecyl-β-d-maltoside. The released phosphate can be detected by a colorimetric assay as described in Tong et al. (J Biol Chem. 2016 Aug 26; 291(35): 18342–18352).

[0185] The activity of CpdB phosphatase can be assessed using any method known to the skilled person. For example, the activity can be assessed by incubating the purified CpdB protein with a cyclic dinucleotide phosphate substrate such as c-di-AMP. The resulting nucleotide product can be detected by HPLC or LC-MS coupled to UV detection.

[0186] Examples of PgpB phosphatases include, but are not limited to, PgpB phosphatases from Escherichia coli (eg, Uniprot Accession No.: P0A924, SEQ ID NO: 55) and Bacillus subtilis (eg, Uniprot Accession No.: O34349, SEQ ID NO: 56).

[0187] Examples of CpdB phosphatases include, but are not limited to, CpdB phosphatases from Escherichia coli (eg, Uniprot Accession No.: P08331, SEQ ID NO: 57) and Salmonella typhimurium (eg, Uniprot Accession No.: P26265, SEQ ID NO: 58).

[0188] The gene encoding the endogenous PgpB or CpdB phosphatase in the host cell can be easily identified using methods well known to the skilled person, for example using known databases or any sequence alignment software applied to the known PgpB and CpdB phosphatases listed above.

[0189] The endogenous PgpB or CpdB activity can be reduced or inhibited using any method known to professionals. Specifically, this activity can be reduced by reducing the expression of endogenous genes, for example, by replacing the endogenous promoter with a weaker promoter. Preferably, the gene encoding endogenous PgpB or CpdB is inactivated by any method known to professionals, for example, by deleting all or part of the gene, by introducing a nonsense codon, an expression cassette, a gene or a mutation that induces a frameshift. In a preferred embodiment, the gene encoding endogenous PgpB and / or the gene encoding endogenous CpdB is inactivated by deleting all or part of the gene. More preferably, the genes encoding endogenous PgpB and CpdB are inactivated by deleting all or part of the gene.

[0190] Improvements to SAM pools

[0191] S-adenosylmethionine (SAM) is a ubiquitous intracellular methyl donor. As a methyl donor, SAM often donates its methyl group in enzyme-catalyzed reactions such as the methylation of GPP by GPP C6-methyltransferase to produce 6-meGPP. SAM is synthesized from methionine and ATP by SAM synthase, which in Escherichia coli is encoded by the metK gene. MetJ is a repressor protein in Escherichia coli that controls the expression of genes involved in methionine biosynthesis and transport. Specifically, when bound to SAM, MetJ represses the expression of the methionine regulon and enzymes involved in SAM synthesis.

[0192] Therefore, the recombinant microbial host cell may be further genetically modified to reduce the activity of the endogenous MetJ repressor protein compared to the unmodified microbial host cell. Preferably, the recombinant microbial host cell is genetically modified to inhibit the activity of the endogenous MetJ repressor protein.

[0193] The terms "MetJ", "MetJ protein" or "MetJ repressor" as used herein refer to the Met repressor protein, which is a ligand-responsive transcriptional repressor protein that naturally regulates methionine and SAM production in response to feedback from SAM accumulation (Cress et al., Microb. Cell Fact. 16 (2017) 1–14). In Escherichia coli, this enzyme is encoded by the gene metJ. The nomenclature of this protein and the encoding gene may vary depending on the organism. However, for clarity, in this specification, these terms are used independently of the origin of the protein or gene.

[0194] The activity of the MetJ repressor protein can be assessed using any method known to the skilled person. For example, the activity can be assessed by studying the interaction of MetJ with a consensus DNA sequence in the presence or absence of SAM using a gel shift assay and / or analytical ultracentrifugation (Augustus et al., Biochemistry 2010, 49, 15, 3289-3295).

[0195] Examples of MetJ repressor proteins include, but are not limited to, MetJ repressor proteins from Escherichia coli (eg, Uniprot Accession No.: P0A8U6, SEQ ID NO: 59) and Salmonella typhimurium (eg, Uniprot Accession No.: P06203, SEQ ID NO: 60).

[0196] The gene encoding the endogenous MetJ repressor protein in the host cell can be easily identified using methods known to the skilled person, for example using known databases or any sequence alignment software applied to the known MetJ repressor proteins listed above.

[0197] The endogenous MetJ activity may be reduced or inhibited using any method known to the skilled person. In particular, this activity may be reduced by reducing the expression of the endogenous gene, for example by replacing the endogenous promoter with a weaker promoter. Preferably, the gene encoding the endogenous MetJ is inactivated by any method known to the skilled person, for example by deleting all or part of the gene, by introducing nonsense codons, expression cassettes, genes or mutations that induce frameshifts. In a preferred embodiment, the gene encoding the endogenous MetJ is inactivated by deleting all or part of the gene.

[0198] Increased pools of DMAPP and IPP precursors

[0199] GPP The substrate of this GPP C6-methyltransferase is synthesized by FPP synthase, which catalyzes the sequential condensation of isopentenyl pyrophosphate (IPP) and dimethylallyl pyrophosphate (DMAPP) to produce GPP. IPP and its isomer DMAPP are synthesized via the mevalonate pathway (MVA) or the non-mevalonate pathway (or the 2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate (MEP / DXP) pathway). The MVA pathway is utilized by most eukaryotes and archaea, while the MEP pathway is present in most bacteria. However, there are some exceptions, such as Staphylococcus aureus, which uses the MVA pathway.

[0200] The recombinant microbial host cells of the present invention can be genetically modified to increase the pools of DMAPP and IPP by:

[0201] (i) introduction of a heterologous MVA pathway or overexpression of one or more enzymes of the MVA pathway; and / or

[0202] (ii) Introducing heterologous MEP pathway or overexpressing one or several enzymes of the MEP pathway.

[0203] Preferably, in embodiments where the recombinant host cell of the present invention comprises an endogenous MVA pathway, the host cell may be genetically modified by introducing a heterologous MEP pathway or overexpressing one or several enzymes of the MVA pathway. On the other hand, in embodiments where the recombinant host cell of the present invention comprises an endogenous MEP pathway, the host cell may be genetically modified by introducing a heterologous MVA pathway or overexpressing one or several enzymes of the MEP pathway.

[0204] The enzyme of the MVA or MEP pathway to be overexpressed may be an endogenous or heterologous enzyme.

[0205] In one embodiment, the recombinant microbial host cell of the present invention has been genetically modified to introduce a heterologous MVA pathway or to overexpress one or more enzymes of the MVA pathway. Preferably, the recombinant microbial host cell of the present invention has been genetically modified to introduce a heterologous MVA pathway.

[0206] The term "mevalonate pathway" or "MVA pathway" is used herein to refer to the biosynthetic pathway that converts acetyl-CoA to IPP. The mevalonate pathway comprises enzymes that catalyze the following steps: (a) condensation of two acetyl-CoA molecules into acetoacetyl-CoA by the action of acetyl-CoA-acetyltransferase (EC 2.3.1.9); (b) condensation of acetoacetyl-CoA with acetyl-CoA to form hydroxymethylglutaryl-CoA (HMG-CoA) by the action of hydroxymethylglutaryl-CoA synthase (EC 2.3.3.10); (c) conversion of HMG-CoA to mevalonate by the action of hydroxymethylglutaryl-CoA reductase (NADPH) (EC 1.1.1.34); (d) phosphorylation of mevalonate to mevalonate 5-phosphate by the action of mevalonate kinase (EC 2.7.1.36); and (e) conversion of mevalonate 5-phosphate to mevalonate 5-pyrophosphate (EC 2.7.4.2). 2.7.4.2); and (f) converting mevalonate 5-pyrophosphate to isopentenyl pyrophosphate by the action of mevalonate diphosphomevalonate decarboxylase (EC 4.1.1.33).

[0207] Genes encoding enzymes of the MVA pathway to be heterologously expressed or overexpressed can be readily identified by a skilled person. Specifically, heterologous genes can be obtained from various sources including plants, fungi and yeast, in particular from Saccharomyces cerevisiae. As an example, the MVA pathway of Saccharomyces cerevisiae comprises hydroxymethylglutaryl-CoA synthase encoded by gene erg13 (Uniprot accession number: P54839), hydroxymethylglutaryl-CoA reductase (NADPH) encoded by gene hmg1 (Uniprot accession number: P12683), mevalonate kinase encoded by gene erg12 (Uniprot accession number: P07277), phosphomevalonate kinase encoded by gene erg8 (Uniprot accession number: P24521) and diphosphomevalonate decarboxylase encoded by gene mvd1 (Uniprot accession number: P32377).

[0208] In a specific embodiment, the recombinant host cell is Escherichia coli and is genetically modified by introduction of a heterologous MVA pathway.

[0209] In another embodiment, the recombinant microbial host cell of the present invention has been genetically modified to introduce a heterologous MEP pathway or to overexpress one or several enzymes of the MEP pathway. Preferably, the recombinant microbial host cell of the present invention has been genetically modified to overexpress one or several enzymes of the MEP pathway.

[0210] As used herein, the terms "MEP pathway," "MEP / DXP pathway," "non-mevalonate pathway," or "2-C-methyl-D-erythritol 4-phosphate / 1-deoxy-D-xylulose 5-phosphate pathway" refer to the biosynthetic pathway that leads to the formation of IPP and DMAPP from the condensation of pyruvate and D-glyceraldehyde 3-phosphate to 1-deoxy-D-xylulose 5-phosphate (DXP). This pathway involves the following enzymes: 1-deoxy-D-xylulose 5-phosphate synthase (EC 2.2.1.7), 1-deoxy-D-xylulose-5-phosphate reductoisomerase (EC 1.1.1.267), 2-C-methyl-D-erythritol 4-phosphate cytidine acyltransferase (EC 2.7.7.60), 4-diphosphocytidine acyl-2-C-methyl-D-erythritol kinase (EC 2.7.1.148), 2-C-methyl-D-erythritol 2,4-cyclodiphosphate synthase (EC 4.6.1.12), 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase (EC 1.17.7.1), 4-hydroxy-3-methylbut-2-enyl diphosphate reductase (EC 1.17.1.2), and isopentenyl diphosphate delta-isomerase (EC 5.3.3.2).

[0211] The genes encoding enzymes of the MEP pathway to be expressed or overexpressed can be easily identified by a skilled person. For example, the MEP pathway of Escherichia coli contains 1-deoxy-D-xylulose 5-phosphate synthase encoded by the gene dxs (Uniprot accession number: P77488), 1-deoxy-D-xylulose 5-phosphate reductoisomerase encoded by the gene dxr (Uniprot accession number: P45568), 2-C-methyl-D-erythritol 4-phosphate cytidine acyltransferase encoded by the gene ispD (Uniprot accession number: Q46893), 4-diphosphocytidine acyl-2-C-methyl-D-erythritol 4-phosphate cytidine acyltransferase encoded by the gene ispE (Uniprot accession number: P62615), and 1-deoxy-D-xylulose 5-phosphate synthase encoded by the gene dxr (Uniprot accession number: P45568). The genes encoding the 2-C-methyl-D-erythritol kinase are 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate synthase, 4-hydroxy-3-methylbut-2-en-1-yl diphosphate reductase, and 4-hydroxy-3-methylbut-2-enyl diphosphate reductase.

[0212] In a specific embodiment, the recombinant host cell is Escherichia coli and is genetically modified to overexpress one or several enzymes of the MEP pathway, preferably at least the idi gene.

[0213] Recombinant nucleic acids and expression cassettes

[0214] Every kind of heterologous nucleic acid as above is all contained in the expression cassette.Preferably, the encoding nucleic acid sequence has been optimized to express in the microbial host cell.The encoding nucleic acid sequence is operably connected with the expression, especially transcription and translation required element of gene.These elements are selected so that there is function in the recombinant microbial host cell.These elements can comprise for example transcription promoter, transcription activator, terminator sequence and start and stop codon.The method for selecting these elements according to the host cell that needs to express therein is well known to those skilled in the art.

[0215] In particular, the present invention also relates to an expression cassette useful for the present invention, i.e. an expression cassette comprising at least one nucleic acid selected from the group consisting of nucleic acids encoding lycopene cyclase, nucleic acids encoding GPP C6-methyltransferase, nucleic acids encoding FPP synthase, nucleic acids encoding GGPP synthase, nucleic acids encoding phytoene synthase, nucleic acids encoding phytoene desaturases that form lycopene, and nucleic acids encoding dioxygenases that cleave carotenoids, and combinations thereof. Preferably, the expression cassette of the present invention comprises at least one nucleic acid encoding lycopene cyclase, and at least one nucleic acid selected from the group consisting of nucleic acids encoding GPP C6-methyltransferase, nucleic acids encoding FPP synthase, nucleic acids encoding GGPP synthase, nucleic acids encoding phytoene synthase, nucleic acids encoding phytoene desaturases that form lycopene, and nucleic acids encoding dioxygenases that cleave carotenoids, and combinations thereof. In this regard, combinations of heterologous nucleic acids disclosed in the sections related to the recombinant microbial host cells of the present invention are also contemplated. The nucleic acid is operably linked to one or more control sequences that direct the expression of the nucleic acid, generally including a transcriptional promoter and a transcriptional terminator.

[0216] Each expression cassette may contain only one coding nucleic acid operably linked to one or more control sequences. Alternatively, an expression cassette useful in the present invention may contain several coding nucleic acids operably linked to one or more control sequences.

[0217] The control sequence generally includes a promoter recognized by the host cell. The promoter contains a transcriptional control sequence that mediates the expression of the encoding nucleic acid. The promoter can be any polynucleotide that exhibits transcriptional activity in the host cell. The promoter can be a native or heterologous promoter. The promoter can be constitutive or inducible, strong or weak.

[0218] Preferably, the nucleic acids encoding lycopene cyclase, GPP C6-methyltransferase, FPP synthase, GGPP synthase, phytoene synthase and lycopene-forming phytoene desaturase are placed under the control of one or several constitutive promoters. Preferably, the nucleic acids encoding carotenoid-cleaving dioxygenases are placed under the control of an inducible promoter.

[0219] For example, if the microbial host cell is prokaryotic, the promoter may be selected from the following promoters: SJM915, Lac1, LacZ, pLacT, ptac, pARA, pBAD, RNA polymerase promoters of bacteriophage T3 or T7, polyhedrin promoters, PR or PL promoters of lambda phage. In a preferred embodiment, the promoter is SJM915. If the microbial host cell is eukaryotic, particularly yeast, the promoter may be selected from the following promoters: promoter pTDH3, promoter pTEF1, promoter pTEF2, promoter pCCW12, promoter pHHF2, promoter pHTB2 and promoter pRPL18B. Examples of inducible promoters that can be used in yeast are promoters tetO-2, GAL10, GAL10-CYC1 and PHO5.

[0220] Optionally, the expression cassette of the present invention may also contain a selectable marker that allows easy selection of recombinant bacteria. Typically, the selectable marker is a gene encoding antibiotic resistance or providing autotrophy.

[0221] The expression cassette of the present invention may be integrated into the genome of the host cell and / or may be maintained in an episomal form in an expression vector. Preferably, the expression cassette is integrated into the genome of the host cell.

[0222] All or part of the expression cassette comprising the heterologous nucleic acids described above, or a combination of some of them, may be contained in a common expression vector or in different expression vectors.

[0223] The present invention also relates to an expression vector comprising one or several expression cassettes according to the present invention.

[0224] Specifically, the expression vector of the present invention may comprise at least one nucleic acid selected from the group consisting of nucleic acids encoding lycopene cyclase, nucleic acids encoding GPPC6-methyltransferase, nucleic acids encoding FPP synthase, nucleic acids encoding GGPP synthase, nucleic acids encoding phytoene synthase, nucleic acids encoding phytoene desaturases that form lycopene, nucleic acids encoding dioxygenases that cleave carotenoids, and combinations thereof. Preferably, the expression vector of the present invention comprises at least one nucleic acid encoding lycopene cyclase, and at least one nucleic acid selected from the group consisting of nucleic acids encoding GPP C6-methyltransferase, nucleic acids encoding FPP synthase, nucleic acids encoding GGPP synthase, nucleic acids encoding phytoene synthase, nucleic acids encoding phytoene desaturases that form lycopene, nucleic acids encoding dioxygenases that cleave carotenoids, and combinations thereof. In this regard, combinations of heterologous nucleic acids disclosed in the sections related to the recombinant microbial host cells of the present invention are also contemplated.

[0225] The expression vector can be used to transform a host cell and express the nucleic acid of interest in the cell. The choice of the vector generally depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be an autonomously replicating vector, i.e., a vector that exists as an extrachromosomal entity, whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an artificial chromosome. The vector can contain any means for ensuring self-replication. Alternatively, the vector can be a vector that is integrated into the genome when introduced into a host cell and replicates together with the chromosome into which it is integrated.

[0226] The vector preferably comprises one or more selectable markers which allow easy selection of host cells comprising the vector. A selectable marker is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc.

[0227] The vector preferably comprises an element that allows the vector to be integrated into the genome of the host cell or the vector to replicate autonomously in the cell independently of the genome. When integrated into the host cell genome, the integration of the sequence in the genome may depend on homology or non-homologous recombination. On the one hand, the vector can contain extra polynucleotides for guiding integration into the genome of the host cell by homologous recombination at the precise position. These extra polynucleotides can be any sequence homologous to the target sequence in the host cell genome. On the other hand, the vector can be integrated into the genome of the host cell by non-homologous recombination.

[0228] For autonomous replication, the vector may also comprise an origin of replication, enabling the vector to replicate autonomously in the host cell in question. The origin of replication may be any plasmid replication factor that functions in the cell and mediates autonomous replication. The term "origin of replication" or "plasmid replication factor" means a polynucleotide that enables a plasmid or vector to replicate in vivo.

[0229] Methods for selecting these elements according to the host cells in which expression is desired are well known to those skilled in the art. The vector can be constructed by classical molecular biology techniques well known to those skilled in the art.

[0230] The present invention also relates to the use of an expression cassette or an expression vector according to the present invention for transforming, transfecting or transducing a microbial host cell. The microbial host cell is as defined above.

[0231] The host cell may be transformed, transfected or transduced in a transient or stable manner. The expression cassette or vector of the invention is introduced into the host cell such that the expression cassette or vector is maintained as a chromosomal integrant or a self-replicating extrachromosomal vector as described above.

[0232] The expression cassette or expression vector according to the invention can be introduced into the host cell by any method known to the skilled person, such as electroporation, conjugation, transduction, transformation of competent cells, protoplast transformation, protoplast fusion, bioballistic "gene gun" transformation, PEG-mediated transformation, lipid-assisted transformation or transfection, chemical-mediated transfection, lithium acetate-mediated transformation, liposome-mediated transformation.

[0233] Optionally, more than one copy of an expression cassette or expression vector of the invention may be inserted into a host cell.

[0234] The present invention also relates to a method for preparing the recombinant microbial host cell of the present invention, the method comprising introducing the expression cassette or expression vector of the present invention into the microbial host cell, and selecting the microbial host cell comprising the expression cassette or vector.

[0235] Method for producing irone compounds

[0236] The present invention also relates to a method for producing β- and / or α-irone, the method comprising culturing the recombinant microbial host cell according to the present invention under conditions suitable for producing the β- and / or α-irone, and optionally recovering the β- and / or α-irone. The present invention also relates to the use of the recombinant microbial host cell according to the present invention for producing β- and / or α-irone.

[0237] In embodiments where production of β-irone is sought, the recombinant microbial host cell preferably comprises a lycopene cyclase selected from the group consisting of a lycopene β-cyclase and a lycopene ɛ-cyclase exhibiting lycopene β-cyclase activity, preferably selected from the group consisting of a wild-type bacterial CrtY-type lycopene β-cyclase, a wild-type heterodimeric lycopene β-cyclase from a Gram-positive bacterium, and a wild-type cyanobacterial CrtL-type lycopene ɛ-cyclase exhibiting lycopene β-cyclase activity, and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases. More preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 6, 8 and 9 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 6, 8 and 9. Even more preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 1 to 3, 8 and 9 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3, 8 and 9.

[0238] In embodiments where production of α-irone is sought, the recombinant microbial host cell preferably comprises a lycopene cyclase selected from the group consisting of lycopene ɛ-cyclases, preferably selected from the group consisting of wild-type cyanobacterial CrtL-type lycopene ɛ-cyclases and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of these cyclases. Preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 16 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any of SEQ ID NOs: 8 to 16. More preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 8 to 11 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 8 to 11. Even more preferably, the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10 and 11 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 10 and 11.

[0239] In embodiments where production of cis-α-irone is sought, the recombinant microbial host cell preferably comprises a lycopene cyclase comprising or consisting of an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, said variants exhibiting lycopene ɛ-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity to SEQ ID NO: 10.

[0240] The recombinant microbial host cell may further exhibit the GPP C6-methyltransferase activity, FPP synthase activity, GGPP synthase activity, phytoene synthase activity, phytoene desaturase activity to form lycopene and / or dioxygenase activity to cleave carotenoids as described above. Preferably, the recombinant microbial host cell at least further exhibits GPP C6-methyltransferase activity, FPP synthase activity, GGPP synthase activity, phytoene synthase activity and phytoene desaturase activity to form lycopene.

[0241] In embodiments wherein the recombinant microbial host cell does not exhibit CCD activity, cleavage of 2-methyl-carotene and / or 2,2'-dimethyl-carotene can be performed in vitro as shown in the experimental section.

[0242] The conditions for culturing the recombinant microbial host cells according to the present invention can be adjusted according to conventional techniques well known to those skilled in the art.

[0243] The recombinant microorganism host cell is cultivated in a suitable substratum. The term "suitable substratum" generally refers to a substratum providing a maintenance and / or growth essential or useful nutrient for the host cell, such as a carbon source, a nitrogen source (such as ammonium sulfate), a phosphorus source (such as potassium dihydrogen phosphate), trace elements (such as copper, iodide, iron, magnesium, zinc or molybdate), vitamins and other growth factors (such as amino acids or other growth promoters). If necessary, a defoamer can be added. According to the present invention, this suitable substratum can be chemically limited or compound. It should be noted that the substratum can comprise a simple carbon source, such as glucose, fructose, xylose, ethanol, glycerol, galactose, sucrose, cellulose, cellobiose, starch, glucose polymers, molasses or the by-products of these sugars.

[0244] In general, the conditions for culturing the microorganism according to the present invention can be easily adjusted by a person skilled in the art according to the microorganism.

[0245] According to the invention, any cultivation method for industrial-scale production of the molecule of interest can be envisaged. Advantageously, the cultivation is carried out in a bioreactor, in particular in batch, fed-batch, chemostat and / or continuous culture mode. The cultivation is usually carried out in a bioreactor, possibly with a step of solid and / or liquid pre-cultivation in conical flasks, and using a suitable culture medium.

[0246] Other aspects and advantages of the present invention will be described in the following examples which are to be regarded as illustrative rather than limiting.

[0247] Example

[0248] Materials and methods

[0249] General reagents, bacterial strains, plasmids, and growth conditions

[0250] Restriction enzymes (New England Biolabs), T4 DNA ligase (Promega), Phusion DNA polymerase (Thermo Scientific) were used for routine cloning methods. The bacterial strains and plasmids used in this study are listed in Tables 1 and 2 below. For routine cloning, Escherichia coli DH5α was used and maintained in Luria-Bertani (LB) medium at 37°C. For carotenoid production, the recombinant Escherichia coli DH5α strain was grown at 37°C on a rotary shaker at 250 rpm for 24 hours and then at 30°C with the same agitation for 24 hours. Chloramphenicol (25 μg / mL) and / or carbenicillin (100 μg / mL) were added as needed.

[0251] Table 1: Plasmid list

[0252]

[0253]

[0254]

[0255]

[0256] Table 2: List of strains

[0257]

[0258] Bioinformatics analysis

[0259] Homologs of CrtL were identified using BLASTp searches using CrtL from Prochlorococcus marinus (WP_011132267) as query sequences. Sequences with significant homology (sequence identity >50%) were selected.

[0260] Construction of expression plasmids using Ecoflex assembly

[0261] Plasmids pAC-LYCipi (Addgene_53279, containing crtE, crtB, crtI, and idi genes from Pantoea agglomerans) and pAC-BETA (Addgene_53272, containing crtE, crtB, crtI, and crtY carotenoid pathway genes from Pantoea agglomerans) were obtained from Addgene (Cunningham et al., Plant Cell. 6 (1994) 1107–1121). Expression plasmids used in this study were constructed on level 1 or level 2 destination plasmids using the procedures described by the MoCloEcoflex kit (Moore et al., ACS Synth. Biol. 5 (2016) 1059–1069) (Addgene kit #1000000080). The two plasmids had the pSB1A2 backbone from the iGEM registry and pMB1 derived from pUC-19 as the replication origin. The Ecoflex assembly program was used as described (Moore et al., supra): for level 0 assembly, 100 fmol of each DNA component was combined with 50 fmol of the target plasmid and incubated with BsmBI and T4 ligase for 25 cycles of 10 min at 37°C and 10 min at 16°C, followed by 5 min at 50°C and 10 min at 80°C. For level 1 assembly, 50 fmol of each DNA component was combined with 50 fmol of the target plasmid and incubated with BsaI and T4 ligase for 25 cycles of 10 min at 37°C and 10 min at 16°C, followed by 5 min at 50°C and 10 min at 80°C. For level 2 assembly, 20 fmol of each DNA component was combined with 20 fmol of the destination plasmid and incubated with BsmBI and T4 ligase for 25 cycles of 10 min at 37°C and 10 min at 16°C, followed by 5 min at 50°C and 10 min at 80°C. 1 μL of the Golden Gate reaction mixture was transformed into 20 μL of chemically competent Escherichia coli DH5α using heat shock transformation. After recovery in 200 μL of LB medium, 150 μL of cells were plated in LB plates with appropriate antibiotics and grown overnight at 37°C. The plasmid backbone pL2k for co-expression of bezA and lycopene cyclase genes was generated from the Ecoflex pTU2-a destination plasmid by changing the resistance to kanamycin using IVA cloning (García-Nafría et al., Sci. Rep. 6 (2016) 27459).

[0262] To obtain genes for cloning, crtM (encoding diapophytoene synthase, Uniprot: Q2FV5), crtN (encoding diapophytoene desaturase, Uniprot: Q2FV60) were PCR amplified from genomic DNA of Staphylococcus aureus, and bezASN (encoding 6-GPP methyltransferase, SEQ ID NO: 17) was PCR amplified from genomic DNA of Streptomyces niveus NRRL2449. Other genes used in this study were codon optimized for Escherichia coli using GeneArt® software and synthesized by Twist Bioscience. The genes encoding lycopene cyclase used were crtYc (SEQ ID NO: 1) and crtYd (SEQ ID NO: 2) from Brevibacterium linens (which forms CrtY BL -β), CrtY from Pantoea agglomerans PA -β (SEQ ID NO: 3), crtL from Vulcanococcus limneticus VL -ɛ (SEQ ID NO: 8), crtL from Synechococcus sp. BS55DK SB -ɛ (SEQ ID NO: 9), crtL from Cyanobium sp. CACIAM 14 CC -ɛ (SEQ ID NO: 10), crtL from P. marinus MED4 PM -ɛ (SEQ ID NO: 7) and crtL from Prochlorococcus sp. HOT208 PH -ɛ (SEQ ID NO: 11). The gene encoding the dioxygenase that cleaves carotenoids is CCD1 from Osmanthus fragrans OF (SEQ ID NO: 50).

[0263] Carotenoid production and extraction

[0264] E. coli DH5α carrying pAC-LYCipi was co-transformed with pL1-BezAsn-2 or pL2k-Bsn2-CXyz to produce methylated lycopene and methylated carotene, respectively. To produce acyclic methylated C30 carotenoids, E. coli DH5α cells were co-transformed with pL2-MN and pL1-BezA. Cells were pelleted from 1 liter of culture by centrifugation (20°C, 4000 rpm), and carotenoids were repeatedly extracted from the pellet with 20 mL of HPLC grade acetone until all pigments were removed from the pellet. The colored supernatant was dried using a vacuum rotary evaporator. The sample was dissolved in 20 mL of 9:2 heptane / dichloromethane and loaded onto a silica gel column pre-equilibrated with 9:2 heptane / dichloromethane. The carotenoids were eluted using the same solvent system. Each eluted sample was dried using a vacuum rotary evaporator and stored at -20 °C under an argon atmosphere until further use. Alternatively, the crude acetone extract of the cell pellet was dried using a vacuum rotary evaporator and stored at -20 °C under an argon atmosphere before analysis by mass spectrometry.

[0265] In vitro CCD1 cleavage reaction

[0266] Carotenoids and CCD1 OF The extraction was performed as described with some modifications (Huang et al., J. Exp. Bot. 60 (2009) 3011–3022). A single colony of a carotenoid-producing E. coli strain from a freshly prepared plate was used to inoculate 50 mL of LB medium containing the appropriate antibiotics and grown at 30°C. OF Constitutive expression of CCD1 OF Escherichia coli DH5α cells were grown overnight at 37°C in 50 mL LB containing carbenicillin. Cells were harvested from the respective cultures by centrifugation (5000 g, 40 min, 4°C) and resuspended in 2 mL 1X phosphate buffered saline (140 mM NaCl, 4.3 mM Na2HPO4, 2.7 mM KCl, 1.47 mM K2HPO4, pH 7.3) containing 5 mM sodium ascorbate. Cells were lysed by sonication on saline ice four times for 30 s each, using 28% and 10% of the maximum amplitude for carotenoids and CCD1, respectively. OFExtraction. A 100 μL aliquot of each homogenized lysate was mixed with 20 μL of a 20% (w / v) β-octylglucoside solution. The mixture was shaken vigorously and incubated at 30°C in the dark for 20 h. The reaction product was extracted once with an equal volume of ethyl acetate (220 μL). The resulting extract was evaporated to dryness and resuspended in 50 μL of LC-MS grade acetonitrile:isopropanol (1:1).

[0267] LC-HR-ESI-MS Analysis of Carotenoids and Apocarotenoids

[0268] Carotenoids eluted from the silica column or from the crude extract were dissolved in 50 μL of LC-MS grade acetonitrile:isopropanol (1:1). 1 to 5 μL were injected onto a UPLC column (Luna Omega 1.6 μm Polar 100 Å, 150 x 2.1 mm, Phenomenex) connected to a system (Ultimate 3000 RSLC, Thermo Scientific) coupled to a HR electrospray ionization quadrupole time-of-flight (ESI-Q-TOF) mass spectrometer (MaXis II ETD, Bruker Daltonics). For carotenoid detection, the gradient of solvent A (0.1% formic acid in water) and solvent B (0.08% formic acid in acetonitrile) was: isocratic elution at 80% B for 10 min, linear increase from 80% B to 100% B in 1 min, then 24 min at 100% B, then reduced to 80% in 5 min, with a total run time of 35 min (flow rate of 0.3 mL / min). For apocarotenoid separation, the gradient of solvent A (0.1% formic acid in water) and solvent B (0.08% formic acid in methanol) was: linear increase from 20% to 65% B in 12 min, then isocratic elution at 65% B for 10 min, then linear increase to 100% B in 10 min, hold at 100% B for 2 min, then reduced to 20% B in 4 min, with a total run time of 62 min (flow rate of 0.25 mL / min). In the first half minute of each run, sodium formate solution was injected directly into the ion source as an internal reference for calibration. The mass range from 100 to 800 m / z was acquired in positive ion mode. The acquisition parameters of the ESI source were set as follows: nebulizer gas 2.4 bar, dry heater 200 °C, drying gas 8.0 L / min, capillary voltage 4500 V, end plate offset 500 V, and charging voltage 2000 V. For LC-MS / MS, the automatic MS / MS mode (collision energy 30.0 eV) was selected, and the parameters were the same as the MS method. Data were processed using Data Analysis 4.3 (Bruker Daltonics).

[0269] result

[0270] Production of acyclic methylated carotenoids in Escherichia coli

[0271] To demonstrate that the non-canonical C generated by BezA 11 The building block 6meGPP can be accepted by downstream carotenoid enzymes. We initially used C 30 Pathway as a model ( Figure 2 The genes crtM and crtN from Staphylococcus aureus (encoding diapophytoene synthase and diapophytoene desaturase, respectively) were assembled on one plasmid, each under the control of the SJM915 promoter and TL9 RBS (pL2-MN). The gene bezA from the benzastatin cluster in Streptomyces niveus was co-expressed in a separate plasmid under the control of the same promoter and RBS as in pL2-MN (pL1-BEZAsn-1).

[0272] UHPLC (ultra-high pressure liquid chromatography) coupled to high-resolution electrospray ionization mass spectrometry (UHPLC-HR-ESI-MS) analysis of acetone extracts of recombinant Escherichia coli cells revealed monomethylated C 31 Two major new carotenoid products, namely 2 or 2'-methyl-4,4'-diapoprotin (3, C 31 H 44 , Rt = 10 min; M + ° 观察值 :m / z is 416.3435; M + ° 理论值 : 416.3130) and 2 or 2'-methyl-4,4'-diap -Carotene (4; C 31 H 46 , Rt = 10.7 min; M + ° 观察值 :m / z is 418.3591; M + ° 理论值 :418.3599)( Figure 3 ). It also produces the main natural C 30 The products 4,4'-diapolycopene (1) and 4,4'-diapolycopene (2) ( Figure 3). Dimethylated products can be detected, but in very low yields. Our results also suggest that methylation affects the desaturation pattern of CrtN, shifting toward products with fewer conjugated double bonds. These experiments confirm that 6meGPP can be incorporated into the terpene pathway by downstream enzymes.

[0273] In order to improve C 31 For carotenoid yields, bezA expression was optimized by screening five different promoter-RBS combinations available in the Ecoflex MoClo kit (Moore et al., ACS Synth. Biol. 5 (2016) 1059–1069). The strongest promoter-RBS pair tested (the resulting plasmid was named pL1-BEZAsn-2) J23100-TL5 resulted in the highest yield of monomethylated 4,4'-diapoprotin (3) used as a readout. This suggests that GPP methylation is at least one of the rate-limiting steps.

[0274] Next, we proceeded to combine bezA encoded in pL1-BEZAsn-2 with C from Pantoa agglomerans encoded in plasmid pAC-LYCipi. 40 The crtE, crtB and crtI of the pathway (Cunningham et al., Plant Cell. 8 (1996) 1613–1626) were co-expressed in Escherichia coli ( Figure 4 UHPLC-HR-ESI-MS analysis of the acetone extract of the recombinant strain confirmed the production of mono- and di-methylated lycopene in addition to the natural product lycopene (5): 6 (C 41 H 58 ; Rt = 20.2 min; M + ° 观察值 :m / z is 550.4533; M + ° 理论值 :550.4538) and 7 (C 42 H 60 ; Rt = 20.9 min; M + ° 观察值 :m / z is 564.4690; M + ° 理论值 :564.4695)( Figure 4Detailed tandem MS analysis allowed the distinction of the Z or (all-E) configuration in these products on the basis of the characteristic fragment ions of each isomer. The position of the methyl group could not be determined by tandem MS analysis at this stage, as the fragmentation pattern of lycopene is a series of fragments differing by 14 Da (-CH2). Notably, the associated lycopene was the only product that accumulated in this system, indicating that the amount of desaturation catalyzed by CrtI is not affected by methylation, which is consistent with the C 30 The opposite was true for CrtN in the pathway. In addition, methylated lycopene was present as the major product, thus facilitating the downstream cyclization and cleavage steps leading to irone production.

[0275] In C 40 -Ironyl ring on the carotene skeleton

[0276] Inspired by the in vivo production of methylated lycopene using bezA, the next step was to incorporate a suitable lycopene cyclase into the pathway. Carotenoids with two β-ionone rings biosynthesized by lycopene β-cyclases are common to all carotenoid-producing organisms. A recent work demonstrated that two bacterial lycopene β-cyclases (CrtY from Brevibacterium linens and CrtY from BL -β and CrtY from Pantoea agglomerans PA -β) are promiscuous and can form unnatural cyclic β-C 30 carotenoids (Kim et al., Sci. Rep. 6 (2016) 21987). Therefore, we combined bezA with crtY BL -β or crtY PA -β were co-expressed in one plasmid (pL2k-Bsn2-CXyz, Figure 5A As revealed by UHPLC-HR-ESI-MS analysis, E. coli cells carrying the lycopene pathway and either co-expression plasmid produced circular C 41 and C 42 Carotene and natural C 40 carotene( Figure 5B Each carotene product family contained a mixture of isomers. Tandem MS analysis confirmed that these new products contained terminal rings. The elimination of xylene (C8H 10, 106 Da) or trimethylcyclohexene (C7H8, 92 Da) motifs, which are characteristic of non-oxidized cyclized carotenoids (Neto et al., Mass Spectrom. 30 (2016) 1540–1548). For monomethylated carotene 9, the number and position of the rings could not be determined accurately. However, tandem MS showed the presence of two fragments corresponding to the ionone (Drummond et al., ACS Synth. Biol. 8 (2019) 1303–1313) and ironone terminal groups (m / z 177.1638 and 191.1789, respectively), indicating that both termini can be cleaved by CrtY BL / PA To provide clear evidence that the methylated termini are cyclized, the cyclic C 42 Carotene was analyzed by tandem MS (10 or 13, C 42 H 56 , M +观察值 :m / z is 564.4690; M +理论值 : 564.4690 m / z). 40 Compared with carotene, only the fragment ion of the terminal group of irone (m / z 191.1789) was detected, while the fragment ion of ionone (m / z 177.1638) was not detected (Köksal et al., Biochemistry. 51 (2012) 3011–3020).

[0277] To generate the α-irone motif (double bond between C4-C5) ( Figure 1 ), which requires the use of lycopene ɛ-cyclase. Carotenoids with an α-ionone ring are mainly found in plants and some cyanobacteria. It is known that the CrtL lycopene cyclase from cyanobacteria mainly produces ɛ-carotene, which is composed of β- and α-ionone rings (Stickforth et al., Arch. Microbiol. 179 (2003) 409–415). The characteristic CrtL (CrtL) from Prochlorococcus marinus was selected. PM-ɛ ) (Stickforth et al., supra) and from Vulcanococcuslimneticus (CrtL VL -ɛ), Synechococcus sp. (CrtL SB -ɛ)、Cyanobium sp.(CrtL CC -ɛ) and Prochlorococcus sp. (CrtL PHEach corresponding gene was cloned into the co-expression plasmid (pL2k-Bsn2-CXyz) of bezA. BL / PA -β, all five cyanobacterial CrtLs were able to cyclize mono- and dimethyl lycopene with varying yields ( Figure 5B As mentioned above, tandem MS analysis of ring 10 or 13 confirmed the presence of the irone ring motif. VL-ɛ Seems to have the best methylated carotene production.

[0278] Given the specificity of the lycopene cyclase used, in the new C42 In support of this, tandem MS analysis of the homologous C40 carotene products (8 or 11) showed different characteristic fragment ions: m / z 307.2421 for 8 (two β-rings produced by CrtY-β) and 321.2565 / 388.3124 for 11 (one β-ring and one α-ring) produced by CrtL-ɛ (Figure S5B). The same fragment ions were observed in C42 carotenes produced by the same cyclase, indicating that their ring structures are the same as those in the natural C40 carotene products.

[0279] Cleavage of 2,2'-dimethyl-carotene to produce β- and α-irone

[0280] Carotenoid cleavage enzymes (CCDs) catalyze the formation of apocarotenoids by double bond cleavage of cyclic carotenoids. This constitutes the last step of the designed irone pathway and requires cleavage of the double bonds between C9-C10 and C9'-C10' of demethylated carotenes, regardless of the configuration of the irone motif. There are five different families of CCDs with different substrate and double bond cleavage specificities (Ahrazem et al., Int. J. Mol. Sci. 2016, 17(11), 1781). Among them, the CCD1 enzyme has a broad substrate specificity and cleaves at multiple double bond positions (Vogel et al., J. Biol. Chem. 283(2008) 11364–11373).

[0281] CCD1 from Osmanthus fragrans (CCD1 OF ) was extracted from the expressing E. coli strain in the presence of β-octylglucoside without further purification. The crude extract of the E. coli strain producing carotene with irone was mixed with CCD1 OFThe mixture was mixed in a 1:1 (v / v) ratio and incubated overnight at 30°C. The reaction products were analyzed by UHPLC-HR-ESI-MS and compared with an irone α standard. Depending on the cyclase, β-, trans-, and / or cis-α-irone was observed only in reactions in which cyclic carotenoids were present (i.e., extracts from cells expressing lycopene cyclase) in addition to ionone. Figure 6 ). Specifically, CrtY-β produced C42 carotene producing β-irone as the sole product. In contrast, the distribution of irone varied depending on the CrtL-ɛ used. VL -ɛ and CrtL SB -ɛ only produces β-irone, while the other cyclases additionally produce α-irone. Interestingly, in CrtL PH The α-irone produced in the -ɛ extract included both the trans and cis configurations, whereas in the CrtL CC Only the cis configuration was observed in the -ɛ extract.

[0282] These experiments demonstrated that CCD1 OF The applicability of these enzymes in cleaving non-natural methylated carotenes to generate irones was investigated. Furthermore, our data provide insights into the substrate specificity and activity of the CrtL-ɛ tested. They showed great variability and thus cyanobacterial CrtL-ɛ represents a promising source that can be exploited to generate specific irone motifs.

[0283] Thus, we provide the first proof of principle of a functional, novel irone biosynthetic pathway in Escherichia coli. This pathway can serve as a versatile platform to produce specific irone isomers at will. Here we demonstrate the production of trans- and cis-α-irone as well as β-irone, which have not been found in natural sources.

Claims

1. A method for producing an irone compound, the method comprising culturing a recombinant microbial host cell comprising a heterologous nucleic acid encoding a lycopene cyclase that catalyzes the β- or ɛ-cyclization of one or both ends of 2-methyl-lycopene and / or the β- or ɛ-cyclization of one or both ends of 2,2'-dimethyl-lycopene under conditions suitable for producing the irone compound, and optionally recovering the irone compound.

2. The method according to claim 1, wherein the lycopene cyclase is selected from the group consisting of a wild-type cyanobacterial CrtL-type lycopene ɛ-cyclase, a wild-type bacterial CrtY-type lycopene β-cyclase and a wild-type heterodimeric lycopene cyclase of a Gram-positive bacterium, and variants thereof, said variants exhibiting lycopene cyclase activity and having at least 70% sequence identity with any of these cyclases.

3. The method according to claim 1 or 2, wherein the lycopene cyclase comprises or consists of an amino acid sequence selected from the group consisting of SEQ ID NOs: 10, 8, 9, 11, 1, 2 and 3 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 10, 8, 9, 11, 1, 2 and 3.

4. The method according to any one of claims 1 to 3, wherein the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 8 to 11 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to any one of SEQ ID NOs: 8 to 11.

5. The method according to any one of claims 1 to 3, wherein the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NO: 10 and variants thereof, said variants exhibiting lycopene cyclase activity and having a sequence identity of at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% to SEQ ID NO:

10.

6. The method according to any one of claims 1 to 3, wherein the lycopene cyclase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 1 to 3 and variants thereof, said variants exhibiting lycopene β-cyclase activity and having at least 70%, preferably at least 80%, more preferably at least 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 3.

7. The method of any one of claims 1 to 6, wherein the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a geranyl pyrophosphate (GPP) C6-methyltransferase.

8. The method according to claim 7, wherein the GPP C6-methyltransferase comprises or consists of an amino acid sequence selected from SEQ ID NOs: 17 to 31 and variants thereof, wherein the variants exhibit GPP C6-methyltransferase activity and have at least 70% sequence identity with SEQ ID NOs: 17 to 31.

9. The method according to any one of claims 1 to 8, wherein the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a farnesyl diphosphate (FPP) synthase and / or a heterologous nucleic acid encoding a geranylgeranyl diphosphate (GGPP) synthase and / or a heterologous nucleic acid encoding a polypeptide exhibiting FPP synthase and GGPP synthase activity.

10. The method of any one of claims 1 to 9, wherein the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a phytoene synthase and a heterologous nucleic acid encoding a lycopene-forming phytoene desaturase.

11. The method of any one of claims 1 to 10, wherein the recombinant microbial host cell further comprises a heterologous nucleic acid encoding a dioxygenase that cleaves carotenoids.

12. The method of any one of claims 1 to 11, wherein the recombinant microbial host has been genetically modified to reduce the activity of an endogenous PgpB phosphatase, an endogenous CpdB phosphatase, and / or an endogenous MetJ repressor protein compared to an unmodified microbial host cell.

13. The method of any one of claims 1 to 12, wherein the recombinant microbial host is a bacterium or a yeast.

14. A recombinant microbial host as defined in any one of claims 1 to 13.

15. Use of the recombinant microbial host cell according to claim 14, for producing an irone compound.

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