EP3924498A1 - Verfahren zur biosynthese von diosmin und/oder hesperidin in einem mikroorganismus - Google Patents

Verfahren zur biosynthese von diosmin und/oder hesperidin in einem mikroorganismus

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Publication number
EP3924498A1
EP3924498A1 EP20703068.5A EP20703068A EP3924498A1 EP 3924498 A1 EP3924498 A1 EP 3924498A1 EP 20703068 A EP20703068 A EP 20703068A EP 3924498 A1 EP3924498 A1 EP 3924498A1
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EP
European Patent Office
Prior art keywords
sequence
seq
exhibiting
polypeptides
activity
Prior art date
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EP20703068.5A
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English (en)
French (fr)
Inventor
Cyrille PAUTHENIER
André Le Jeune
Hélène SCORNEC
Célia ROUSSEL
Laetitia JOUBERT
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Laboratoires Servier SAS
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Laboratoires Servier SAS
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Publication of EP3924498A1 publication Critical patent/EP3924498A1/de
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    • C12P19/60—Preparation of O-glycosides, e.g. glucosides having an oxygen of the saccharide radical directly bound to a non-saccharide heterocyclic ring or a condensed ring system containing a non-saccharide heterocyclic ring, e.g. coumermycin, novobiocin
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Definitions

  • the present invention relates to a method for producing diosmin and hesperidin.
  • Dation is a mixture of flavonoids with vaso-tonic and vasculoprotective effects. This mixture is mainly composed of Diosmin ⁇ 85%, Hesperidin ⁇ 8% and traces of different flavones and their respective oxidized forms.
  • the current production method of the drug is the acid / base extraction from the peels of small oranges, a mixture of flavonoids containing mainly Hesperidin (94%) and a mixture of Isonaringine ( ⁇ 3%) , Neoponcirin ( ⁇ 2%) and Hesperetin ( ⁇ 1%). This mixture then undergoes controlled oxidation by a chemical process, transforming approximately 90% of the hesperidin into diosmin, and minority flavonoids in their oxidized form.
  • Daflon is linked to the supply of purified extract of orange flavonoids, which may vary due to climatic variations, fluctuations in currency prices and the difficulty of obtaining supplies from dozens of sites in several countries (mainly Mexico, Mediterranean Basin countries and China).
  • the inventors have developed a method for the biosynthesis of diosmin and hesperidin in a recombinant microorganism.
  • the present invention relates to a recombinant microorganism comprising: a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding a glucose at position 7 of hesperetin and / or diosmetin; and a heterologous nucleic acid sequence encoding a 6 "-0- rhamnosyltransferase (RhaT) capable of transferring a rhamnose to position 6 of the glucose of hesperetin-7-O-glucoside and / or of diosmetin-7-O- glucoside; and a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose -reductase (RHM) capable
  • the flavanone 7-O-beta-D-glucosyltransferase is an enzyme from Citrus sinensis, Citrus clementina, Arubidopsis thaliana, Scuteiiaria baicolensis or Homo sapiens.
  • the flavanone 7-O-beta-D-glucosyltransferase (UGT) can be an enzyme from Arabidopsis thaliana, Scuteiiaria baicolensis or Homo sapiens, preferably Arabidopsis thaliana or Scuteiiaria baicolensis.
  • the flavanone 7-O-beta-D-glucosyltransferase (UGT) is from Citrus sinensis or from Scuteiiaria baicolensis.
  • the flavanone 7-O-beta-D-glucosyltransferase can be selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from the enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95 and 97 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone activity 7 -O-beta-D-glucosyltransferase.
  • enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95 and 97 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity.
  • the flavanone 7-O-beta-D-glucosyltransferase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity
  • the 6 "-O-rhamnosyltransferase (RhaT) is a plant enzyme, preferably of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus
  • the 6 "-O-rhamnosyltransferase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting a 6 "-0-rhamnosyltransferase activity.
  • the 6" -0-rhamnosyltransferase is selected from the enzymes comprising a sequence chosen from SEQ ID NOs: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting 6 "-O-rhamnosyltransferase activity.
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase (RHM) is a plant enzyme, preferably from Citrus sinensis or Arabidopsis thaliana.
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase is selected from the enzymes comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting UDP- activity glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase.
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP-glucose activity 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase.
  • the microorganism according to the invention further comprises: a heterologous nucleic acid sequence encoding a tyrosine ammonia lyase (TAL); and / or a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H);
  • TAL tyrosine ammonia lyase
  • PAL phenylalanine ammonia lyase
  • C4H cinnamate 4-hydroxylase
  • CHS naringenin chalcone synthase
  • CH I chalcone isomerase
  • F3'H flavonoid 3'-monooxygenase
  • OMT O-methyltransferase
  • heterologous nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase capable of converting tyrosine to L-Dopa and also p coumaric acid to caffeic acid; or a heterologous nucleic acid sequence encoding a p-coumarate 3-hydroxylase capable of converting p-coumaric acid to caffeic acid.
  • the microorganism comprises
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d 'sequence identity with one of these sequences and exhibiting tyrosine ammonia lyase activity, and preferably a tyrosine ammonia lyase (TAL) comprising a sequence chosen from SEQ ID NO: 41 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting tyrosine ammonia lyase activity; and
  • a heterologous nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL) comprising a sequence chosen from SEQ ID NOs: 123, 125, 43, 45, 47 and 49 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity, preferably a 4CL comprising a sequence selected from SEQ ID NOs: 123, 125 and 45 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity, and in particular a heterologous nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL) comprising a sequence selected from SEQ ID NO: 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95%
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) comprising a sequence chosen from SEQ ID NOs: 53, 51, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, and preferably a CHS comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with this sequence and exhibiting chalcone synthase activity; and
  • CHS chalcone synthase
  • a heterologous nucleic acid sequence encoding a chalcone isomerase comprising a sequence chosen from SEQ ID NOs: 61 and 59 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with one of these sequences and exhibiting chalcone isomerase activity, and preferably a CHI comprising a sequence chosen from SEQ ID NO: 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting chalcone isomerase activity.
  • CH I chalcone isomerase
  • the microorganism comprises a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) from Callistephus chinensis, Perilla frutescens var. crispa, Petunia x hybrida, Gerbera hybrida, Citrus sinensis, Arabidopsis thaliana, Pilosella officinarum, Osteospermum hybrid cultivar, Phanerochaete chrysosporium, Citrus clementina or Streptomyces avermitilis, in particular Callistephus chinensis, Perilla frutescens var.
  • F3'H flavonoid 3'-monooxygenase
  • an enzyme comprising a sequence chosen from SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 121 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, preferably selected from the enzymes of SEQ ID NOs: 7, 11, 17 , and 121 and polypeptides having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity.
  • the microorganism comprises a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) comprising a sequence chosen from SEQ ID NOs: 7, 17, and 121 and polypeptides having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, preferably a flavonoid 3'-monooxygenase (F3'H) comprising a sequence chosen from SEQ ID NOs: 7 and polypeptides having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3'H flavonoid 3'-monooxygenase
  • the microorganism comprises a heterologous nucleic acid sequence encoding an O-methyl-transferase (OMT) of Citrus, in particular Citrus clementino or Citrus sinensis, Homo sapiens or Arabidopsis thaliana, preferably an enzyme comprising a sequence chosen from SEQ ID NOs: 119, 117, 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting the O-methyltransferase activity.
  • OMT O-methyl-transferase
  • it comprises a heterologous nucleic acid sequence encoding an O-methyl-transferase (OMT) comprising a sequence chosen from SEQ ID NOs: 119, 117 and 89 and the polypeptides comprising a sequence having at least 60 , 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity, preferably a sequence selected from SEQ ID NOs: 119 and 117 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity.
  • OMT O-methyl-transferase
  • the microorganism comprises
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65 and 77, preferably SEQ ID NOs: 65 and 77, and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a phenylalanine ammonia lyase activity, and more particularly preferably a phenylalanine ammonia lyase (PAL) comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting phenylalanine ammonia lyase activity; and
  • PAL phenylalanine ammonia lyase
  • C4H a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity; and very particularly preferably a cinnamate 4-hydroxylase (C4H) comprising a sequence chosen from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting cinnamate 4-hydroxylase activity.
  • C4H cinnamate 4-hydroxylase
  • the microorganism may further comprise a heterologous nucleic acid sequence encoding a flavone synthase (FNS), in particular a flavone synthase capable of producing luteolin from erodictyol, preferably from Arobidopsis thaliana, Petroselinum crispum, Zea mays, Lonicero japonica, Lonicera macranthoides, Callistephus chinensis, Apium graveolens, Medicago truncatula, Cuminum cyminum, Aethusa cynapium, Angelica archangelica, Conium maculatum, Camellia sinensis, Cynara cardunculus var scolymus, Saussureo barorcatuarum, Scirrocirrinumus, Scirrushusellinum, Scirrocirratus, Saussureo barhusellinum, Scirrushirratus, Scirrushusellinum, Scirrushirratus, Scir
  • Flavone synthase can be selected from enzymes comprising a sequence chosen from SEQ ID NOs: 33, 35, 37, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 , 151, 153, 155, 157 and 159 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone synthase activity.
  • the FNS may be an FNS comprising a sequence chosen from SEQ ID NOs: 33, 35, 37 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and presenting a flavone synthase activity, and preferably a flavone synthase (FNS) comprising a sequence selected from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavone synthase activity.
  • FNS flavone synthase
  • CPR cytochrome P450 reductase
  • SAMT S-adenosylmethionine synthetase
  • the CPR comprises a sequence selected from SEQ ID NOs: 25, 23, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, preferably a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, and in particular a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with this sequence and exhibiting cytochrome P450 reductase activity.
  • the microorganism is a yeast or a bacterium, preferably a yeast of the genus Saccharomyces, in particular Saccharomyces cerevisiae, or a bacterium such as Escherichia coli.
  • the present invention also relates to the use of a microorganism as described in the present document for producing diosmin and / or hesperidin.
  • diosmin and / or hesperidin relates to a method of producing diosmin and / or hesperidin comprising culturing a microorganism as described herein, and optionally harvesting diosmin and / or hesperidin.
  • microorganism is meant a unicellular organism.
  • the microorganism is a bacterium or a yeast.
  • recombinant microorganism is understood a microorganism which is not found in nature and which contains a genome modified following an insertion, modification or deletion of one or more heterologous genetic elements.
  • nucleic acid is meant a nucleic acid which has been modified and does not exist in a naturally occurring microorganism.
  • this term can denote a coding sequence or gene which is operably linked to a promoter which is not the natural promoter. It can also refer to a coding sequence in which introns have been deleted for genes comprising exons and introns.
  • heterologous is understood that the gene has been introduced by genetic engineering into the cell. It can be present there in episomal or chromosomal form. The origin of the gene may be different from the cell into which it is introduced. However, the gene can also come from the same species as the cell into which it is introduced but it is considered heterologous due to its environment which is not natural.
  • the gene or nucleic acid sequence is heterologous because he / she is under the control of a promoter other than his / her natural promoter, he / she is introduced in a different place from this one where he / she is naturally located.
  • the host cell may contain a copy of the endogenous gene prior to the introduction of the heterologous gene or it may not contain an endogenous copy.
  • the nucleic acid sequence may be heterologous in the sense that the coding sequence has been optimized for expression in the host microorganism.
  • a heterologous nucleic sequence encodes a protein which is heterologous to the host cell, that is to say which is not naturally present in yeast.
  • the term "native" or "endogenous", relative to the host microorganism refers to a genetic element or protein naturally present in said microorganism.
  • gene denotes any nucleic acid encoding a protein.
  • the term gene encompasses DNA, such as cDNA or gDNA, as well as RNA.
  • the gene can first be prepared by recombinant, enzymatic and / or chemical techniques, and then replicated in a host cell or an in vitro system.
  • the gene typically comprises an open reading frame encoding a desired protein.
  • the gene may contain additional sequences such as a transcription terminator or a signal peptide. Due to the degeneration of the genetic code, several nucleic acids can encode a particular polypeptide.
  • the codons in the coding sequence for a given polypeptide can be altered such that optimal expression in a particular microorganism is obtained, for example by using codon translation tables appropriate for that microorganism.
  • Nucleic acids can also be optimized to a GC content preferable for the particular yeast and / or to reduce the number of repeat sequences.
  • the heterologous nucleic acids have been optimized by codon for expression in the relevant microorganism. Optimization of codons can be accomplished by routine methods known in the art (see, eg, Welch, M., et al. (2011), Methods in Enzymology 498: 43-66).
  • 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 expression of the coding sequence.
  • control sequences denotes the nucleic acid sequences necessary for the expression of a gene.
  • the control sequences can be native or heterologous. Control sequences well known and currently used by those skilled in the art will be preferred. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter, a signal peptide sequence and a transcription terminator. Preferably, the control sequences comprise a promoter and a transcription terminator.
  • expression cassette denotes a nucleic acid construct comprising a coding region, ie a gene, and a regulatory region, ie comprising one or more control sequences. , linked in an operational manner. Preferably, the control sequences are suitable for the host microorganism.
  • the term "expression vector” refers to a DNA or RNA molecule which comprises an expression cassette.
  • the expression vector is a linear or circular double stranded DNA molecule.
  • the vector may further include an origin of replication, a selection marker, etc.
  • percentage identity between two nucleic acid or amino acid sequences within the meaning of the present invention, is meant a percentage of nucleotides or of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and over their entire length.
  • the best alignment or optimal alignment is the alignment for which the percentage identity between the two sequences to be compared, as calculated below, is the highest.
  • Sequence comparisons between two nucleic acid or amino acid sequences are traditionally carried out by comparing these sequences after having optimally aligned them, said comparison being carried out by segment or by comparison window to identify and compare the local regions of sequence similarity.
  • Alignment for the purpose of determining percent amino acid sequence identity can be accomplished in various ways which are well known in the art, for example using computer software available on the web on the Internet such as http: //blast.ncbi.nlm. Nih.gov/ or http://www.ebi.ac.uk/Tools/emboss/). Those skilled in the art can determine the appropriate parameters for measuring alignment, including any algorithm necessary to achieve maximum alignment along the length of the sequences being compared.
  • percent amino acid sequence identity values refer to values generated using the EMBOSS Needle pair sequence alignment program which creates an optimal overall alignment of two sequences.
  • all identity percentages mentioned in this application can be set at at least 60%, at least 70%, at least 80%, at least 85%, preferably at least 90% identity, more preferably at at least 95% identity.
  • all the percentages of sequence identity of the enzymes are at least 80% or at least 85%, preferably at least 90% or at least 95%, are considered as described. sequence identity.
  • the polypeptides may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additions, substitutions or deletions relative to the sequences described in SEQ ID Nos.
  • these additions, substitutions or deletions are introduced at the N-terminus, C-terminus or at both ends.
  • the polypeptides can optionally be in the form of a fusion protein.
  • overexpression and “increased expression” as used herein are used interchangeably and mean that the expression of a gene or an enzyme is increased relative to an unmodified microorganism, e.g. a wild-type microorganism or not comprising the genetic modifications described herein.
  • wild is meant an unmodified microorganism existing in nature.
  • the increased expression of an enzyme is usually achieved by increasing the expression of the gene encoding said enzyme.
  • the terms "overexpression” and “expression” can be used interchangeably.
  • a person skilled in the art can use all the known techniques such as increasing the number of copies of the gene in the microorganism, using a promoter inducing a high level of expression of the gene, that is, a strong promoter, using elements stabilizing the corresponding messenger RNA or ribosome binding site (RBS) sequestration sequences and surrounding sequences.
  • overexpression can be obtained by increasing the number of copies of the gene in the microorganism.
  • One or more copies of the gene can be introduced into the genome by recombination methods known to those skilled in the art, including gene replacement or multicopy insertion (see for example international patent application WO 2015/092013 ).
  • an expression cassette comprising the gene, preferably placed under the control of a promoter strong, is integrated into the genome.
  • the gene can be carried by an expression vector, preferably a plasmid, comprising an expression cassette with the gene of interest placed preferably under the control of a strong promoter.
  • the expression vector may be present in the microorganism in one or more copies, depending on the nature of the origin of replication.
  • Overexpression of the gene can also be achieved by using a promoter inducing a high level of expression of the gene.
  • the promoter of an endogenous gene can be replaced by a stronger promoter, that is to say a promoter inducing a higher level of expression.
  • heterologous nucleic acid The endogenous gene under the control of a promoter which is not the natural promoter is called heterologous nucleic acid.
  • Promoters suitable for use in the present invention are known to those skilled in the art and can be constitutive or inducible, and be endogenous or heterologous.
  • the microorganism according to the present invention can be a eukaryotic or prokaryotic microorganism.
  • the microorganism is a eukaryote.
  • it is a yeast of the order of Saccharomycetales, Sporidiobolales and Schizosaccharomycetales.
  • the yeast may for example be selected from Pichia, Kluyveromyces, Soccharomyces, Schizosaccharomyces, Candida, Lipomyces, Rhodotorula, Rhodosporidium, Yarrowia, or Dehoryomyces.
  • the yeast is selected from Pichia pastoris, Kluyveromyces lactis, Kluyveromyces marxianus, Soccharomyces cerevisiae, Soccharomyces carlshergensis, Soccharomyces diastaticus, Saccharomyces douglasii, Soccharomyces kluyveri, Soccharomyces kluyveriomyces, Schidaharomyces ovisoccyceshyceshyida, Soccisoccomyces norceshidomyces norcesidomyces norcesidaceshidomyces albida, Soccidaomyces albidae, Soccidaomyces albida, Soccidaomyces albidae Rhodotorula glutinis, Rhodosporidium toruloides, Yarrowia lipolytica, Dehoryomyces hansenii and Lipomyces starkeyi.
  • the microorganism is a Saccharomyces yeast, preferably a Saccharomyces cerevisiae yeast.
  • the microorganism can be a fungus, from preferably a filamentous fungus. Preferably, it is chosen from Aspergillus, Trichoderma, Neurospora, Podospora, Endothia, Mucor, Cochiobolus or Pyricularia.
  • the fungus is chosen from Aspergillus nidulans, Aspergillus niger, Aspergillus awomari, Aspergillus oryzae, Aspergillus terreus, Neurospora crassa, Trichoderma reesei, and Trichoderma viride.
  • the microorganism is a prokaryote.
  • it is a bacterium, in particular chosen from among the phyla Acidohacteria, Actinobacteria, Aguificae, Bacterioidetes, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrutesobacteresobacteres, Firmicmatiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrutesobacteresobacteria, Firmicmatiae, Nectrobacteria, Nectarmatiae, Firmicmatiae, Chlorobi, , Proteobacteria, Spirochaetes, Thermodesulfobacter
  • the bacterium belongs to the genus Acaryochloris, Acetobacter, Actinobacillus, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Anaerobiospirillum, Aguifex, Arthrobacter, Arthrospira, Azobacter, Bacillus, Brevibacterium, Burkholderia, Chlorobriobidium, Chlororybacterium, Burkholderia, Chlorobriobidium, Chromatobacterium, Burkholderia, Chlorobriobidium, Chromatobacterium, Burkholderia, Chlorobriobidium, Chromatobacterium, Burkholderia, Chlorobriobidium, Chromatobacterium, Burkholderia, Chlorobriobidium, Chromatobacterium, Burkholderia, Chlorobriobidium, Chromatos, , Cyanothece, Enterobacter, Deinococcus, Erwinia, Escherichia, Geohacter, Gloeobacter, Glu
  • the bacterium is chosen from the species Agrobacterium tumefaciens, Anaerobiospirillum succiniciproducens, Actinobacillus succinogenes, Aguifex aeolicus, Aguifex pyrophilus, Bacillus subtilis, Bacillus amyloliguefacines, Brevibacterium ammoniagenes, Closumrevibickiidium, Ammonium, Closumidicidium, Brevibacterium ammoniagenes, Closumrevibickiidium, Closumidicidium, Bacillus amyloliguefacins, Brevibacterium ammoniagenes, Closumidicidium, Brevibacterium ammoniagenes, Closumidicidium, Brevibacterium ammoniagenes, Closumidicidium, Brevibacterium, Clostricidium, Closumidium, Bacillus amyloliguefacins, Brevibacterium.
  • the microorganism is a bacterium of the genus Streptomyces, in particular Streptomyces venezuelae.
  • the microorganisms may have been modified to increase the production of tyrosine and / or phenylalanine, preferably tyrosine.
  • the genes responsible for the feedback inhibition of the production of tyrosine and / or phenylalanine, preferably tyrosine can be inactivated.
  • the pathway for the biosynthesis of tyrosine and / or phenylalanine, preferably tyrosine can be optimized, in particular by redirecting the flow of carbon from other metabolic pathways towards that of tyrosine and / or of phenylalanine, preferably tyrosine.
  • the microorganism produces significant amounts of tyrosine and / or phenylalanine, in particular from a simple carbon source such as glucose.
  • the recombinant microorganism according to the present invention has been modified to produce hesperidin and / or diosmin.
  • the microorganism has been modified to introduce the enzymes necessary for the glycosylation of hesperetin and / or diosmetin in position 7 and the transfer of a rhamnose in position 6 of glucose from hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside.
  • the recombinant microorganism is capable of producing hesperetin and / or diosmetin, in particular it has been modified for this purpose.
  • hesperetin and / or diosmetin can be supplied to the microorganism, for example by adding these compounds to the culture medium.
  • the microorganism produces hesperidin.
  • Diosmin can then be prepared from hesperidin by chemical conversion, in particular by oxidation.
  • the microorganism produces hesperidin and diosmin.
  • the recombinant microorganism comprises:
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding glucose at position 7 of hesperetin and / or diosmetin;
  • UGT flavanone 7-O-beta-D-glucosyltransferase
  • a heterologous nucleic acid sequence encoding a 6-0-rhamnosyltransferase (RhaT) capable of transferring a rhamnose to position 6 of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside; and
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase ( RHM) capable of producing UDP-rhamnose.
  • flavanone 7-O-beta-D-glucosyltransferase (UGT), 6 "-0-rhamnosyltransferase (RhaT), and UDP-glucose 4,6-dehydratase / UDP-4-keto-6 -deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase (RHM) are enzymes heterologous to the microorganism.
  • UGT is an enzyme which transfers glucose to position 7 of hesperetin and / or diosmetin.
  • the name of the UGT is UDP-glucose: flavanone 7-O-beta-D-glucosyltransferase or flavanone 7-O-beta-D-glucosyltransferase. It is also called by the following names: uridine diphosphoglucose-flavanone 7-0-glucosyltransferase, naringenin 7-O-glucosyltransferase, and hesperetin 7-O-glucosyl-transferase. This enzyme belongs to the class of EC 2.4.1.185.
  • the inventors had to identify and select enzymes capable of accepting hesperetin and / or diosmetin as a substrate and of adding glucose at position 7 of these compounds.
  • the enzyme is selected so as to exhibit a preference for glycosylation at the 7-position of hesperetin and / or diosmetin.
  • the enzyme is specific for the 7-position of hesperetin and / or diosmetin.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding glucose at position 7 of hesperetin and diosmetin.
  • UGT flavanone 7-O-beta-D-glucosyltransferase
  • 7-O-beta-glycosyltransferase activity is meant a UGT enzyme capable of adding glucose at position 7 of a flavonoid.
  • an enzymatic test can be performed which consists of the in vitro incubation of the flavanone 7-O-beta-D-glucosyltransferase enzyme in the presence of NAD ( P) H, O2, and of a flavonoid, under optimal conditions (pH, ions ...), and the observation in UPLC-MS and in comparison with the expected standard of the appearance of a flavonoid having a glucose additional at position 7.
  • the flavonoid is hesperetin or diosmetin and the flavonoid having additional glucose at position 7 is their form with additional glucose at position 7, i.e. hesperetin 7- O-glucoside and diosmetin 7-0 glucoside.
  • the enzyme is only present in higher eukaryotes, especially in plants.
  • the enzyme can be obtained from plants of the genus Citrus, in particular Citrus maxima, Citrus sinensis, Citrus ciementina, Citrus mitis and Citrus x paradisi, Lysium, in particular Lysium harharum, Petunia, in particular Petunia x hybrida, Arabidopsis, in especially Arabidopsis thaliana, or Scutellaria, in particular Scutellaria baicalensis.
  • the UGT is an enzyme from Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens.
  • the UGT is an enzyme from Arabidopsis thaliana or from Scutellaria baicalensis.
  • the UGT is an enzyme from Citrus sinensis, Citrus ciementina, Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Citrus sinensis or Scutellaria baicalensis.
  • the UGT is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95 and 97 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavanone 7-O-beta-D-glucosyltransferase activity, in particular with hesperetin and / or diosmetin as substrate.
  • the UGT is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95 and 97 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O activity -beta-D-glucosyltransferase.
  • the UGT is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d 'sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity
  • the UGT may be Arabidopsis tholiono.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers NM_119576 and N P_567955.1, respectively, and more particularly in SEQ ID NO: 91.
  • the protein is also described in UniProtKB / Swiss Prot under the reference number UGT73B1.
  • the UGT is Scutellario baicalensis.
  • the nucleic acid sequences encoding a first UGT and protein sequences are described in NCBI under the reference numbers KU712253 and AMK52071.1, respectively, and more particularly in SEQ ID NO: 93.
  • the protein is described in UniProtKB / Swiss Prot under the reference number A0A140DPB7.
  • the nucleic acid sequences encoding a second UGT and protein sequences are described in NCBI under the reference numbers KU712254 and AMK52072.1, respectively, and more particularly in SEQ ID NO: 95.
  • the protein is described in UniProtKB / Swiss Prot under the reference number A0A140DPB8.
  • nucleic acid sequences encoding a third UGT and protein sequences are described in NCBI under the reference numbers KU712255 and AMK52073.1, respectively, and more particularly in SEQ ID Nos: 97.
  • the protein is described in UniProtKB / Swiss Prot under the reference number A0A140DPB9.
  • the UGT can be Homo sapiens.
  • the UGT is UGT1A6 (UDP glucuronosyltransferase family 1 member A6).
  • the protein is described in UniProtKB / Swiss Prot under the reference number P19224.
  • the consensus coding sequence is described in NCBI under the number CCDS2507.1.
  • the sequence of this enzyme is described in SEQ ID NO: 99.
  • the UGT is UGT1A7 (UDP glucuronosyltransferase family 1 member A7).
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q9HAW7.
  • the consensus coding sequence is described in NCBI under the number CCDS2506.1.
  • the sequence of this enzyme is described in SEQ ID NO: 101.
  • the UGT can also be from Citrus, in particular from Citrus sinensis or Citrus clementina.
  • Citrus sinensis UGT is described in SEQ ID NO: 113.
  • a nucleotide sequence encoding this enzyme is described in SEQ ID NO: 114.
  • the Citrus clementina UGT is described in SEQ ID NO: 115.
  • a nucleotide sequence encoding this enzyme is described in SEQ ID NO: 116.
  • the UGT is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and polypeptides comprising a sequence having at least 60 , 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity.
  • RhaT 6-O-rhamnosyltransferase
  • RhaT is an enzyme which carries out the transfer of u n rhamnose to the 6-position of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside.
  • RhaT is a 6-0-rhamnosyltransferase. This enzyme belongs to the class of EC 2.4.1.B53. The inventors had to identify and select enzymes capable of accepting rhesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside as substrate and of adding a rhamnose at position 6 of the glucose of these compounds.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding a 6-O-rhamnosyltransferase (RhaT) capable of transferring a rhamnose to position 6 of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7- O-glucoside.
  • RhaT 6-O-rhamnosyltransferase
  • This enzyme is only present in higher eukaryotes, especially in plants.
  • 6-O-rhamnosyltransferase activity is meant the addition of a rhamnose at position 6 of glucose by the enzyme RhaT.
  • an enzymatic test can be performed which consists of in vitro incubation of the 6-O-rhamnosyltransferase enzyme in the presence of NAD (P) H, O2, and of a flavonoid, under optimal conditions (pH, ions ...), and the observation in UPLC-MS and in comparison to the expected standard of the appearance of a flavonoid where a rhamnose is added at position 6 of the glucose .
  • the flavonoid is hesperetin 7-0-glucoside or diosmetin 7-O-glucoside and the flavonoid to which a rhamnose is added at position 6 of glucose are hesperidin and diosmin.
  • this enzyme is an enzyme produced by a plant of the genus Citrus or of Petunia hybrida, preferably of the species Citrus sinensis, Citrus maxima, or Citrus clementina.
  • the enzyme is an enzyme from Citrus sinensis or Citrus clementina.
  • 6-O-rhamnosyltransferase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting 6-O-rhamnosyltransferase activity.
  • the RhaT can be from Citrus clementina. It is described in the Genbank database of NCBI under the number XM_006420965 for the nucleic sequence and under the number XP_006421028 for the protein sequence, and more particularly in SEQ ID NO: 103. The protein is described in UniProtKB / Swiss Prot under the reference number V4RJL6.
  • the RhaT can also be from Citrus sinensis. It is described in the NCBI Genbank database under number DQ119035 for the nucleic sequence and under number ABA18631.1 for the protein sequence, and more particularly in SEQ ID NO: 105. The protein is described in UniProtKB / Swiss Prot under the reference number A7ISD3.
  • the RhaT is from Citrus sinensis and is an enzyme comprising the sequence SEQ ID NO: 105 or a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequenced therewith and exhibiting 6 "-O-rhamnosyltransferase activity.
  • the RhaT is selected from enzymes comprising a sequence selected from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting 6 "-O-rhamnosyltransferase activity.
  • RH M UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP- 4-keto-L-rhamnose-reductase
  • RHM is a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase trifunctional enzyme. This enzyme is able to produce UDP-rhamnose from UDP-glucose. This enzyme belongs to the class of EC 4.2.1.76. UDP-rhamnose is required for 6-0-rhamnosyltransferase (RhaT) activity.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L -rhamnose-reductase (RHM) capable of producing UDP-rhamnose.
  • This enzyme is only present in higher eukaryotes, especially in plants.
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase activity is meant the transformation of 'UDP-glucose to UDP-rhamnose.
  • a enzymatic test can be carried out which consists of the in vitro incubation of the UDP-glucose enzyme of NAD (P) H, O2, under optimal conditions (pH, ions ...), and observation in UPLC-MS and in comparison to the expected standard of the appearance of UDP-rhamnose.
  • this enzyme is an enzyme produced by a plant of the genus Citrus, in particular Citrus sinensis, or by Arabidopsis thaliana.
  • LIDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 107, 109, 111 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase activity.
  • RHM can be from Citrus sinensis. It is described in the NCBI Genbank database under the number XM_006477756 for the nucleic acid sequence and under the number XP_006477819.1 for the protein sequence, and more particularly in SEQ ID NO: 107.
  • RHM can also be Arabidopsis thaliana.
  • the protein is described in the Genbank database of NCBI under the number AY081471 for the nucleic sequence and under the number AAM10033.1 for the protein sequence, and more particularly in SEQ ID NO: 109.
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q9SYM5.
  • the protein is described in the NCBI Genbank database under the number AJ565874 for the nucleic acid sequence and under the number CAD92667.1 for the protein sequence, and more particularly in SEQ ID NO: 111.
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q9LPG6.
  • the RHM an enzyme comprising a sequence selected from SEQ ID NOs: 107, 109 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase activity.
  • the RHM is selected from enzymes comprising a sequence selected from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase activity.
  • the recombinant microorganism comprises: a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis, preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; in particular a flavanone 7-O-beta-D-glucosyltransfera
  • UGT fla
  • a heterologous nucleic acid sequence encoding a 6-0-rhamnosyltransferase (RhaT) of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus clementina, of preferably a 6-0-rhamnosyltransferase (RhaT) comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting a 6-O-rhamnosyltransferase activity, and preferably an RhaT comprising a sequence chosen from SEQ ID NOs: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting 6 "-O-rhamnosyltransfera
  • the recombinant microorganism comprises a heterologous nucleic acid sequence encoding a 6-O-rhamnosyltransferase (RhaT) and a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose- reductase (RHM) as defined in the previous embodiment and a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably from Arabidopsis thaliana or from Scutellaria baicalensis, preferably from flavanone 7-O-beta - D-glucosyltransferase (UGT) selected
  • the microorganism comprises
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115 and 95 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from among SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta activity -D-glucosyltransferase, and more particularly preferably, selected from enzymes comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85
  • heterologous nucleic acid sequence encoding a 6 "-O-rhamnosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with this sequence and exhibiting 6 "-O-rhamnosyltransferase activity; and
  • heterologous nucleic acid sequence encoding a selected UDP-glucose 4,6- dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose- reductase from enzymes comprising a sequence chosen from SEQ ID NOs: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP-glucose activity 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase.
  • the enzymes described above are enzymes from higher eukaryotes, preferably plant enzymes.
  • the enzymes originate from plants of the same genus, for example of the same species. Modifications allowing the production of hesperetin and / or diosmetin
  • either hesperetin and / or diosmetin are supplied to the microorganism, or the microorganism is capable of producing hesperetin and / or diosmetin.
  • the microorganism is, or has been, modified to be capable of producing hesperetin and / or diosmetin.
  • the inventors have therefore further developed a biosynthetic pathway allowing the microorganism to produce hesperetin and / or diosmetin.
  • Hesperetin and / or diosmetin can be obtained from naringenin and apigenin.
  • the flavonoid 3'-monooxygenase (F3'H) is an enzyme which carries out the addition of a hydroxyl group at the 3 'position of naringenin and / or apigenin. This enzyme belongs to the class of EC 1.14.14.82. It is also called flavone 3'-hydroxylase.
  • the inventors had to identify and select enzymes capable of accepting naringenin and / or apigenin as substrate and of adding a hydroxyl group at the 3 'position of these compounds.
  • the enzyme is selected so as to exhibit a preference for hydroxylation at the 3 'position of naringenin and / or apigenin.
  • the enzyme is specific for the 3 'position of naringenin and / or apigenin, in particular with respect to the 5' position in order to avoid double hydroxylation at the 3 'and 5 positions. ', and preferably also avoid hydroxylation at the 5' position.
  • the flavonoid 3'-monooxygenase (F3'H) is an enzyme which carries out the addition of a hydroxyl group at the 3 'position of naringenin and / or apigenin. This enzyme belongs to the class of EC 1.14.14.82. It is also called a flavonoid 3'-hydroxylase.
  • 3'-monooxygenase flavonoid activity is meant the transformation of a flavonoid into a 3 'hydroxylated flavonoid by an F3'H, CPR dependent enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of the flavonoid 3 'monooxygenase enzyme in the presence of NAD (P) H, O2, and d 'a flavonoid, under optimal conditions (pH, ions, etc.), and observation in UPLC-MS and in comparison with the expected standard of the appearance of a 3'-hydroxylated flavonoid.
  • the flavonoid is naringenin or apigenin and the 3'-hydroxylated flavonoid is their 3'-hydroxylated form, i.e. Eriodictyol or Luteolin.
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) capable of adding a hydroxyl in position 3 'of naringenin and / or apigenin.
  • F3'H flavonoid 3'-monooxygenase
  • F3'H is a plant enzyme, in particular plants of the genus Allium, Arabidopsis, Brassica, Callistephus, Coiumnea, Citrus, Dianthus, Gentiana, Gerbera, Glycine, Fragaria, Ipomoea, Malus, Matthiola, Osteospermum , Oryza, Phanerochaete, Perilla, Petroselinum, Pelargonium, Pilosella, Petunia, Sinningia, Sorghum, Torenia, Vitis or Zea, for example of Allium cepa, Arabidopsis thaliana, Brassica napus, Coiumnea hybrida, Callistephus chinensis, Citrus sinensis, Dianthus clement caryophyllus, Fragaria vesca, Fragaria x ananassa, Gerbera hybrida, Glycine max, Gentiana triflora, Ipomoea nil, Ipomoe
  • F3'H is an enzyme from plants of the genus Allium, Brassica, Callistephus, Coiumnea, Citrus, Dianthus, Gentiana, Gerbera, Glycine, Fragaria, Ipomoea, Malus, Matthiola, Osteospermum, Oryza, Phanerochaete , Perilla, Petroselinum, Pelargonium, Pilosella, Petunia, Sinningia, Sorghum, Torenia, Vitis or Zea, for example of Allium cepa, Brassica napus, Coiumnea hybrida, Callistephus chinensis, Citrus sinensis, Citrus clementina, Dianthus caryophyllus, Fragaria vesca, Fragaria x
  • the F3′H is an enzyme from Perilla frutescens var. crispa, Petunia x hybrida, Callistephus chinensis, Gerbera hybrida, Citrus clementina, Osteospermum hybrid cultivar, Phanerochaete chrysosporium, Streptomyces avermitilis, Citrus sinensis, Arahidopsis thaliana or Pilosella officinarum.
  • F3'H can be an enzyme from Perilla frutescens var. crispa, Petunia x hybrida, Callistephus chinensis, Gerbera hybrida, Citrus sinensis and Pilosella officinarum.
  • F3'H is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 121, in particularly among SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21, and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d 'sequence identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 1, 5, 7, 11, 17, 19 and 121, in particularly among SEQ ID NOs: 1, 5, 7, 11, 17 and 19, and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting the 3'-monooxygenase flavonoid activity, in particular with naringenin and / or apigenin as substrate and with hydroxylation at the 3 'position.
  • F3'H is an enzyme comprising a sequence selected from SEQ ID NOs: 5, 7, 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3'H is an enzyme comprising a sequence selected from SEQ ID NOs: 7, 11, 17 and 121, and polypeptides comprising a sequence having at least 60, 70, 75, 80, 85 , 90 or 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity.
  • the F3′H can be an enzyme comprising a sequence selected from SEQ ID NOs: 7, 17 and 121 and polypeptides comprising a sequence having at least 60, 70, 15, 80, 85, 90 or 95%, identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity
  • the F3'H may be from Perilla frutescens var. twitched.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AB045593.1 and BAB59005.1, respectively, and more particularly in SEQ ID Nos: 2 and 1.
  • the F3'H may be from Phanerochaete chrysosporium.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AB597870.1 and BAL05157.1, respectively, and more particularly in SEQ ID Nos: 4 and 3.
  • the F3'H can be from Petunia x hybrida.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AF155332.1 and AAD56282.1, respectively, and more particularly in SEQ ID Nos: 6 and 5.
  • the F3'H may be from Callistephus chinensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AF313488.1 and AAG49298.1, respectively, and more particularly in SEQ ID Nos: 8 and 7.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AF313489.1 and AAG49299.1, respectively, and more particularly in SEQ ID Nos: 10 and 9.
  • the F3'H can be from Gerbera hybrida.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers DQ218417.1 and ABA64468.1, respectively, and more particularly in SEQ ID Nos: 12 and 11.
  • the F3'H can be from Osteospermum hybrid cultivar.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers DQ250711.1 and ABB29899.1, respectively, and more particularly in SEQ ID Nos: 14 and 13.
  • the F3'H can be from Citrus clementina.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers XM_006440673.1 and XP_006440736.1, respectively, and more particularly in SEQ ID Nos: 16 and 15.
  • the F3'H can be from Citrus sinensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers XM_006477592.2 and XP_006477655.1, respectively, and more particularly in SEQ ID Nos: 18 and 17.
  • the F3'H may be from Pilosella officinarum.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers DQ319866.2 and ABC47161.1, respectively, and more particularly in SEQ ID Nos: 20 and 19.
  • the F3'H can be from Streptomyces avermitilis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers SAV_4539 and WP_010985964.1, respectively, and more particularly in SEQ ID Nos: 22 and 21.
  • the F3'H can be Arabidopsis thaliana.
  • a nucleic acid sequence encoding this enzyme and the protein sequence are described in NCBI under the reference numbers NM_120881.2 and N P_196416.1, respectively and more particularly in SEQ ID NOs: 122 and 121.
  • F3′H is an enzyme comprising a sequence selected from SEQ ID NOs: 7, 11, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3′H is an enzyme comprising a sequence selected from SEQ ID NOs: 7, 17 and 121, and the polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%. , at least 90% or at least 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3′H is an enzyme comprising a sequence chosen from SEQ ID NO: 7 and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% sequence identity with the sequence SEQ ID NO: 7 and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3'H is an enzyme comprising a sequence chosen from SEQ ID NO: 17 and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90 % or at least 95% identity of sequence with the sequence SEQ ID NO: 17, and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3'H is an enzyme comprising a sequence chosen from SEQ ID NO: 121 and polypeptides comprising a sequence having at least 70%, at least 75%, at least 80%, at least 85 %, at least 90% or at least 95% sequence identity with the sequence SEQ ID NO: 121, and exhibiting the flavonoid 3'-monooxygenase activity.
  • F3′H is an enzyme comprising a sequence chosen from SEQ ID NO: 11 and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90 % or at least 95% sequence identity with the sequence SEQ ID NO: 11, and exhibiting the flavonoid 3'-monooxygenase activity.
  • the flavonoid 3'-monooxygenase requires the presence of NADPH to effect the addition of the hydroxyl group.
  • the microorganism comprises a heterologous nucleic acid encoding a cytochrome P450 reductase, a NADPH-cytochrome P450 reductase.
  • This enzyme belongs to the class of EC 1.6.2.4.
  • Cytochrome P450 reductase originates from a eukaryote, in particular from a yeast, for example of the genus Saccharomycetales, or from a plant, for example a plant of the genus Arabidopsis, Ammi, Avicennia, Comellia, Camptotheca, Cotharanthus, Citrus, Glycine, Heiianthus, Lotus, Mesembryanthemum, Phaseolus, Physcomitrella, Pinus, Populus, Ruta, Saccharum, Solanum, Vigna, Vitis or Zea.
  • the cytochrome P450 reductase originates from a eukaryote, for example from yeast, in particular from Saccharomyces cerevisiae, or from a plant, for example from Cotharanthus roseus or Arabidopsis thaliana.
  • cytochrome P450 reductase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity.
  • cytochrome P450 reductase can be selected from enzymes comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with this sequence and exhibiting cytochrome P450 reductase activity.
  • the cytochrome P450 reductase can be from Cathoronthus roseus. It is described in the NCBI Genbank database under the number X69791.1 for the nucleic acid sequence and under the number CAA49446.1 for the protein sequence, and more particularly in SEQ ID Nos: 24 and 23, respectively. The protein is described in UniProtKB / Swiss Prot under the reference number Q05001.
  • the cytochrome P450 reductase can be from Saccharomyces cerevisiae. It is described in the Genbank database of NCBI under the number N M_001179172.1 for the nucleic sequence and under the number N PJD11908.1 for the protein sequence, and more particularly in SEQ ID Nos: 26 and 25, respectively. The protein is described in UniProtKB / Swiss Prot under the reference number P16603.
  • Cytochrome P450 reductase can be chimeric. It is described in the article Aigrain et al (2009, EM BO reports, 10, 742-747. The nucleic acid sequence encoding this enzyme and the protein sequence are described in SEQ ID Nos: 28 and 27, respectively.
  • the cytochrome P450 reductase can be from Arabidopsis thaliana.
  • cytochrome P450 comes from Arabidopsis thaliana, it can be called ATR. It is described in the Genbank database from NCBI under the number NM_118585.4 for the nucleic acid sequence and under the number N P_194183.1 for the protein sequence, and more particularly in SEQ ID Nos: 30 and 29, respectively.
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q9SB48.
  • cytochrome P450 reductase can be from Arabidopsis thaliana and be described in the NCBI Genbank database under the number NM_179141.2 for the nucleic acid sequence and under the number N P_849472.2 for the protein sequence, and more particularly in SEQ ID Nos: 32 and 31, respectively.
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q9SUM3.
  • a new copy of a coding sequence for CPR as defined above is introduced into the yeast.
  • the yeast is Saccharomyces cerevisiae and the CPR is from the same yeast, the promoter of the endogenous gene encoding CPR is replaced by a strong promoter.
  • the expression of CPR is increased compared to wild yeast; CPR is therefore overexpressed in modified yeast.
  • the F3′H and the CPR come from the same origin, the same species.
  • OMT O-methyltransferase
  • O-methyltransferases are a very large family of enzymes with targets that are difficult to pinpoint. The inventors had to identify and select O-methyltransferases capable of methylating erodictyol and / or luteolin in the 4 'position (para position).
  • the enzyme has been selected so as to exhibit a preference for methylation at the 4 'position of erodictyol and / or luteolin.
  • the enzyme is specific for the 4 'position of erodictyol and / or luteolin.
  • the methyl group introduced by the enzyme on erodictyol and / or luteolin is found in position 4 'in 60% of cases, the remainder being introduced in position 3', preferably in 70% of cases, and more preferably in 80% of cases.
  • 4'O-methyltransferase activity is meant the transformation of a 4'-hydroxyflavonoid into a 4'-methoxyflavonoid by a 4'-O-methyltransferase enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme 4'-0-methyltransferase, a 4 ' - hyd roxy f I a vo n o ⁇ d e, of S-adenosyl-L-methionine, under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of 4'-methoxyflavonoid is observed in UPLC-MS in comparison with the expected standard.
  • the 4 '- h y d roxyf I a vo n o ⁇ d e is erodictyol or luteolin and will be transformed respectively into their 4'-methoxyflavonoid form, that is to say into hesperetin or into diosmetin.
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding an O-methyltransferase capable of methylating erodictyol and / or luteolin in position 4 '.
  • This enzyme is only present in higher eukaryotes, especially in plants.
  • O-methyltransferase is an enzyme from Arabidopsis thaliana.
  • O-methyltransferase is from a higher eukaryote, preferably a mammal.
  • O-methyltransferase (OMT) is of human origin (Homo sapiens).
  • GOMT is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and methylation in the 4 'position.
  • GOMT is selected from the enzyme comprising a sequence selected from SEQ ID NO 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting O-methyltransferase activity.
  • GOMT is selected from the enzyme comprising a sequence selected from SEQ ID NO 87 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting O-methyltransferase activity.
  • OMT can be Arabidopsis thaliana.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers NM_118755.4 and N P_567739.1, respectively.
  • the protein is also described in UniProtKB / Swiss Prot under the reference number Q9C5D7, and more particularly in SEQ ID No: 87.
  • GOMT is Homo sapiens.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers NM_007310.2 and N PJ309294.1, respectively.
  • the protein is also described in UniProtKB / Swiss Prot under the reference number P21964, and more particularly in SEQ ID No: 89.
  • Homo sapiens OMT has the advantage of accepting erodictyol and luteolin as a substrate for methylation, while Arabidopsis thaliana GOMT has a strong preference for erodictyol. Conversely, if the synthesis of hesperetin is to be promoted by compared to that of diosmetin, OMT of Arabidopsis thaliana may have an advantage.
  • GOMT is an OMT of Citrus, in particular Citrus clementina or Citrus sinensis.
  • GOMT is selected from an enzyme comprising a sequence selected from SEQ ID NO 117 and 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with one of these sequences and exhibiting O-methyl-transferase activity.
  • the OMT is selected from an enzyme comprising a sequence chosen from SEQ ID NO 117 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting the O-methyltransferase activity.
  • OMT is selected from an enzyme comprising a sequence chosen from SEQ ID NO 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting O-methyl-transferase activity.
  • the OMTs of Citrus and Arahidopsis thaliana described above have the advantage of specifically methylating erodictyol at the 4 'position.
  • the microorganism further comprises a heterologous or endogenous sequence encoding an enzyme synthesizing S-adenosyl-L-methionine, an S-adenosylmethionine synthetase (SAMT).
  • SAMT S-adenosylmethionine synthetase
  • the microorganism comprises a heterologous nucleic acid sequence encoding an O-methyl-transferase (OMT), in particular capable of methylating erodictyol and / or luteolin at the 4 'position and a sequence of ′.
  • OMT O-methyl-transferase
  • SAMT S-adenosylmethionine synthetase
  • the SAMT originate from a yeast, in particular from Saccharomyces cerevisiae, most particularly when the microorganism is a yeast.
  • S-adenosylmethionine synthetase is an enzyme comprising a sequence chosen from SEQ ID NO: 81 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity with this sequence and exhibiting S-adenosylmethionine synthetase activity.
  • the S-adenosylmethionine synthetase can be from Saccharomyces cerevisiae. It is described in the NCBI Genbank database under the number NM_001180810.3 for the nucleic acid sequence and under the number NPJD10790.3 for the protein sequence. The protein is described in UniProtKB / Swiss Prot under the reference number P19358.
  • a new copy of a coding sequence for SAMT as defined above is introduced into the microorganism.
  • the microorganism is Saccharomyces cerevisiae
  • the promoter of the endogenous gene encoding SAMT is replaced by a strong promoter.
  • the expression of SAMT is increased compared to the wild microorganism; SAMT is therefore overexpressed in the modified microorganism.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding an O-methyltransferase capable of methylating erodictyol and / or luteolin in position 4 'and a heterologous or endogenous sequence encoding a S-adenosylmethionine synthetase (SAMT) capable of producing S-adenosyl-L-methionine.
  • SAMT S-adenosylmethionine synthetase
  • FNS Flavone synthase
  • Diosmetin can be produced from luteolin. It can also be obtained from erodictyol, either by transforming it into luteolin then by preparing diosmetin from luteolin, or by transforming it into hesperetin and then by preparing diosmetin from hesperetin.
  • the enzyme capable of converting erodictyol into luteolin and / or hesperetin into diosmetin is a flavone synthase (FNS).
  • the flavone synthase is also capable of converting erodictyol into luteolin.
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a flavone synthase, in particular a flavone synthase capable of to produce luteolin from erodictyol and / or diosmetin from hesperetin.
  • flavone synthase activity is meant the transformation of a flavanone into a flavone by an FNSI enzyme (independent CPR) or an FNSII (dependent CPR).
  • an enzymatic test can be performed which consists of in vitro incubation in the case of FNSI of a mixture composed of the enzyme flavone synthase (FNSI), a flavanone, 2-oxoglutarate, C> 2, under optimal conditions (pH, temperature, ions ...) and in the case of FNSII of a mixture composed of the FNSII enzyme, of a flavanone, of NAD (P) H, of 02, under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of the flavone corresponding to the flavanone is observed in UPLC-MS in comparison with the expected standard.
  • the flavanone is erodictyol or hesperetin and will be converted respectively into their flavone form, that is to say into luteolin or diosmetin.
  • the microorganism comprises a heterologous nucleic acid sequence encoding an O-methyl-transferase (OMT), in particular capable of methylating erodictyol and / or luteolin in position 4 '; and a heterologous nucleic acid sequence encoding a flavone synthase, in particular a flavone synthase capable of producing luteolin from erodictyol and / or diosmetin from hesperetin.
  • OMT O-methyl-transferase
  • the flavone synthase is an enzyme derived from plants, for example of the genus Aethusa, Angelica, Antirrhinum, Apium, Arabidopsis, Callistephus, Camellia, Conium, Cuminum, Cynara, Dahlio, Dorcoceras, Erythranthe, Lonicera, Medicago, Oryzo, Perilla , Petroselinum, Plectranthus, Populus, Saussurea, Scutellaria or Zea, in particular of the genus Arabidopsis, Lonicera, Medicago, Oryza, Petroselinum, Populus or Zea, in particular of Arabidopsis thaliana, Lonicera japonica, Lonicera macranthoides, Medicago truncatula, Petrosum sativa, Oryza sativa , Populus deltoids, Zea mays, Callistephus chinensis, Apium graveo
  • the flavone synthase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 33, 35, 37, 127, 129, 131, 133, 135, 137, 139, 141, 143 , 145, 147, 149, 151, 153, 155, 157 and 159 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavone synthase activity.
  • the flavone synthase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 33, 35, 37, and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting flavone synthase activity.
  • the FNS is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 37 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavone synthase activity.
  • FNS flavone synthase
  • FNSI flavone synthase 1
  • FNSIII flavone synthase 2
  • the FNS is a type I flavone synthase. In another embodiment, the FNS is a type II flavone synthase. In a further embodiment, the microorganism comprises a type I flavone synthase and a type II flavone synthase.
  • the microorganism comprises a heterologous nucleic acid sequence encoding type I flavone synthase (FNSI).
  • FNSI heterologous nucleic acid sequence encoding type I flavone synthase
  • FNSI can be a plant flavone synthase such as Petroselinum crispum, Oryza sativa, Populus delto ⁇ des, Medicago truncatula, Apium graveolens, Cuminum cyminum, Aethusa cynapium, Angelica orchangelica, or Conium maculatum, in particular from Petroselinum crispum, Populus deltiva, Medicago truncatula, preferably from Petroselinum crispum.
  • a plant flavone synthase such as Petroselinum crispum, Oryza sativa, Populus delto ⁇ des, Medicago truncatula, Apium graveolens, Cuminum cyminum, Aethusa cynapium, Angelica orchangelica, or Conium maculatum, in particular from Petroselinum crispum, Populus deltiva,
  • the FNSI can be an enzyme comprising a sequence chosen from SEQ ID NOs: 37, 127, 137, 141, 143 and 145 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting flavone synthase activity.
  • the FNSI can be an enzyme comprising a sequence selected from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence. and exhibiting flavone synthase activity.
  • the FNSI can be from Petroselinum crispum. It is described in the NCBI Genbank database under number AY817680.1 for the nucleic acid sequence and under number AAX21541.1 for the protein sequence. The protein is described in UniProtKB / Swiss Prot under the reference number Q7XZQ8. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 37 and 38, respectively.
  • the FNSI can also be Angeüca orchongelica. It is described in the NCBI Genbank database under number DQ683352.1 for the nucleic acid sequence and under number ABG78793.1 for the protein sequence. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 127 and 128, respectively.
  • FNSI can also be Apium graveolens. It is described in NCBI's Genbank database under number AY817676.1 for the nucleic acid sequence and under number AAX21537.1 for the protein sequence. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 137 and 138, respectively.
  • the FNSI can also be from Cuminum cyminum. It is described in the NCBI Genbank database under number DQ683349.1 for the nucleic acid sequence and under number ABG78790.1 for the protein sequence. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 141 and 142, respectively.
  • the FNSI can also be Aethuso cynapium. It is described in the NCBI Genbank database under number DQ683350.1 for the nucleic acid sequence and under number DQ683350.1 for the protein sequence. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 143 and 144, respectively.
  • the FNSI can also be from Conium maculatum. It is described in the NCBI Genbank database under number DQ683354.1 for the nucleic acid sequence and under number ABG78795.1 for the protein sequence. The amino acid and nucleic acid sequences are described in SEQ ID Nos: 145 and 146, respectively.
  • the microorganism comprises a heterologous nucleic acid sequence encoding a type II flavone synthase (FNSII).
  • FNSII can be a plant flavone synthase, for example of Arabidopsis thaliana, Zea mays, of the genus Lonicera such as for example Lonicera japonica and Lonicera mocronthoides, Callistephus chinensis, Medicago truncatula, Camellia sinensis, Cynara cardunculus var scolymus, Saussureo medronthuso, Saussureo medronthuso barbatus, Scutellaria baicalensis, Dorcoceros hygrometricum, Antirrhinum majus, Perilla frutescens var crispa, Dahlia pinnata or Erythranthe lewisii, in particular a flavone synthase of Arabidopsis thaliana, Zea mays or of the genus Lonicera such as for example Lonicera macranthoides and Loniceraides.
  • the genus Lonicera such as
  • the flavone synthase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 33, 35, 129, 131, 133, 135, 139, 147, 149, 151, 153, 155 , 157 and 159 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 33 and 35 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone synthase activity.
  • the FNS flavone synthase is an FNSII from Lonicera japonica.
  • the enzyme may be an enzyme described in the Genbank database of NCBI under the number KU127576.1 for the nucleic sequence and under the number AMQ91109.1 for the protein sequence, and more particularly in SEQs. ID Nos: 34 and 33, respectively.
  • the FNS flavone synthase is an FNSII from Lonicera macranthoides.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers KU127580.1 and AMQ91113.1, respectively, and more particularly in SEQ ID Nos: 36 and 35, respectively.
  • the FNS flavone synthase is an FNSII from Cynara cardunculus var scolymus.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers JN825735.1 and AFG31000.1, respectively, and more particularly in SEQ ID Nos: 130 and 129, respectively.
  • the FNS flavone synthase is an FNSII from Perilla frutescens var crispa.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AB045592.1 and BAB59004.1, respectively, and more particularly in SEQ ID Nos: 132 and 131, respectively.
  • the FNS flavone synthase is FNSII from Dahlia pinnata.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AB769842.1 and BAM72335.1, respectively, and more particularly in SEQ ID Nos: 134 and 133, respectively.
  • the FNS flavone synthase is an FNSII from Callistephus chinensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AF188612.1 and AAF04115.1, respectively, and more particularly in SEQ ID Nos: 136 and 135, respectively.
  • the FNS flavone synthase is an FNSII from Medicago truncatula.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers DQ354373.1 and ABC86159.1, respectively, and more particularly in SEQ ID Nos: 140 and 139, respectively.
  • the FNS flavone synthase is an FNSII from Camellia sinensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers FJ169499.1 and ACH99109.1, respectively, and more particularly in SEQ ID Nos: 148 and 147, respectively.
  • the FNS flavone synthase is an FNSII from Saussurea medusa.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers KF170286.1 and AGV40781.1, respectively, and more particularly in SEQ ID Nos: 150 and 149, respectively.
  • the FNS flavone synthase is FNSII from Plectranthus barbatus.
  • the nucleic acid sequences encoding this enzyme and sequences proteins are described in NCBI under the reference numbers KF606861.1 and AHJ89438.1, respectively, and more particularly in SEQ ID Nos: 152 and 151, respectively.
  • the FNS flavone synthase is an FNSII from Scutellaria boicalensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers KT963454.1 and AMW91729.1, respectively, and more particularly in SEQ ID Nos: 154 and 153, respectively.
  • the FNS flavone synthase is FNSII from Dorcoceras hygrometricum.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers KV013332.1 and KZV23934.1, respectively, and more particularly in SEQ ID Nos: 156 and 155, respectively.
  • the FNS flavone synthase is FNSII from Antirrhinum majus.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers AB028151.1 and BAA84071.1, respectively, and more particularly in SEQ ID Nos: 158 and 157, respectively.
  • the FNS flavone synthase is FNSII from Erythronthe lewisii.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers KX710102.1 and AOR81894.1, respectively, and more particularly in SEQ ID Nos: 160 and 159, respectively.
  • the microorganism comprises a heterologous nucleic acid sequence encoding a type II flavone synthase (FNSII) and a type I flavone synthase, for example a sequence chosen from SEQ ID NOs: 33, 35, 129, 131, 133, 135, 139, 147, 149, 151, 153, 155, 157 and 159 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone synthase activity and an enzyme comprising a sequence chosen from SEQ ID NOs: 37, 127, 137, 141, 143 and 145 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavone synthase activity, preferably a sequence chosen from SEQ ID NOs: 33 and 35 and polypeptides comprising a sequence having at least 60
  • FNS type II, FNSII requires the presence of cytochrome P450 reductase (CPR). If the microorganism does not include cytochrome P450 reductase, it will therefore be necessary to introduce a heterologous cytochrome P450 reductase. If the microorganism already includes one, it is possible to consider either the overexpression of an endogenous cytochrome P450 reductase (for example by replacing the promoter with a strong promoter or by adding one or more copies of the coding sequence) or also introduce a heterologous cytochrome P450 reductase.
  • CPR cytochrome P450 reductase
  • the type II FNS and the CPR come from the same origin, the same species.
  • the microorganism preferably comprises the enzymes allowing the production of the hesperitin and / or diosmetin from naringenin and / or apigenin.
  • the recombinant microorganism comprises:
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding glucose at position 7 of hesperetin and / or diosmetin; preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arabidopsis thaliana, from Citrus sinensis, from Citrus clementino, from Scutellaria hoicalensis or from Homo sapiens, preferably from Citrus sinensis or from Scutellaria baicalensis; preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase ( RHM) capable of producing UDP-rhamnose; preferably an RHM of Citrus sinensis or Arabidopsis thaliana, preferably an RHM comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto- L-rhamnose reductase, in particular an RHM comprising a sequence selected from SEQ ID NOs: 107
  • an F3'H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 1, 9, 11, 13, 15, 17 , 19, 21 and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 3'-monooxygenase flavonoid activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 1, 5, 7, 11, 17, 19 and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with one of these sequences and exhibiting flavonoid 3'-monooxygenase activity, in particular an F3'H comprising a sequence selected from SEQ ID NOs: 7, 11, 17 and 121 and polypeptides comprising a sequence having at least 60, 70,
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharanthus roseus or of Arabidopsis thaiiana; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity;
  • CPR cytochrome P450 reductase
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position; preferably an OMT from Arabidopsis thaiiana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117, 119, 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and a methylation in position 4 ', preferably selected from the enzyme comprising a sequence chosen from SEQ ID NO: 117, 119 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with one of these sequences
  • the recombinant microorganism comprises: a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding glucose at position 7 of hesperetin and / or diosmetin; preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arobidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis; preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60 , 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-
  • a heterologous nucleic acid sequence encoding a 6-0-rhamnosyltransferase (RhaT) capable of transferring a n rhamnose to the 6 position of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside; preferably a RhaT of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus clementina, preferably a 6-O-rhamnosyltransferase (RhaT) comprising a selected sequence from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 6-O-rhamnosyltransferase activity;
  • RhaT 6-O-rhamnosyltransfer
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase ( RHM) capable of producing UDP-rhamnose; preferably an RHM of Citrus sinensis or Arabidopsis thaliana, preferably a RHM comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting activity UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase, in particular an RHM comprising a sequence selected from SEQ ID NOs:
  • an F3′H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavonoid 3'-monooxygenase activity, preferably selected from among enzymes comprising a sequence chosen from SEQ ID NOs: 1, 5, 7, 11, 17 and 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting a flavonoid 3'-monooxygenase activity, in particular an F3'H comprising a sequence selected from SEQ ID NOs: 5, 7, 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharanthus roseus or of Arabidopsis thaliana; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity;
  • CPR cytochrome P450 reductase
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position; preferably an OMT from Arabidopsis thaliana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and methylation in position 4 ', preferably selected from the enzyme comprising a sequence SEQ ID NO 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting O-methyltransferase activity; and
  • the microorganism comprises - a heterologous nucleic acid sequence encoding a flavanone 7-0-beta-D-glucosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, and very particularly preferably selected from enzymes comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80
  • heterologous nucleic acid sequence encoding a 6 "-O-rhamnosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting 6 "-O-rhamnosyltransferase activity; and
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6- dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose - reductase selected from enzymes comprising a sequence chosen from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting activity UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase; and
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) comprising a sequence chosen from SEQ ID NOs: 23, 25, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, and very particularly preferably from enzymes comprising a sequence chosen from SEQ ID NO : 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cytochrome P450 reductase activity; and
  • CPR cytochrome P450 reductase
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in position 4 'and comprising a sequence chosen from SEQ ID NOs: 117, 119 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and a methylation in position 4 ', preferably selected from the enzyme comprising a sequence chosen from SEQ ID NOs: 117 and 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with one of these sequences and exhibiting O-methyltransferase activity; and
  • O-methyltransferase O-methyltransferase
  • a heterologous nucleic acid sequence encoding a flavone synthase (FNS) capable of producing a flavone from a flavanone, in particular capable of transforming naringenin into apigenin, and / or erodictyol into luteolin, of preferably transforming erodictyol into luteolin and comprising a sequence chosen from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting flavone synthase activity.
  • FNS flavone synthase
  • the microorganism comprises each of these heterologous nucleic acid sequences.
  • the recombinant microorganism comprises: a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding glucose at position 7 of hesperetin and / or diosmetin;
  • UGT flavanone 7-O-beta-D-glucosyltransferase
  • heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) capable of transferring a rhamnose to the 6-position of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside ;
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase ( RHM) capable of producing U DP-rhamnose;
  • a heterologous nucleic acid sequence encoding a flavonoid 3′-monooxygenase (F3′H) capable of hydroxylating in the 3 ′ position naringenin and / or apigenin;
  • F3′H flavonoid 3′-monooxygenase
  • heterologous nucleic acid sequence encoding an O-methyltransferase capable of methylating erodictyol and / or luteolin in the 4 'position;
  • a heterologous nucleic acid sequence encoding a flavone synthase (FNS) capable of producing a flavone from a flavanone, in particular capable of transforming naringenin into apigenin, erodictyol into luteolin and / or hesperetin into diosmetin , preferably to convert erodictyol to luteolin.
  • FNS flavone synthase
  • the recombinant microorganism further comprises a heterologous or endogenous nucleic acid sequence encoding an S-adenosylmethionine synthetase (SAMT), in particular from Saccharomyces cerevisiae, for example a SAMT comprising a sequence chosen from SEQ ID NO: 81 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting S-adenosylmethionine synthetase activity.
  • SAMT S-adenosylmethionine synthetase
  • Each enzyme can be chosen from the enzymes described above.
  • naringenin and apigenin are known in plants, in particular from glucose, tyrosine or phenylalanine.
  • Microorganisms notably E. coli and Saccharomyces cerevisiae, have been modified to produce naringenin and / or and apigenin (Hwang El, et al. 2003. Appl Environ Microbiol. 2003, 69 (5): 2699-2706 ; Jiang Hl, et al. 2005. Appl Environ Microbiol. 2005, 71 (6): 2962-9; Pandey et al, 2016, Biotechnol Adv., 34, 634-662).
  • naringenin and apigenin can be that described in Figure 1.
  • the microorganism comprises the enzymes necessary for the synthesis of naringenin and / or apigenin from tyrosine. In a second embodiment, the microorganism comprises the enzymes necessary for the synthesis of naringenin and / or apigenin from phenylalanine.
  • the microorganism comprises the enzymes necessary for the synthesis of naringenin and / or apigenin from tyrosine and phenylalanine.
  • TAL is a tyrosine ammonia lyase. This enzyme is able to produce p-coumaric acid from tyrosine. This enzyme belongs to the class of EC 4.3.1.23.
  • phenylalanine ammonia lyase activity is meant the transformation of phenylalanine into frans-cinnamic acid by an enzyme Phenylalanine ammonia lyase.
  • Phenylalanine ammonia lyase activity an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme phenylalanine ammonia lyase and phenylalanine under optimal conditions (pH, temperature , ions ). After a certain incubation time, the appearance of trans-cinnamic acid is observed in UPLC-MS in comparison with the expected standard.
  • a tyrosine ammonia lyase can also exhibit a phenylalanine ammonia lyase (PAL) activity as defined above and / or a dihydroxyphenylalanine ammonia-lyase (DAL) activity.
  • PAL phenylalanine ammonia lyase
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a tyrosine ammonia lyase.
  • this enzyme is an enzyme produced by a bacterium of the genus Rhodobacter or a bacterium of the genus Flavobacteriaceae.
  • this enzyme is produced by a bacterium Rhodobacter capsulatus, or Rhodobacter sphaeroides.
  • this enzyme is produced by a Flavobacterium johnsoniae bacterium.
  • this enzyme is an enzyme produced by a yeast, in particular a yeast of the genus Rhodotorula, for example Rhodotorula glutinis.
  • Other organisms also produce such an enzyme, for example Camellia sinensis, Fragaria x ananassa, Ralstonia metallidurans, or Zea mays.
  • tyrosine ammonia lyase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 39 and 41 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with one of these sequences and exhibiting tyrosine ammonia lyase activity.
  • the TAL is from Flavobacterium johnsoniae. It is described in the Genbank database from NCBI under the number KR095306.1 for the nucleic acid sequence and under the number AKE50827.1 for the protein sequence, and more particularly in SEQ ID Nos: 40 and 39.
  • the TAL is from Rhodotorula glutinis. It is described in the Genbank database of NCBI under the number KF765779.1 for the nucleic acid sequence and under the number AGZ04575.1 for the protein sequence, and more particularly in SEQ ID Nos: 42 and 41, respectively.
  • the tyrosine ammonia lyase is selected from the enzyme comprising a sequence chosen from SEQ ID NO: 41 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with this sequence and exhibiting tyrosine ammonia lyase activity.
  • 4CL 4-Coumarate-CoA ligase
  • 4CL is a 4-coumarate-CoA ligase. This enzyme is able to produce 4-coumaroyl-CoA from p-coumaric acid and Coenzyme A and to produce caffeoyl-CoA from caffeic acid and Coenzyme A. This enzyme belongs to the class of EC 6.2.1.12.
  • 4-coumarate-CoA ligase activity is meant the conversion of p-coumaric acid to p-coumaroyl-CoA or caffeic acid to caffeoyl-CoA by a 4-coumarate CoA ligase enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme 4-coumarate-CoA ligase, acid p -coumaric or caffeic acid, ATP and CoA under optimal conditions (pH, temperature, ions ).
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a 4-coumarate-CoA ligase.
  • this enzyme is an enzyme produced by a plant, for example Abies, Arabidopsis, Agastache, Amorpho, Brassica, Citrus, Cathayo, Cedrus, Crocus, Larix, Festuca, Glycine, Jugions, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon , Molus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Phaseolus, Pelargonium, Petroselinum, Physcomitrella, Piceo, Prunus, Pseudolarix, Pseudotsuga, Roso, Rubus, Ryzo, Saccharum, Suaeda, Pi nus, Populus, Solanum, Tritellicung Tsuga, Vitis or Zea.
  • a plant for example Abies, Arabidopsis, Agastache, Amorpho, Brassica, Citrus, Cathayo, Cedrus, Crocus, La
  • this enzyme is an enzyme produced by a microorganism, for example Aspergillus, Mycosphaerella, Mycobacterium, Neisseria, Neurospora, Streptomyces, Rhodobacter or Yarrowia.
  • a microorganism for example Aspergillus, Mycosphaerella, Mycobacterium, Neisseria, Neurospora, Streptomyces, Rhodobacter or Yarrowia.
  • this enzyme is an enzyme produced by a plant, preferably Arabidopsis thaliana, Citrus clementina or Petroselinum crispum, in particular Arabidopsis thaliana or Petroselinum crispum, or by a bacterium, preferably of the genus Streptomyces, in particular Streptomyces clavuligerus.
  • the 4-coumarate-CoA ligase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 43, 45, 47, 49, 123 and 125 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity.
  • 4-coumarate-CoA ligase is an enzyme comprising a sequence selected from SEQ ID NOs: 43, 45, 47 and 49, preferably SEQ ID NOs: 45 and 49, and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity.
  • the 4CL is Arabidopsis tholiana. It is described in the Genbank database from NCBI under the number AY099747.1 for the nucleic sequence and under the number AAM20598.1 for the protein sequence, and more particularly in SEQ ID Nos: 44 and 43, respectively.
  • the 4CL is from Petroselinum crispum. It is described in the NCBI Genbank database under number X13324.1 or X13325.1 for the nucleic acid sequence and under the number CAA31696.1 or CAA31697.1 for the protein sequence, respectively.
  • the proteins are described in UniProtKB / Swiss Prot under the reference number P14912 and P14913, respectively, and more particularly in SEQ ID Nos: 46 and 45, and 48 and 47, respectively.
  • 4CL is from Petroselinum crispum and is described in the Genbank database of NCBI under the number X13324.1 for the nucleic sequence and under the number CAA31696.1 for the protein sequence, and in UniProtKB / Swiss Prot under the number reference number P14912, and more particularly in SEQ ID Nos: 46 and 45, respectively.
  • the 4CL is from Streptomyces clavuiigerus. It is described in the Genbank database of NCBI under the number CP016559.1 for the nucleic sequence and under the number ANW18832.1 for the protein sequence, and more particularly in SEQ ID Nos: 50 and 49, respectively.
  • the 4CL is Arabidopsis tholiana.
  • a nucleotide sequence as well as the protein sequence of this enzyme are described respectively in SEQ ID NOs: 124 and 123.
  • the 4CL is from Citrus clementina and a nucleotide sequence as well as the protein sequence of this enzyme are described respectively in SEQ ID NOs: 126 and 125.
  • 4CL is an enzyme comprising a sequence selected from SEQ ID NOs: 45, 123 and 125, preferably SEQ ID NOs: 123 and 45, and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity.
  • 4CL is an enzyme comprising a sequence selected from SEQ ID NO: 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequenced and exhibiting 4-coumarate-CoA ligase activity.
  • CHS is a chalcone synthase.
  • This enzyme is able to produce naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA and to produce erodictyol-chalcone from caffeoyl-CoA and malonyl-CoA.
  • This enzyme belongs to the class of EC 2.3.1.74.
  • chalcone synthase activity is meant the transformation of p-coumaroyl-CoA and malonyl-CoA ⁇ to naringenin chalcone or of caffeoyl-CoA and malonyl-CoA-to eriodictyol chalcone by a chalcone synthase enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme Chalcone synthase, coumaroyl-CoA or caffeoyl-CoA and malonyl -CoA under optimal conditions (pH, temperature, ions ). After a certain incubation time, the appearance respectively of Naringenin chalcone or erodictyol chalcone is observed in HPLC-MS in comparison with the expected standard.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding a chalcone synthase.
  • This enzyme can be an enzyme produced by a plant, in particular of the genus Arabidopsis, Aveno, Cosmos, Citrus, Daucus, Fagopyrum, Freesia, Glycine, Glycyrrhiza, Humulus, Hypericum, Flordeum, Jugions, Medicogo, Phaseolus, Physcomitrella, Plagiochasmo, Petroselinum, Pueraria, Rubus, Secale, Scutellaria, Silene, Sinopis, Spinacia, Stellaria, Triticum, Tulipa, Verbeno, Vitis, or Xanthisma, e.g.
  • this enzyme is an enzyme produced by a plant, for example of the genus Citrus, in particular Citrus sinensis, or of Hordeum vulgare, or by a bacterium, preferably of the genus Streptomyces, in particular Streptomyces clavuligerus.
  • the chalcone synthase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 51, 53, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 53 and 55 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity.
  • the chalcone synthase is an enzyme comprising a sequence selected from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting chalcone synthase activity.
  • the CHS is from Hordeum vulgare. It is described in the Genbank database of NCBI under the number Y09233.1 for the nucleic acid sequence and under the number CAA70435.1 for the protein sequence, and more particularly in SEQ ID Nos: 52 and 51, respectively.
  • the protein is described in UniProtKB / Swiss Prot under the reference number Q96562.
  • the CHS is from Citrus sinensis. It is described in the Genbank database from NCBI under the number AB009351.1 for the nucleic acid sequence and under the number BAA81664.1 for the protein sequence, and more particularly in SEQ ID Nos: 54 and 53, respectively.
  • the CHS is from Citrus sinensis. It is described in the Genbank database of NCBI under the number XM_006489733.1 for the nucleic sequence and under the number XPJD06489796.1 for the protein sequence, and more particularly in SEQ ID Nos: 56 and 55, respectively.
  • the CHS is from Streptomyces clavuligerus. It is described in the Genbank database from NCBI under the number CP016559.1 for the nucleic sequence and under the number ANW16917.1 for the protein sequence, and more particularly in SEQ ID Nos: 58 and 57, respectively.
  • the microorganism can be modified to increase the synthesis of malonyl-CoA.
  • CHI is a chalcone isomerase. It is able to produce naringenin from Naringenin chalcone and produce eriodictyol from eriodictyol chalcone. This enzyme belongs to the class of EC 5.5.1.6.
  • Chalcone isomerase activity is meant the transformation of naringenin chalcone or erodictyol chalcone into naringenin or eriodictyol by a chalcone isomerase enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme Chalcone isomerase, naringenin chalcone or erodictyol chalcone under optimal conditions. (pH, temperature, ions ). After a certain incubation time, the appearance of Naringenin or erodictyol respectively is observed in H PLC-MS compared to the expected standard.
  • the microorganism therefore comprises a heterologous nucleic acid sequence encoding a chalcone isomerase.
  • This enzyme can come from a plant, in particular of the genus Arabidopsis, Ginkgo, Oncidium, Perilla, Citrus or Trigoneiia, for example Arabidopsis thaliana, Ginkgo biloba, Oncidium Gower Ramsey, Perilla frutescens, Citrus Sinensis or Trigoneiia foenum-graecum.
  • this enzyme is an enzyme produced by a plant, for example Arabidopsis thaliana or by a bacterium, preferably of the genus Streptomyces, in particular Streptomyces clavuligerus.
  • the chalcone isomerase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 59 and 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting chalcone isomerase activity.
  • the chalcone isomerase is selected from enzymes comprising a sequence selected from SEQ ID NO: 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting chalcone isomerase activity.
  • the CH I is from Streptomyces clavuligerus. It is described in the Genbank database of NCBI under the number CP016559.1 for the nucleic acid sequence and under the number ANW16918.1 for the protein sequence, and more particularly in SEQ ID Nos: 60 and 59, respectively.
  • the CHI is Arabidopsis thaliano. It is described in the Genbank database of NCBI under the number N M_115370.4 for the nucleic acid sequence and under the number NP_191072.1 for the protein sequence, and more particularly in SEQ ID Nos: 62 and 61, respectively.
  • FNS Flavone synthase
  • Apigenin can be prepared from naringenin using a flavone synthase (FNS). It is able to produce apigenin from naringenin.
  • FNS flavone synthase
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a flavone synthase, in particular capable of producing apigenin from naringenin and / or a heterologous nucleic acid sequence encoding a flavone synthase, in particular capable of producing luteolin from erodictyol, and / or a heterologous nucleic acid sequence encoding a flavone synthase, in particular capable of producing diosmetin from hesperetin.
  • the SNSF can be chosen from those described above.
  • the microorganism can also comprise the enzymes necessary for the synthesis of p-coumaric acid from phenylalanine.
  • the microorganism may further comprise a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H).
  • PAL belongs to the class of EC 4.3.1.24. It is able to produce cinnamic acid from phenylalanine.
  • phenylalanine ammonia lyase activity is meant the transformation of phenylalanine into frans-cinnamic acid by an enzyme Phenylalanine ammonia lyase.
  • Phenylalanine ammonia lyase activity an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme phenylalanine ammonia lyase and phenylalanine under optimal conditions (pH, temperature, ions, etc.) ⁇ After a certain incubation time, the appearance of trans-cinnamic acid is observed in UPLC-MS compared to the expected standard.
  • the enzyme is from a plant, for example a plant of the genus Arobidopsis, Agastache, Ananas, Asparagus, Brassica, Bromheadia, Bambusa, Beta, Betula, Citrus, Cucumis, Came Ilia, Capsicum, Cassia, Catharanthus, Cicer , Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Gly
  • phenylalanine ammonia lyase may further exhibit tyrosine ammonia lyase (TAL) activity and / or dihydroxyphenylalanine ammonia-lyase (DAL) activity as described below.
  • TAL tyrosine ammonia lyase
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the phenylalanine ammonia lyase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting phenylalanine ammonia lyase activity.
  • PAL is selected from enzymes comprising a sequence selected from SEQ ID NOs: 65 and 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with one of these sequences and exhibiting phenylalanine ammonia lyase activity.
  • PAL is selected from enzymes comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequences and exhibiting phenylalanine ammonia lyase activity.
  • the PAL of Citrus sinensis is described in the Genbank database of NCBI under the number XM_006481431.2 for the nucleic sequence and under the number XP_006481494.1 for the protein sequence, and more particularly in SEQs ID Nos: 64 and 63, respectively.
  • the PAL of Citrus sinensis is described in the Genbank database of NCBI under the number XM_006488000.2 for the nucleic acid sequence and under the number XP_006488063.1 for the protein sequence, and more particularly in the SEQ ID Nos: 66 and 65, respectively.
  • the PAL of Arabidopsis tholiona is described in the Genbank database of NCBI under the number NM_115186.4 for the nucleic sequence and under the number N P_190894.1 for the protein sequence, and more particularly in SEQ ID Nos: 78 and 77, respectively.
  • PTAL phenylalanine / tyrosine ammonia lyase
  • C4H belongs to the class of EC 1.14.13.11. It is able to produce p-coumaric acid from cinnamic acid.
  • trans-cinnamate 4-monooxygenase activity is meant the conversion of trans-cinnamic acid to p-coumaric acid by a trans-cinnamate 4-monooxygenase (CPR dependent) enzyme.
  • CPR dependent trans-cinnamate 4-monooxygenase activity
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme trans-cinnamate 4-monooxygenase, cinnamic acid , NADPH, H + and O2 under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of 4-hydroxycinnamate (p-coumaric acid) is observed in UPLC-MS in comparison with the expected standard.
  • the enzyme is from a plant, for example a plant of the genus Arobidopsis, Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Citrus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitreila, Phaseoius, Pi nus, Populus, Ruta , Saccharum, Solonum, Vitis, Vigna or Zea.
  • the cinnamate 4-hydroxylase (C4H) is from Citrus sinensis or Arabidopsis thaliana.
  • the cinnamate 4-hydroxylase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity.
  • C4H is selected from enzymes comprising a sequence selected from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cinnamate 4-hydroxylase activity.
  • the C4H of Citrus sinensis is described in the Genbank database of NCBI under the number NMJD01288840.1 for the nucleic sequence and under the number NP_001275769.1 for the protein sequence, and more particularly in SEQs. ID Nos: 68 and 67, respectively.
  • the C4H of Citrus sinensis is described in the Genbank database of NCBI under the number NM_001288895.1 for the nucleic sequence and under the number NP_001275824.1 for the protein sequence, and more particularly in the SEQ ID Nos: 70 and 69, respectively.
  • the C4H of Arabidopsis thaliana is described in the Genbank database of NCBI under the number NM_128601.3 for the nucleic sequence and under the number N P_180607.1 for the protein sequence, and more particularly in SEQ ID Nos: 80 and 79, respectively.
  • the biosynthesis of erodictyol can also comprise the synthesis of L-DOPA (3,4-dihydroxy-L-phenylalanine) from tyrosine followed by caffeic acid from L -DOPA (3,4-dihydroxy-L-phenylalanine).
  • L-DOPA 3,4-dihydroxy-L-phenylalanine
  • caffeic acid L -DOPA (3,4-dihydroxy-L-phenylalanine).
  • the following enzymes are required.
  • To convert tyrosine to L-DOPA (3,4-dihydroxy-L-phenylalanine) two subunits are needed, HpaB and HpaC.
  • HpaB is a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit.
  • this enzyme is an enzyme produced by a bacterium, preferably Escherichia coii.
  • 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) is an enzyme comprising a sequence chosen from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 4-hydroxyphenylacetate 3-monooxygenase activity.
  • the HpaB is from Escherichia coli. It is described in the Genbank database of NCBI under the number CAQ34705.1 for the protein sequence, and more particularly in the sequence SEQ ID No: 83. A nucleic sequence encoding this enzyme is described in the sequence SEQ ID No: 84. The protein is described in UniProtKB / Swiss Prot under the reference number A0A140NG21.
  • HpaC is a 4-hydroxyphenylacetate 3-monooxygenase reductase subunit.
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase (HpaC) subunit.
  • p-coumarate 3 hydroxylase activity is meant the conversion of p-coumaric acid to caffeic acid and / or L-tyrosine to L-Dopa using an enzyme complex composed of HpaB (4-hydroxyphenylacetate 3-hydroxylase oxidase) and HpaC (4-hydroxyphenylacetate 3-hydroxylase reductase).
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzymes HpaB, HpaC, p-coumaric acid or L- Tyrosine, FAD and NADH under optimal conditions (pH, temperature, ions ). After a certain incubation time, the appearance of caffeic acid or L-Dopa is observed by HPLC-MS in comparison with the expected standard.
  • this enzyme is an enzyme produced by a bacterium, preferably Escherichia coli.
  • HpaC 4-hydroxyphenylacetate 3-monooxygenase reductase
  • SEQ ID NO: 85 is an enzyme comprising a sequence chosen from SEQ ID NO: 85 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 4-hydroxyphenylacetate 3-monooxygenase activity.
  • the HpaC is from Escherichia coli. It is described in the Genbank database of NCBI under the number CAQ34704.1 for the protein sequence, and more particularly in the sequence SEQ ID No: 85. A sequence nucleic acid encoding this enzyme is described in the sequence SEQ ID No: 86. The protein is described in UniProtKB / Swiss Prot under the reference number A0A140NG67. Together, HpaB and HpaC are able to produce L-DOPA (3,4-dihydroxy-L-phenylalanine) from tyrosine.
  • the microorganism can therefore comprise a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) and a heterologous nucleic acid sequence encoding 4-hydroxyphenylacetate 3-monooxygenase reductase (HpaC).
  • HpaB 4-hydroxyphenylacetate 3-monooxygenase oxygenase
  • HpaC 4-hydroxyphenylacetate 3-monooxygenase reductase
  • this route also requires the presence of an enzyme capable of synthesizing caffeic acid from L-DOPA (3,4-dihydroxy-L-phenylalanine), a dihydroxyphenylalanine ammonia-lyase (DAL).
  • L-DOPA 3,4-dihydroxy-L-phenylalanine
  • DAL dihydroxyphenylalanine ammonia-lyase
  • dihydroxyphenylalanine ammonia lyase activity is meant the conversion of L-Dopa to trans-caffeic acid by a dihydroxyphenylalanine ammonia lyase enzyme.
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme dihydroxyphenylalanine ammonia lyase and L-dopa (levodopa) under the conditions optimal (pH, temperature, ions, etc.). After a certain incubation time, the appearance of trans-caffeic acid is observed in UPLC-MS in comparison with the expected standard.
  • DAL dihydroxyphenylalanine ammonia-lyase
  • TAL tyrosine ammonia lyase
  • PAL phenylalanine ammonia lyase
  • the microorganism can thus comprise a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit, a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase subunit ( HpaC) and a heterologous nucleic acid sequence encoding a dihydroxyphenylalanine ammonia-lyase (DAL).
  • HpaB 4-hydroxyphenylacetate 3-monooxygenase oxygenase
  • HpaC 4-hydroxyphenylacetate 3-monooxygenase reductase subunit
  • DAL dihydroxyphenylalanine ammonia-lyase
  • an enzyme for converting tyrosine into L-Dopa and an enzyme for converting p-coumaric acid into acid caffeic. It is respectively a 4-methoxybenzoate O-demethylase, also called 4-methoxybenzoate monooxygenase (O-demethylant) which has the L-tyrosine hydroxylase activity belonging to the class of EC 1.14.99.15 and a p- coumarate-3-hydroxylase possessing p-coumarate 3-hydroxylase activity belonging to the class of EC 1.14.13.
  • O-demethylase also called 4-methoxybenzoate monooxygenase (O-demethylant) which has the L-tyrosine hydroxylase activity belonging to the class of EC 1.14.99.15 and a p- coumarate-3-hydroxylase possessing p-coumarate 3-hydroxylase activity belonging to the class of EC 1.14.13.
  • L-tyrosine hydroxylase activity is meant the conversion of p-coumaric acid to caffeic acid and / or L-tyrosine to L-Dopa using a p-coumarate 3 hydroxylase (CPR dependent) enzyme.
  • CPR dependent p-coumarate 3 hydroxylase
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme p-coumarate 3 hydroxylase, p-coumaric acid or of L-Tyrosine, the necessary co-factors under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of caffeic acid or L-Dopa is observed by HPLC-MS in comparison with the expected standard.
  • p-coumarate 3 hydroxylase activity is meant the conversion of p-coumaric acid to caffeic acid and / or L-tyrosine to L-Dopa using a p-coumarate 3 hydroxylase (CPR dependent) enzyme.
  • CPR dependent p-coumarate 3 hydroxylase
  • an enzymatic test can be performed which consists of the in vitro incubation of a mixture composed of the enzyme p-coumarate 3 hydroxylase, p-coumaric acid or L-Tyrosine, under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of caffeic acid or L-Dopa is observed by HPLC-MS in comparison with the expected standard.
  • the recombinant microorganism can therefore comprise a heterologous nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase (CYP) capable of converting tyrosine into L-Dopa and also p coumaric acid into caffeic acid.
  • CYP 4-methoxybenzoate O-demethylase
  • 4-methoxybenzoate O-demethylase is an enzyme from bacteria, in particular from Rhodopseudomonas palustris, Pseudomonas putida, or Escherichia coli, from plants, in particular from Beta vulgaris, from mammals, in particular from Oryctolagus cunicuius, or from fungus, especially Rhodotorula glutinis.
  • 4-methoxybenzoate O-demethylase is an enzyme from Rhodopseudomonas palustris, Saccharothrix espanaensis, or Beta vulgaris.
  • 4-methoxybenzoate O-demethylase is selected from enzymes comprising a sequence chosen from SEQ ID NOs: 73 and 75 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting L-tyrosine hydrolase activity.
  • the 4-methoxybenzoate O-demethylase can also be from Beta vulgaris.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in SEQ ID Nos: 74 and 73, respectively.
  • the protein is described in UniProtKB / Swiss P rot under the reference number P0DKI2.
  • the 4-methoxybenzoate O-demethylase can be from Saccharothrix espanaensis.
  • the nucleic acid sequences encoding this enzyme and protein sequences are described in NCBI under the reference numbers NC_005296.1 and WP_011157377.1, respectively, and more particularly in SEQ ID Nos: 76 and 75.
  • the protein is described in UniProtKB / Swiss P rot under the reference number Q6N8N2.
  • the microorganism may include a heterologous nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase and a heterologous nucleic acid sequence encoding a dihydroxyphenylalanine ammonia-lyase (DAL).
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the recombinant microorganism can therefore comprise a heterologous nucleic acid sequence encoding a coumarate 3-hydroxylase (Coum3H) capable of converting p-coumaric acid into caffeic acid.
  • a heterologous nucleic acid sequence encoding a coumarate 3-hydroxylase (Coum3H) capable of converting p-coumaric acid into caffeic acid.
  • the coumarate 3-hydroxylase is an enzyme from bacteria, in particular from Saccharothrix,
  • the 4-methoxybenzoate O-demethylase is selected the enzyme comprising a sequence chosen from SEQ ID NO: 71 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting coumarate 3-hydroxylase activity.
  • nucleic acid sequence encoding this enzyme and protein sequence is described in NCBI under the reference numbers DQ357071.1 and ABC88666.1, respectively, and more particularly in SEQ ID Nos: 72 and 71.
  • the microorganism may comprise a heterologous nucleic acid sequence encoding a coumarate 3-hydroxylase and a heterologous nucleic acid sequence encoding a dihydroxyphenylalanine ammonia-lyase (DAL). Additional enzyme combination
  • the microorganism preferably comprises the enzymes allowing the production of naringenin and / or or apigenin from tyrosine and / or phenylalanine, preferably from tyrosine.
  • the microorganism comprises
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) capable of adding a glucose in position 7 of hesperetin and / or diosmetin,
  • UGT flavanone 7-O-beta-D-glucosyltransferase
  • heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) capable of transferring a rhamnose to position 6 of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O- glucoside,
  • RhaT rhamnosyltransferase
  • O-methyltransferase capable of methylating erodictyol and / or luteolin in position 4 '
  • heterologous nucleic acid sequence encoding an FNS enzyme and a heterologous nucleic acid sequence encoding a CPR enzyme and further comprises
  • TAL tyrosine ammonia lyase
  • CL 4-coumaroyl-CoA ligase
  • CHS chalcone synthase
  • CHI chalcone isomerase
  • the microorganism comprises
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in position 4 '; preferably an OMT from Citrus clementina, Citrus sinensis, Arahidopsis tholiano or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117, 119, 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and a methylation in position 4 ', preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117 and 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95%
  • a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) capable of hydroxylating naringenin and / or apigenin in position 3 'and comprising a sequence selected from SEQ ID NOs: 7, 11, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of these sequences and exhibiting the '3'-monooxygenase flavonoid activity, preferably an enzyme comprising a sequence selected from SEQ ID NOs: 7, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, and very particularly preferably an enzyme comprising a sequence selected from SEQ ID NO: 7, and polypeptides
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) and comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70 , 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, and in particular from enzymes comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity in sequence with this sequence and exhibiting cytochrome P450 reductase activity; and
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a TAL comprising a sequence chosen from SEQ ID NOs: 41 and 39 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a tyrosine ammonia lyase activity
  • TAL comprising a sequence chosen from SEQ ID NO: 41 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting tyrosine ammonia lyase activity
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) of Citrus sinensis, of Hordeum vulgore or of Streptomyces clavuligerus, in particular a CHS comprising a sequence chosen from SEQ ID NOs: 53, 51, 55 and 57 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably a CHS comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting chalcone synthase activity; and
  • a heterologous nucleic acid sequence encoding a chalcone isomerase (CH I) of Arabidopsis thaliana or Streptomyces clavuligerus in particular a CHI comprising a sequence chosen from SEQ ID NOs: 61 and 59 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone isomerase activity; preferably a CHI comprising a sequence chosen from SEQ ID NO: 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting chalcone isomerase activity .
  • CH I chalcone isomerase
  • the microorganism comprises one of the combinations of UGT, RhaT and RHM enzymes described above, in particular
  • - flavanone 7-O-beta-D-glucosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d 'sequence identity with one of these sequences and exhibiting a flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; and
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase selected from enzymes comprising a selected sequence from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP-glucose 4,6-dehydratase / UDP activity -4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase.
  • the microorganism further comprises
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65 and 77, preferably SEQ ID NOs: 65 and 77, and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a phenylalanine ammonia lyase activity, and more particularly preferably a phenylalanine am monia lyase (PAL) comprising a sequence selected from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting phenylalanine ammonia lyase activity; and
  • PAL phenylalanine ammonia lyase
  • C4H a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity; and very particularly preferably a cinnamate 4-hydroxylase (C4H) comprising a sequence chosen from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting cinnamate 4-hydroxylase activity.
  • C4H cinnamate 4-hydroxylase
  • the microorganism comprises: - a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) comprising a sequence chosen from SEQ ID NOs: 117 and 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting an O-methyltransferase activity, and very particularly preferably an OMT comprising a sequence chosen from SEQ ID NO: 117 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting O-methyltransferase activity; and
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) capable of hydroxylating naringenin and / or apigenin in position 3 'and comprising a sequence selected from SEQ ID NOs: 7, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of these sequences and exhibiting the activity flavonoid 3'-monooxygenase, preferably an enzyme comprising a sequence selected from SEQ ID NO: 7, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at less 95% identity with this sequence and exhibiting the flavonoid 3'-monooxygenase activity; and
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) and comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, preferably chosen from enzymes comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cytochrome P450 reductase activity; and
  • CPR cytochrome P450 reductase
  • a heterologous nucleic acid sequence encoding a flavone synthase comprising a sequence chosen from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavone synthase activity; and
  • TAL tyrosine ammonia lyase
  • chalcone synthase comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity in sequence with this sequence and exhibiting chalcone synthase activity; and
  • CHI chalcone isomerase
  • the microorganism comprises one of the combinations of UGT, RhaT and RHM enzymes described above, in particular
  • flavanone 7-O-beta-D-glucosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d 'sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; and
  • UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / U DP-4-keto-L-rhamnose-reductase selected from enzymes comprising a selected sequence from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP-glucose 4,6-dehydratase / UDP activity -4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase.
  • the microorganism further comprises a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H) as described. in the previous embodiment.
  • PAL phenylalanine ammonia lyase
  • C4H cinnamate 4-hydroxylase
  • the microorganism further comprises a heterologous or endogenous nucleic acid sequence encoding an S-adenosylmethionine synthetase (SAMT); in particular of Saccharomyces cerevisiae, for example a SAMT comprising a sequence chosen from SEQ ID NO: 81 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting S-adenosylmethionine synthetase activity.
  • SAMT S-adenosylmethionine synthetase
  • the microorganism comprises:
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis, preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70 , 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; in particular a flavanone 7-O-beta-D-glucosyltransferase (UGT) comprising a sequence chosen from SEQ ID NOs: 91, 93
  • a heterologous nucleic acid sequence encoding a 6 "-O-rhamnosyltransferase (RhaT) of the genus Citrus or Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus ciementina, even more preferably Citrus sinensis or Citrus ciementina, preferably a 6 "-O-rhamnosyltransferase (RhaT) comprising a sequence selected from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a 6 "-O-rhamnosyltransferase activity, and preferably an RhaT comprising a sequence chosen from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting 6 "-O-rhamnosyltrans
  • keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase a heterologous nucleic acid sequence encoding a flavonoid 3′-monooxygenase (F3′H) capable of hydroxylating in the 3 ′ position naringenin and / or apigenin; preferably Perilla frutescens var.
  • an F3'H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19 and 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 3'-monooxygenase flavonoid activity, preferably selected from among enzymes comprising a sequence selected from SEQ ID NOs: 1, 5, 7, 11, 17 and 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and having a flavonoid 3'-monooxygenase activity, in particular an F3'H comprising a sequence selected from SEQ ID NOs: 5, 7, 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharanthus rose us or of Arabidopsis thaliana; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, in particular a CPR comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position; preferably an OMT from Arabidopsis thaliana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting an O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and methylation in position 4 ', preferably selected from the enzyme comprising a sequence chosen from SEQ ID NO 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting O-methyltransferase activity; optionally, a heterologous or endogenous nucleic acid sequence en
  • a heterologous nucleic acid sequence encoding a flavone synthase (FNS) capable of converting naringenin to apigenin, erodictyol to luteolin and / or hesperetin to diosmetin, preferably of converting eriodictyol to luteolin; preferably an SNF of Arabidopsis thaliana, Lonicera japonica, Lonicera macranthoides, Medicago truncatula, Oryza sativa, Petroselinum crispum, Pop u lus delto ⁇ des, Zea mays, Callistephus chinensis, Apium graveolens, Medicago truncatula, Cuminum cyminapelium, Aethusa cynica Conium maculatum, Camellia sinensis, Cynara cardunculus var scolymus, Saussurea formuladusa, Plectrant
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) capable of producing coumaryl-CoA from p-coumaric acid and Coenzyme A; preferably Arabidopsis thaliana, Petroselinum crispum or Streptomyces clavuligerus; a 4CL comprising a sequence chosen from SEQ ID NOs: 43, 45, 47 and 49 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity, preferably a 4CL comprising a sequence selected from SEQ ID NO: 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 4-coumarate-CoA activity ligase;
  • 4CL 4-coumarate-CoA ligase
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) capable of producing naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA; preferably from Citrus sinensis, from Hordeum vulgare or from Streptomyces clavuligerus, in particular a CHS comprising a sequence chosen from SEQ ID NOs: 51, 53, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 53 and 55 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, and more particularly preferably a CHS comprising a sequence chosen from SEQ ID NO: 53 and polypeptid
  • the microorganism comprises the heterologous nucleic acid sequences encoding the enzymes UGT, RhaT, RHM, F3'H, OMT, 4CL, CHS, CHI, and optionally for the enzymes CPR, FNS and SAMT , as described in the previous embodiment and further comprises: a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting phenylalanine ammonia lyase activity, in particular a PAL comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70 , 80, 85, 90 or 95% sequence identity with this sequences and exhibiting phenylalanine ammonia lyase
  • the microorganism comprises the heterologous nucleic acid sequences encoding the enzymes UGT, RhaT, RHM, F3'H, O MT, 4CL, CHS, CHI, and optionally for the enzymes CPR, FNS and SAMT, as described in the previous embodiment and further comprises:
  • DAL dihydroxyphenylalanine ammonia-lyase
  • heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit, preferably comprising a sequence selected from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase subunit ( HpaC), preferably comprising a sequence chosen from SEQ ID NO: 85 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity; or a heterologous nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase capable of
  • the microorganism comprises the heterologous nucleic acid sequences encoding the enzymes UGT, RhaT, RHM, F3'H, OMT, 4CL, CHS, CHI, and optionally for the enzymes CPR, FNS and SAMT , as described in the previous embodiment and further comprises:
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence selected from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a phenylalanine ammonia lyase activity, preferably a PAL comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with this sequence and exhibiting phenylalanine ammonia lyase activity; and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60
  • C4H
  • a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit preferably comprising a sequence selected from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60, 70 , 80, 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity
  • a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase subunit reductase (HpaC) preferably comprising a sequence chosen from SEQ ID NO: 85 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the latter and exhibiting a 4-hydroxyphenylacetate 3-monooxygenase reductase activity
  • a heterologous nucleic acid sequence encoding a dihydroxyphenylalanine ammonia-lyase (DAL) capable of producing caffeic acid from L-DOPA (3,4-dihydroxy-L-phenylalanine).
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the microorganism comprises:
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity;
  • UGT flavanone 7-O-beta-D-glucosyltransferase
  • heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) comprising a sequence chosen from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 6 "-O-rhamnosyltransferase activity; and
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6- dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose- reductase (RH M) comprising a sequence chosen from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP- activity.
  • PAL phenylalanine ammonia lyase
  • C4H cinnamate 4-hydroxylase
  • TAL tyrosine ammonia lyase
  • chalcone synthase comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity in sequence with this sequence and exhibiting chalcone synthase activity;
  • CHS chalcone synthase
  • CHI chalcone isomerase
  • F3'H flavonoid 3'-monooxygenase
  • F3'H a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) comprising a sequence chosen from SEQ ID NO: 7 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 7 and exhibiting the flavonoid 3'-monooxygenase activity;
  • a heterologous nucleic acid sequence encoding a flavone synthase comprising a sequence chosen from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting flavone synthase activity
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity
  • CPR cytochrome P450 reductase
  • OMT O-methyltransferase
  • the microorganism further comprises a heterologous or endogenous nucleic acid sequence encoding an S-adenosylmethionine synthetase (SAMT), in particular a SAMT comprising a sequence chosen from SEQ ID NO: 81 and a polypeptide comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting S-adenosylmethionine synthetase activity.
  • SAMT S-adenosylmethionine synthetase
  • the origin of the enzymes or of a set of enzymes may be chosen so that their origin is the same or is close.
  • the enzymes or the set of enzymes can be derived from bacteria, for example bacteria of the same genus or of the same species.
  • the enzymes or the set of enzymes may be derived from plants, for example plants of the same genus or of the same species. Indeed, these common origins allow the enzymes to work together optimally.
  • the microorganisms comprise a metabolic pathway for the biosynthesis of tyrosine.
  • the microorganisms may have been modified to have an increased production of tyrosine compared to a wild strain.
  • the microorganisms may have been modified so that the carbon flow is redirected towards the biosynthesis of tyrosine.
  • the microorganisms may have been modified to reduce or eliminate feedback inhibitions of tyrosine biosynthesis.
  • the microorganisms comprise a metabolic pathway for the biosynthesis of phenylalanine.
  • the microorganisms may have been modified to have an increased production of phenylalanine compared to a wild strain.
  • the microorganisms may have been modified so that the carbon flow is redirected towards the biosynthesis of phenylalanine.
  • the microorganisms may have been modified to reduce or eliminate feedback inhibitions of phenylalanine biosynthesis.
  • the microorganisms comprise a metabolic pathway for the biosynthesis of phenylalanine and tyrosine.
  • the microorganisms may have been modified to have an increased production of phenylalanine and tyrosine compared to a wild strain.
  • the microorganisms may have been modified so that the carbon flow is redirected towards the biosynthesis of phenylalanine and tyrosine.
  • the microorganisms may have been modified to reduce or eliminate feedback inhibitions of phenylalanine and tyrosine biosynthesis.
  • Each nucleic acid sequence encoding an enzyme as described above is included in an expression cassette.
  • the coding nucleic sequences have been optimized for expression in the host microorganism.
  • the coding nucleic sequence is operably linked to the elements necessary for the expression of the gene, in particular for transcription and translation. These elements are chosen so as to be functional in the host recombinant microorganism. These elements can include, for example, transcription promoters, transcription activators, terminator sequences, initiation and termination codons. The methods for selecting these elements as a function of the host cell in which expression is desired are well known to those skilled in the art.
  • the promoter is a strong promoter.
  • the promoter can optionally be inducible.
  • the promoter can be selected from the following promoters: Lacl, LacZ, pLacT, ptac, pARA, pBAD, the bacteriophage T3 or T7 RNA polymerase promoters, the polyhedrin promoter, the lambda phage PR or PL promoter.
  • the promoter is pLac.
  • the promoter can be selected from the following promoters: the pTDH3 promoter, the pTEF1 promoter, the pTEF2 promoter, the pCCW12 promoter, the pH HF2 promoter, the pHTB2 promoter and the pRPL18B promoter.
  • inducible promoters which can be used in yeast are the tetO-2, GAL10, GAL10-CYC1, PH05 promoters.
  • All or part of the expression cassettes comprising the nucleic acid sequences encoding the enzymes as described or a combination of some of these can be included in a common expression vector or in different expression vectors.
  • the present invention therefore relates to a vector comprising two nucleic acid sequences chosen from a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT), a heterologous nucleic acid sequence encoding for a 6 ′′ -0-rhamnosyltransferase (RhaT) and a heterologous nucleic acid sequence encoding a UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase (RHM), preferably a vector comprises these three sequences.
  • the present invention therefore relates to a vector comprising two nucleic acid sequences chosen from
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arabidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis , preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; in particular a flavanone 7-O-beta-D-glucosyltransferase (UGT) comprising a sequence chosen from SEQ ID NOs
  • a heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus clementina, preferably a 6 "- O-rhamnosyltransferase (RhaT) comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting a 6 "-0-rhamnosyltransferase activity, and preferably a RhaT comprising a sequence chosen from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 6 "-O-rham
  • RHM M UDP-glucose 4,6- dehydratase / U DP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L- rhamnose reductase (RH M) from Citrus sinensis or Arabidopsis thaliana, preferably an RHM comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4 activity -keto-L-rhamnose-reductase, in particular an RHM comprising a sequence selected from SEQ ID NOs: 107, 109 and polypeptid
  • a heterologous nucleic acid sequence encoding a flavanone 7-0-beta-D-glucosyltransferase (UGT) of Arabidopsis thaliana, Scutelloria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis , preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; in particular a flavanone 7-O-beta-D-glucosyltransferase (UGT) comprising a sequence chosen from SEQ ID NOs: 91,
  • a heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus clementina, preferably a 6 "- O-rhamnosyltransferase (RhaT) comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting 6 "-O-rhamnosyltransferase activity; and
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6- dehydratase / U DP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L- rhamnose reductase (RH M) from Citrus sinensis or from Arabidopsis thaliana, preferably an RHM comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and the polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D- activity glucose 3,5-epimerase / UDP-4-keto-L-rhamnose-reductase, in particular an RHM comprising a sequence selected from SEQ ID NOs: 107, 109 and polypeptides
  • the vector comprises two nucleic acid sequences chosen from
  • a heterologous nucleic acid sequence encoding a flavanone 7-0-beta-D-glucosyltransferase selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) comprising a sequence chosen from SEQ ID NO: 113 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity sequenced with this sequence and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity;
  • a heterologous nucleic acid sequence encoding a 6 "-O-rhamnosyltransferase (RhaT) comprising a sequence selected from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting 6 "-O-rhamnosyltransferase activity; and
  • a heterologous nucleic acid sequence encoding a UDP-glucose 4,6- dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose -reductase (RHM) comprising a sequence chosen from SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting UDP activity glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose
  • the microorganism comprises all three.
  • the coding nucleic sequences and the sequences of the enzymes are as described above.
  • By comprising a nucleic acid sequence is also understood to include an expression cassette comprising the nucleic acid sequence.
  • the vector may further comprise one or more nucleic acid sequences selected from: a nucleic acid sequence encoding an O-methyltran sera (OMT), a nucleic acid sequence encoding an F3'H, a nucleic acid sequence encoding a CPR, a nucleic acid sequence encoding an FNS, a nucleic acid sequence encoding a SAMT, a nucleic acid sequence encoding a TAL, a nucleic acid sequence encoding a 4CL, a nucleic acid sequence encoding a CHS, a nucleic acid sequence encoding a CHI, a nucleic acid sequence encoding a PAL, a nucleic acid sequence encoding a C4H, a nucleic acid sequence encoding an HpaB, and a nucleic acid sequence encoding a DAL, each of these enzymes being as defined above, as well as their combinations.
  • OMT O-methyltran sera
  • F3'H a nu
  • the vector further comprises one or more sequences chosen from a nucleic acid sequence encoding an OMT, a nucleic acid sequence encoding an F3'H, a nucleic acid sequence encoding a CPR, a nucleic acid sequence encoding an FNS, a nucleic acid sequence encoding a TAL, a nucleic acid sequence encoding a 4CL, a nucleic acid sequence encoding a CHS, a nucleic acid sequence encoding for a CHI, a nucleic acid sequence encoding a PAL and a nucleic acid sequence encoding a C4H.
  • the vector can also comprise one or more nucleic acid sequences chosen from:
  • a heterologous nucleic acid sequence encoding a flavonoid 3′-monooxygenase (F3′H) capable of hydroxylating in the 3 ′ position naringenin and / or apigenin; preferably Perilla frutescens var. crispa, Petunia x hybrida, Callistephus chinensis, Gerbera hybrida, Citrus sinensis, Arabidopsis thaliana, Citrus clementina, Osteospermum hybrid cultivar, Phanerochaete chrysosporium, Streptomyces avermitilis or Pilosella officinarum, in particular Perilla frutescens var.
  • F3′H flavonoid 3′-monooxygenase
  • an F3'H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19, 21 and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting 3'-monooxygenase flavonoid activity, preferably selected from enzymes comprising a sequence chosen from SEQ ID NOs: 1, 5, 7, 11, 17, 19 and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 3'-monooxygenase flavonoid activity, in particular an F3'H comprising a sequence selected from SEQ ID NOs: 5, 7, 17 and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85
  • cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharanthus roseus or of Arabidopsis thaliona; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, and preferably a CPR comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95 % sequence identity
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position; preferably an OMT from Arabidopsis thaliana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117, 119, 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting an O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and a methylation in position 4 ', preferably selected from the enzyme comprising a sequence chosen from SEQ ID NO: 117, 119 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting O-methyl
  • a heterologous nucleic acid sequence encoding a flavone synthase (FNS) capable of converting naringenin into apigenin, erodictyol into luteolin and / or hesperetin into diosmetin, preferably of converting erodictyol into luteolin; preferably an SNF of Arabidopsis thaliana, Lonicera joponica, Lonicera macranthoides, Medicago truncatula, Oryza sativa, Petroselinum crispum, Populus delto ⁇ des, Zea mays, Callistephus chinensis, Apium graveolens, Medicago truncatula, Cuminum cyminapelium, Aethusa cynica arcelica, Cuminum cyminapelium, Aethusa cynica maculatum, Camellia sinensis, Cynara cardunculus
  • SAMT S-adenosylmethionine synthetase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) capable of producing naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA; preferably from Citrus sinensis, from Hordeum vulgare or from Streptomyces clovuligerus, in particular a CHS comprising a sequence chosen from SEQ ID NOs: 51, 53, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 53 and 55 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, and preferably a CHS comprising a sequence chosen from SEQ ID NO: 53 and polypeptides
  • a heterologous nucleic acid sequence encoding a chalcone isomerase capable of producing naringenin from naringenin chalcone; preferably Arabidopsis thaliana or Streptomyces clovuligerus, in particular a CH I comprising a sequence chosen from SEQ ID NOs: 59 and 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with one of these sequences and exhibiting chalcone isomerase activity, and preferably a CHI comprising a sequence chosen from SEQ ID NO: 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting chalcone isomerase activity;
  • CH I chalcone isomerase
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting phenylalanine ammonia lyase activity, preferably a PAL comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting phenylalanine ammonia lyase activity; and
  • a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity, capable of producing p-coumaric acid from phenylalanine, and preferably a C4H comprising a sequence chosen from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cinnamate 4-hydroxylase activity;
  • nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (H paB) subunit preferably comprising a sequence chosen from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity
  • a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase (HpaC) subunit preferably comprising a sequence chosen from SEQ ID NO: 85 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity therewith and exhibiting 4- activity hydroxyphenylacetate 3-monooxygenase reductase; or a nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase capable of converting tyrosine into
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the vector can also comprise one or more nucleic acid sequences chosen from:
  • a heterologous nucleic acid sequence encoding a flavonoid 3′-monooxygenase (F3′H) capable of hydroxylating in the 3 ′ position naringenin and / or apigenin; preferably Perilla frutescens var.
  • F3′H flavonoid 3′-monooxygenase
  • an F3′H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17, 19 and 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavonoid 3'-monooxygenase activity, preferably selected from among enzymes comprising a sequence chosen from SEQ ID NOs: 1, 5, 7, 11, 17 and 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting flavonoid 3'-monooxygenase activity, in particular an F3'H comprising a sequence selected from SEQ ID NOs: 5, 7, 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharanthus roseus or of Arabidopsis thaliana; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity; a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating ero
  • a heterologous nucleic acid sequence encoding a flavone synthase (FNS) capable of converting naringenin into apigenin, erodictyol into luteolin and / or hesperetin into diosmetin, preferably of converting erodictyol into luteolin; preferably an SNF of Arabidopsis thaliana, Lonicera japonica, Lonicera macranthoides, Medicago truncatula, Oryza sativa, Petroselinum crispum, Populus delto ⁇ des, or Zea mays, preferably of Lonicera japonica, Lonicera macranthoides, and Petroselinum crispum; preferably an FNS comprising a sequence chosen from SEQ ID NOs: 33, 35, 37 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone syntha
  • SAMT S-adenosylmethionine synthetase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) capable of producing coumaryl-CoA from p-coumaric acid and Coenzyme A; preferably Arabidopsis thaliana, Petroselinum crispum or Streptomyces clavuligerus; a 4CL comprising a sequence chosen from SEQ ID NOs: 43, 45, 47 and 49 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity;
  • 4CL 4-coumarate-CoA ligase
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) capable of producing naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA; preferably from Citrus sinensis, from Hordeum vulgare or from Streptomyces clavuligerus, in particular a CHS comprising a sequence chosen from SEQ ID NOs: 51, 53, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 53 and 55 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity;
  • CHS chalcone synthase
  • a heterologous nucleic acid sequence encoding a chalcone isomerase capable of producing naringenin from naringenin chalcone; of preference of Arabidopsis thaliana or Streptomyces clavuligerus, in particular a CH I comprising a sequence chosen from SEQ ID NOs: 59 and 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity in sequence with one of these sequences and exhibiting chalcone isomerase activity;
  • CH I chalcone isomerase
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting phenylalanine ammonia lyase activity; and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity, capable of producing p-coumaric acid from phenylalanine;
  • PAL phenylalanine ammonia lyase
  • nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit preferably comprising a sequence chosen from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity
  • a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase (HpaC) subunit preferably comprising a sequence chosen from SEQ ID NO: 85 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity therewith and exhibiting 4-hydroxyphenylacetate activity 3-monooxygenase reductase; or a nucleic acid sequence encoding a 4-methoxybenzoate O-demethylase capable of converting tyrosine into L
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the vector can also comprise one or more nucleic acid sequences chosen from:
  • a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase comprising a sequence chosen from SEQ ID NOs: 7, 11, 17, and 121 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavonoid 3'-monooxygenase activity, preferably selected from the enzymes comprising a sequence chosen from SEQ ID NOs: 7, 17 and 121 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a 3'-monooxygenase flavonoid activity, and very particularly preferably an F3'H comprising a sequence selected from SEQ ID NO: 7 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting flavonoid 3'-monooxygen
  • a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, and very particularly preferably from enzymes comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cytochrome P450 reductase activity;
  • CPR cytochrome P450 reductase
  • SAMT S-adenosylmethionine synthetase
  • a heterologous nucleic acid sequence encoding a tyrosine ammonia lyase (TAL) capable of producing p-coumaric acid from tyrosine and comprising a sequence selected from SEQ ID NO: 41 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting tyrosine ammonia lyase activity;
  • TAL tyrosine ammonia lyase
  • 4CL 4-coumarate-CoA ligase capable of producing coumaryl-CoA from p-coumaric acid and from Coenzyme A and comprising a sequence chosen from SEQ ID NOs: 123, 125 and 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity, preferably comprising a sequence selected from SEQ ID NO: 45 and
  • heterologous nucleic acid sequence encoding a chalcone synthase (CHS) capable of producing naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA and comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting chalcone synthase activity;
  • CHS chalcone synthase
  • a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H) and comprising a sequence chosen from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% d sequence identity with this sequence and exhibiting cinnamate 4-hydroxylase activity, capable of producing p-coumaric acid from phenylalanine.
  • C4H cinnamate 4-hydroxylase
  • the vector comprises a nucleic acid sequence encoding an O-methyltransferase, in particular capable of methylating erodictyol and / or luteolin in position 4 'and a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H ), in particular capable of adding a hydroxyl at the 3 'position of naringenin and / or apigenin; or
  • heterologous nucleic acid sequence encoding an O-methyltransferase capable of methylating erodictyol and / or luteolin in the 4 'position; a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) capable of hydroxylating in the 3 'position naringenin and / or apigenin; a heterologous nucleic acid sequence encoding a cytochrome P450 reductase; and a heterologous nucleic acid sequence encoding flavone synthase (FNS) capable of converting naringenin to apigenin, erodictyol to luteolin and / or hesperetin to diosmetin, preferably of converting eriotdictyol to luteolin.
  • FNS flavone synthase
  • the vector can thus comprise several nucleic acid sequences chosen from among them, in particular 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 nucleic acid sequences chosen from among them.
  • the vector can in particular comprise combinations of particular coding sequences as described above.
  • the vectors comprise heterologous coding sequences as long as the coding sequences can be optimized for the host microorganism, be under the control of a heterologous promoter and / or can combine coding sequences which are not from the same organism of origin and / or which are not present in the same arrangement.
  • the vector can be any DNA sequence into which it is possible to insert foreign nucleic acids, the vectors allowing the introduction of foreign DNA into the host microorganism.
  • the vector can be for example a plasmid, a phagemid, a cosmid, an artificial chromosome, in particular a YAC, or a BAC.
  • Expression vectors can include nucleic acid sequences encoding selection markers.
  • the selection markers can be genes for resistance to one or more antibiotics or genes for auxotrophy.
  • the auxotrophy gene can for example be URA3, LEU2, HIS3 or TRP1.
  • the antibiotic resistance gene may for example preferably be a resistance gene to ampicillin, kanamycin, hygromycin, geneticin and / or nureseothricin.
  • the host microorganism may be transiently or stably transformed / transfected and the nucleic acid, cassette or vector may be contained therein as an episome or as integrated into the genome of the host microorganism.
  • the expression vector can also comprise one or more sequences allowing the targeted insertion of the vector, of the expression cassette or of the nucleic acid into the genome of the host microorganism.
  • the expression cassettes comprising the nucleic acid sequences encoding the enzymes as described above or a combination of some of these can be inserted into the / a chromosome of the recombinant microorganism.
  • all or part of the expression cassettes comprising the nucleic acid sequences encoding the enzymes as described or a combination of some of these can be preserved in episomal form, in particular in the form of a plasmid.
  • the microorganism can comprise several copies of nucleic acid sequences encoding an enzyme as described above. In particular, it can comprise 2 to 10 copies, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a nucleic acid sequence encoding an enzyme as described above.
  • the present invention relates to a method for preparing a microorganism according to the present invention comprising the introduction of nucleic acid sequences encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT), in particular capable of add glucose in position 7 of hesperetin and diosmetin; for a 6 "-O-rhamnosyltransferase (RhaT), in particular capable of transferring a rhamnose to the 6-position of the glucose of hesperetin-7-O-glucoside and / or diosmetin-7-O-glucoside; and for a UDP -glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP- 4-keto-L-rhamnose-reductase (RHM), in particular capable of producing UDP-rhamnose in the microorganism and the selection of microorganism
  • the method can further comprise the introduction of one or more nucleic acid sequences chosen from:
  • a heterologous nucleic acid sequence encoding a flavonoid 3′-monooxygenase (F3′H) capable of hydroxylating in the 3 ′ position naringenin and / or apigenin; preferably Perilla frutescens var. crispa, Petunia x hybrida, Callistephus chinensis, Gerbera hybrida, Citrus sinensis, Citrus clementina, Osteospermum hybrid cultivar, Phanerochaete chrysosporium, Streptomyces avermitilis or Pilosella officinarum, in particular Perilla frutescens var.
  • F3′H flavonoid 3′-monooxygenase
  • an F3'H comprising a sequence chosen from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 15, 17 , 19 and 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 3'-monooxygenase flavonoid activity, preferably selected from among enzymes comprising a sequence selected from SEQ ID NOs: 1, 5, 7, 11, 17 and 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavonoid 3'-monooxygenase activity, in particular an F3'H comprising a selected sequence from SEQ ID NOs: 5, 7, 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90
  • cytochrome P450 reductase preferably a CPR of Saccharomyces cerevisiae, or of a plant, for example of Catharonthus roseus or of Arabidopsis thaliono; preferably a CPR comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with a of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, in particular a CPR comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least
  • a heterologous nucleic acid sequence encoding an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position; preferably an OMT from Arabidopsis thaliana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 87 and 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95 % sequence identity with one of these sequences and exhibiting an O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and methylation in position 4 ', preferably selected from the enzyme comprising a sequence chosen from SEQ ID NO 89 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting O-methyltransferase activity; a heterologous nucleic acid sequence encoding a flavone synth
  • SAMT S-adenosyl methionine synthetase
  • a SAMT comprising a sequence chosen from SEQ ID NO: 81 and a polypeptide comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting S-adenosyl methionine synthetase activity
  • TAL tyrosine ammonia lyase
  • a heterologous nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) capable of producing coumaryl-CoA from p-coumaric acid and Coenzyme A; preferably Arahidopsis thaliana, Petroselinum crispum or Streptomyces clavuligerus; a 4CL comprising a sequence chosen from SEQ ID NOs: 43, 45, 47 and 49 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity, and preferably a 4CL comprising a sequence selected from SEQ ID NO: 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity sequenced with this sequence and exhibiting 4-coumarate-CoA ligase activity;
  • 4CL 4-coumarate-CoA ligase
  • a heterologous nucleic acid sequence encoding a chalcone synthase (CHS) capable of producing naringenin-chalcone from 4-coumaroyl-CoA and malonyl-CoA; preferably from Citrus sinensis, from Hordeum vulgare or from Streptomyces clavuligerus, in particular a CHS comprising a sequence chosen from SEQ ID NOs: 51, 53, 55 and 57 and polypeptides comprising a sequence having at least 60, 70, 80, 85 , 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 53 and 55 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity, and more particularly preferably a CHS comprising a sequence selected from SEQ ID NO: 53 and polypeptid
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65, 77 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a phenylalanine ammonia lyase activity, in particular a PAL comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70 , 80, 85, 90 or 95% sequence identity with this sequence and exhibiting phenylalanine ammonia lyase activity; and
  • a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity, capable of producing p-coumaric acid from phenylalanine, in particular a C4H comprising a selected sequence from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting cinnamate 4-hydroxylase activity; a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) subunit, preferably comprising a sequence chosen from SEQ ID NO: 83 and polypeptides comprising a sequence having at least 60
  • DAL dihydroxyphenylalanine ammonia-lyase
  • the method comprises the introduction:
  • a heterologous nucleic acid sequence encoding a flavanone 7-O-beta-D-glucosyltransferase (UGT) from Arahidopsis thaliana, Scutellaria baicalensis or Homo sapiens, preferably Arabidopsis thaliana or Scutellaria baicalensis , preferably a flavanone 7-O-beta-D-glucosyltransferase (UGT) selected from enzymes comprising a sequence chosen from SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavanone 7-O-beta-D-glucosyltransferase activity; in particular a flavanone 7-O-beta-D-glucosyltransferase (UGT) comprising a sequence chosen from SEQ ID NO
  • a heterologous nucleic acid sequence encoding a 6 "-0-rhamnosyltransferase (RhaT) of the genus Citrus or of Petunia hybrida, preferably Citrus sinensis, Citrus maxima, or Citrus clementina, even more preferably Citrus sinensis or Citrus clementina, preferably an RhaT comprising a sequence chosen from SEQ ID NOs: 103, 105 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a 6 "-0-rhamnosyltransferase activity, in particular a 6" -0-rhamnosyltransferase (RhaT) comprising a sequence chosen from SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting 6 "-O-rhamnosyl
  • RHM M UDP-glucose 4,6- dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4-keto-L-rhamnose -reductase (RH M) of Citrus sinensis or Arabidopsis thaliana, preferably an RHM comprising a sequence chosen from SEQ ID NOs: 107, 109 and 111 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting UDP-glucose 4,6-dehydratase / UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase / UDP-4- activity keto-L-rhamnose reductase, in particular an RHM comprising a sequence selected from SEQ ID NO: 107 and polypeptides comprising a sequence having
  • OMT O-methyltransferase
  • a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT) capable of methylating erodictyol and / or luteolin in the 4 'position preferably, an OMT from Citrus clementina, Citrus sinensis, Arabidopsis thaüana or Homo sapiens, preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117, 119, 87 and 89 and polypeptides comprising a sequence having at least at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting O-methyltransferase activity, in particular with erodictyol and / or luteolin as substrate and methylation in the 4 'position , preferably an OMT comprising a sequence chosen from SEQ ID NOs: 117 and 119 and polypeptides comprising a sequence having at least 60, 70, 80, 85
  • a heterologous nucleic acid sequence encoding a flavonoid 3'-monooxygenase (F3'H) capable of hydroxylating naringenin and / or apigenin in position 3 'and comprising a sequence selected from SEQ ID NOs: 7, 11, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85%, at least 90% or at least 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, preferably an enzyme comprising a sequence selected from SEQ ID NOs: 7, 17 and 121, and polypeptides comprising a sequence having at least 75%, at least 80%, at least 85 %, at least 90% or at least 95% identity with one of these sequences and exhibiting the flavonoid 3'-monooxygenase activity, and very particularly preferably an enzyme comprising a sequence selected from SEQ ID NO: 7, and polypeptides comprising
  • cytochrome P450 reductase a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) and comprising a sequence chosen from SEQ ID NOs: 23, 25, 27, 29 and 31 and polypeptides comprising a sequence having at least 60, 70, 80 , 85, 90 or 95 % sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, preferably from enzymes comprising a sequence chosen from SEQ ID NOs: 23, 25 and 29 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cytochrome P450 reductase activity, in particular a CPR comprising a sequence chosen from SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting cytochrome P450 reductase activity; and
  • a heterologous nucleic acid sequence encoding an FNS comprising a sequence chosen from SEQ ID NOs: 33, 35, 37, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149 , 151, 153, 155, 157 and 159 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting flavone synthase activity, in particular a flavone synthase (FNS) and comprising a sequence selected from SEQ ID NOs: 33, 35, 37 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a flavone synthase activity, preferably a flavone synthase (FNS) comprising a sequence chosen from SEQ ID NO: 37 and a polypeptide comprising a sequence having at least
  • SAMT S-adenosylmethionine synthetase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • a TAL comprising a sequence chosen from SEQ ID NOs: 41 and 39 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a tyrosine ammonia lyase activity
  • TAL comprising a sequence chosen from SEQ ID NO: 41 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% of sequence identity with this sequence and exhibiting tyrosine ammonia lyase activity
  • a heterologous nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL) from Arabidopsis thaliana, Citrus clementina, Petroselinum crispum or Streptomyces clavuligerus; in particular, a 4CL comprising a sequence chosen from SEQ ID NOs: 123, 125, 45, 43, 47 and 49 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequenced with one of these sequences and exhibiting 4-coumarate-CoA ligase activity; preferably a 4CL comprising a sequence selected from SEQ ID NOs: 123, 125 and 45 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4-coumarate-CoA ligase activity; very particularly preferably a 4CL comprising a sequence selected from SEQ ID NO: 45 and polypeptide
  • chalcone synthase of Citrus sinensis, of Hordeum vulgare or of Streptomyces clavuligerus
  • a CHS comprising a sequence chosen from SEQ ID NOs: 53, 51, 55 and 57 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone synthase activity
  • a CHS comprising a sequence chosen from SEQ ID NO: 53 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with that sequence and exhibiting chalcone synthase activity
  • a heterologous nucleic acid sequence encoding a chalcone isomerase (CHI) of Arabidopsis thaliana or Streptomyces clavuligerus in particular a CHI comprising a sequence chosen from SEQ ID NOs: 61 and 59 and polypeptides comprising a sequence having at least 60 , 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting chalcone isomerase activity; preferably a CHI comprising a sequence chosen from SEQ ID NO: 61 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting chalcone isomerase activity .
  • the method comprises the introduction of all of these sequences.
  • the method further comprises introducing:
  • a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase in particular a PAL comprising a sequence chosen from SEQ ID NOs: 63, 65 and 77, preferably SEQ ID NOs: 65 and 77, and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting a phenylalanine ammonia lyase activity, and more particularly preferably a phenylalanine ammonia lyase (PAL) comprising a sequence chosen from SEQ ID NO: 65 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with this sequence and exhibiting phenylalanine ammonia lyase activity; and
  • PAL phenylalanine ammonia lyase
  • C4H a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), in particular a C4H comprising a sequence chosen from SEQ ID NOs: 67, 69, 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting cinnamate 4-hydroxylase activity; and very particularly preferably a cinnamate 4-hydroxylase (C4H) comprising a sequence chosen from SEQ ID NO: 79 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with this sequence and exhibiting cinnamate 4-hydroxylase activity.
  • C4H cinnamate 4-hydroxylase
  • the method comprises the introduction of combinations of particular coding sequences as described above.
  • the present invention relates to the use of a microorganism according to the present invention for the production of diosmin and / or hesperidin.
  • it relates to the use of a microorganism according to the present invention for the production of diosmin.
  • it relates to the use of a microorganism according to the present invention for the production of hesperidin.
  • it relates to the use of a microorganism according to the present invention for the production of diosmin and hesperidin.
  • the present invention also relates to a method of producing diosmin and / or hesperidin comprising the cultivation of a microorganism according to the present invention, in particular under conditions allowing or favorable to the production of diosmin and / or hesperidin and optionally the recovery and / or purification of the diosmin and / or hesperidin produced.
  • the conditions for culturing the microorganism according to the invention can be adapted according to conventional techniques, well known to those skilled in the art.
  • the microorganism is cultivated in an appropriate culture medium.
  • suitable culture medium generally designates a culture medium providing nutrients which are essential or beneficial for the maintenance and / or growth of said microorganism, such as carbon sources; nitrogenous sources such as ammonium sulfate; sources of phosphorus, for example, potassium phosphate monobasic; trace elements, for example copper, iodide, iron, magnesium, zinc or molybdate salts; vitamins and other growth factors such as amino acids or other growth promoters.
  • a defoamer can be added as needed.
  • this suitable culture medium can be chemically defined or complex.
  • the culture medium can thus be of identical or similar composition to a synthetic medium, as defined by Verduyn et al., (Yeast.
  • the culture medium can comprise a simple carbon source, such as glucose, fructose, xylose, ethanol, glycerol, galactose, sucrose, cellulose, cellobiose, starch, polymers of glucose, molasses, or byproducts of these sugars.
  • a simple carbon source such as glucose, fructose, xylose, ethanol, glycerol, galactose, sucrose, cellulose, cellobiose, starch, polymers of glucose, molasses, or byproducts of these sugars.
  • the production of diosmin and / or hesperidin by the microorganism according to the invention is obtained without supplying naringenin, apigenin, erodictyol, luteolin, hesperetin and / or diosmetin in the medium of culture, preferably without addition of naringenin, apigenin, erodictyol, luteolin, hesperetin and diosmetin in the culture medium.
  • any mode of culture allowing the production on an industrial scale of molecules of interest can be envisaged.
  • the culture is carried out in bioreactors, in particular in batch, fed-batch, chemostat and / or continuous culture mode. Controlled vitamin supply during the process may also benefit productivity (Alfenore et al., Appl Microbiol Biotechnol. 2002. 60: 67-72).
  • the culture is generally carried out in bioreactors, with possible steps of solid and / or liquid precultures in Erlenmeyer flasks, with an appropriate culture medium.
  • the culture conditions of the microorganisms according to the invention are easily adaptable by a person skilled in the art, depending on the microorganism.
  • the culture temperature is in particular for yeasts between 20 ° C and 40 ° C, preferably between 28 ° C and 35 ° C, and more particularly around 30 ° C for cerevisiae.
  • the microorganism according to the present invention can be cultured for 1 to 30 days, and preferably 1 to 10 days.
  • a microorganism according to the present invention is capable of producing diosmin and / or hesperidin in a minimum amount of 1 mg / l of culture medium, preferably 10, 20, 25, 30, 35, 40, 45 , 50, 55, 60, 65, 70, 75, 80, 85, 90, 95, 100 mg / l of culture medium, optionally 150, 200, 250, 300, 350, 400, 450, 500, 600, 700, 800, 900 or 1000 mg / l of culture medium.
  • Figure 1 Description of the metabolic pathways of hesperidin and diosmin production.
  • Figure 2 Production of erodictyol from naringenin by the strain FL_405 (F3'H4 + CPR2). Control strain: CF235. Observation of the disappearance of the naringenin peak and the appearance of an erodictyol peak in the FL-405 strain.
  • Figure 3 Production of luteolin from apigenin by the strain FL_405 (F3′H4 + CPR2). Control strain: CF235. Observation of the disappearance of the apigenin peak and appearance of a luteolin peak in strain FL-405.
  • Figure 4 Production of Apigenin from naringenin by strain SC744 (FNSII1 + CPR2). Control strain: CF234. Observation of the disappearance of the naringenin peak and appearance of an apigenin peak in the strain.
  • Figure 5 Production of luteolin from erodictyol SC744 (FNSII1 + CPR2). Control strain: CF234. Observation of the disappearance of the erodictyol peak and appearance of a luteolin peak in the strain.
  • Figure 6 Production of erodictyol and luteolin by strain SC1500. Control strain: CF237. Observation of erodictyol and luteolin peaks.
  • Figure 7 Production of hesperetin from erodictyol by strains SC 1612 (MET + SAM) and SC 1614 (MET + SAM). Control strain: CF235. Observation of the disappearance of the erodictyol peak and appearance of a hesperetin peak in the strains.
  • Figure 8 Production of diosmetin from luteolin by strain SC 1612 (MET + SAM) and SC 1614 (MET + SAM). Control strain: CF235. Observation of the disappearance of the luteolin peak and appearance of a diosmetin peak in the strains.
  • Figure 9 Production of diosmetin from hesperetin by strain SC744 (FNSII + CPR). Control strain: CF234. Observation of the disappearance of the hesperetin peak and appearance of a diosmetin peak in the strain.
  • Figure 10 Production of hesperetin from erodictyol by E. coli EC26 (MET + SAM).
  • Control strain E. coli MH1. Observation of the disappearance of the erodictyol peak and appearance of a hesperetin peak in the strain.
  • FIG. 11 Production of diosmetin from luteolin by E. coli EC26 (MET + SAM).
  • Control strain E. coli MH1. Observation of the disappearance of the luteolin peak and appearance of a diosmetin peak in the strain.
  • Figure 12 Production of diosmetin from hesperetin by E. coli EC30 (FNSII).
  • Control strain E. coli MH1. Observation of the disappearance of the hesperetin peak and appearance of a diosmetin peak in the strain.
  • Figure 13 Production of hesperetin and diosmetin by strain SC1508. Control strain: CF237. Observation of hesperetin peaks in diosmetin.
  • Figure 14 Production of hesperidin from hesperetin by strain FL 547 (GT + RHM + RHAT). Control strain: CF 233. Observation of the disappearance of the hesperetin peak and appearance of the hesperidin peak.
  • Figure 15 Production of diosmin from diosmetin by strain FL 547 (GT + RHM + RHAT). Control strain: CF 233. Observation of the disappearance of the disometin peak and appearance of the diosmin peak.
  • Figure 16 Production of hesperedin from hesperetin by E. coli EC38, EC45 and EC47 (GT + RHM + RHAT). Control strain: E. coli MH1. Observation of the disappearance of the hesperetin peak and appearance of the hesperidin peak.
  • Figure 17 Production of diosmin from diosmetin by E. coli EC38, EC45 and EC47 (GT + RH M + RHAT). Control strain: E. coli MH1. Observation of the disappearance of the diosmetin peak and appearance of the diosmin peak.
  • Figure 18 Production of hesperidin and diosmin by strains SC1509, SC1530, SC1529, SC1568 and SC2410. Control strain: CF237. Observation of hesperidin and diosmin peaks.
  • Figure 19 Production of erodictyol and luteolin by strains SC2424, SC2425, SC2426, SC2427, SC2428 and SC1500.
  • Control strain CF237.
  • Figure 20 Production of hesperetin and homoeriodictyol by strains SC2147, SC2151, SC1612 and SC1614. Control strain: CF235.
  • Figure 21 Production of diosmetin and chrysoeriol by strains SC2147, SC2151, SC1612 and SC1614. Control strain: CF235.
  • Figure 22 Production of hesperetin from erodictyol by E. coli EC41 (M ET + SAM).
  • Control strain E. coli MH1. Observation of the disappearance of the erodictyol peak and appearance of a hesperetin peak in the strain.
  • Figure 23 Production of hesperetin from erodictyol by E. coli EC43 (M ET + SAM).
  • Control strain E. coli MH1. Observation of the disappearance of the erodictyol peak and appearance of a hesperetin peak in the strain.
  • Figure 24 Production of diosmetin from luteolin by E. coli EC43 (MET + SAM).
  • Control strain E. coli MH1. Observation of the disappearance of the luteolin peak and appearance of a diosmetin peak in the strain.
  • Figure 25 Production of hesperetin and diosmetin by strain SC2408. Control strain: CF237. Observation of hesperetin peaks in diosmetin.
  • Figure 26 Production of hesperetin and diosmetin by strain SC2409. Control strain: CF237. Observation of hesperetin peaks in diosmetin.
  • Figure 27 Production of hesperetin and diosmetin by strains SC2408, SC2409 and SC1508. Control strain: CF237.
  • Figure 28 Production of hesperidin and diosmin by strains SC1579, SC1584, SC1621 and SC1626.
  • yeasts used in the examples were obtained from Saccharomyces cerevisiae FY1679-28A (Tettelin et al., 1995 https://doi.org/10.1016/S1067-
  • This yeast has a quadri auxotrophy for uracil, tryptophan, histidine and leucine.
  • the strains of bacteria used in the examples were obtained from Escherichia coli MH1.
  • the genes optimized for expression in yeast have been synthesized by Eurofins genomics, Ebersberg, Germany or Biomatik, Cambridge, Canada or Twist Biosciences, San Francisco, USA or DC Biosciences, Dundee, UK.
  • the cpr2 gene SEQ ID NO: 26 was amplified from genomic DNA.
  • the genes obtained by synthesis or by PCR comprise, at the 5 'and 3' end, a BbsI (GAAGAC) or Bsal (GGTCTC) restriction site.
  • the pSBK vector comprises a yeast selection marker URA, or LEU or TRP or HIS and the vector pSB1K3 comprises a kanamycin resistance marker.
  • the strains were cultured in 1 ml of minimum nitrogen base medium (Dutscher, Brumath, Fr) supplemented with glucose at 20 g / l for the yeasts and in 1 ml of M9 supplemented with glucose at 4 gl 1 for E. coli in a 24-well plate (Starlab, Orsay, Fr) at 30 ° C. for 72 h with continuous stirring at 200 RPM.
  • minimum nitrogen base medium Dutscher, Brumath, Fr
  • M9 supplemented with glucose at 4 gl 1 for E. coli in a 24-well plate (Starlab, Orsay, Fr) at 30 ° C. for 72 h with continuous stirring at 200 RPM.
  • naringenin or apigenin was added at a concentration of 100 mg.l 1 to determine the activity of F3'H
  • naringenin or erodictyol was added at a concentration of 100 mg.l 1 to determine the activity of FNSII
  • erodictyol or luteolin was added at a concentration of 100 mg.l 1 to determine the activity of METs
  • hesperetin or diosmetin was added added at a concentration of 100 mg.l 1 to determine the activity of GTs
  • hesperetin 7-O-glucioside and or diosmetin 7-O-glucoside was determined the activity of RFIM and RFIAT.
  • Each strain was inoculated at OD 0.2 from a 24 hour pre-culture cultivated under the same conditions.
  • the final concentrations of the internal standards are: Diosmin C13 0.5 mg / L
  • Mobile phase A is a solution of 0.1% formic acid in LC / MS grade water and mobile phase B is a solution of 0.1% formic acid in pure acetonitrile of LC / MS quality.
  • the temperature of the column is 50 ° C and the temperature of the autosampler is 10 ° C.
  • Two chromatographic conditions could be used for the detection of the flavonoids of interest:
  • the ions monitored and the fragmentation conditions for the molecules of interest are:
  • F3′H Constructs for each of the F3′H were carried out in a vector carrying the URA selection marker (Table 6). Constructs each comprising SAM2 and only one of the different CPRs were created in a vector carrying the LEU selection marker (Table 7). Two vectors comprising only the URA or LEU selection marker were also created as a control. The marker genes make it possible to identify and select the cells which have integrated the gene of interest. [Table 6] List of the different F3′H constructions tested.
  • strain FL 405 has the constructs FL 26 and FL 401.
  • the control strain (without the genes) having the TT URA and TT LEU constructs is called CF235.
  • FNSII For each of the following FNSII, constructs in a TRP vector were performed (Table 8). The same vectors with the LEU selection marker each containing SAM2 and a different CPR were used to test FNSII (Table 9).
  • strain SC 744 has the constructs FL 620 and FL 401.
  • the control strain (without the genes) possessing the TT TRP and TT LEU constructs is named CF234.
  • strain SC1500 comprises the constructions FL 26, FL 602, FL 808 and FL 822;
  • strain SC2424 comprising the constructions FL 1031 + FL 602 + FL 822 + TT HIS;
  • strain SC2425 comprising the constructions FL 26 + FL 602 + FL 822 + TT HIS;
  • strain SC2426 comprising the constructions FL 31 + FL 602 + FL 822 + TT HIS;
  • the strain SC2427 comprises the constructions FL 1031, FL 602, FL 808 and FL 822; and - strain SC2428 comprising the constructions FL 31 + FL 602 + FL 808 + FL 822.
  • the control strain (without the genes) possessing the constructions TT LIRA, TT TRP, TT HIS and TT LEU is named CF237.
  • SC2429 FL 1111 + FL 1031 + FL 121 SC2451: FL 1113 + FL 26 + FL 121 SC2430: FL 1112 + FL 1031 + FL 121 SC2452: FL 1114 + FL 26 + FL 121 SC2431: FL 1113 + FL 1031 + FL 121 25 SC2453: FL 1115 + FL 26 + FL 121 SC2432: FL 1114 + FL 1031 + FL 121 SC2459: FL 1111 + FL 26 + TT LEU SC2433: FL 1115 + FL 1031 + FL 121 SC2460: FL 1112 + FL 26 + TT LEU SC2439: FL 1111 + FL 1031 + TT LEU SC2461: FL 1113 + FL 26 + TT LEU SC2440: FL 1112 + FL 1031 + TT LEU SC2462: FL 1114 + FL 26 + TT LEU SC2441: FL 1113 + FL 1031 + TT LEU 30 SC2463: FL 1115 + FL 26 + TT LEU SC2442: FL 1114 + FL 1031 + FL
  • SC2450 FL 1112 + FL 26 + FL 121
  • the control strain (without the genes) possessing the constructions TT LIRA, TT TRP, TT HIS and TT LEU is named CF237.
  • the strain SC1508 comprises the constructions FL 121 + FL 268 + FL 602 + FL 808 of table 14.
  • the strain SC2408 comprises the constructions FL 121 + FL 469 + FL 602 + FL 808 of table 14.
  • the strain SC2409 comprises the constructions FL 121 + FL 475 + FL 602 + FL 808 from table 14.
  • control strain (without the genes) having the constructions TT LEU, TT URA, TT TRP and
  • TT HIS is named CF237.
  • the control strain (without the genes) having the TT URA construct is called CF233.
  • the control strain (without the genes) having the TT URA construct is called CF233.
  • the different assemblies carried out with the different RHATs will make it possible to verify the enzymatic activity of the RHATs and also make it possible to determine the most effective RHATs.
  • the control strain (without the genes) having the TT URA construct is called CF233.
  • the strain SC1509 comprises the constructs FL 121 + FL 511 + FL 602 + FL 808.
  • the strain SC1530 comprises the constructs FL 121 + FL 603 + FL 602 + FL 808.
  • the strain SC1529 comprises the constructs FL 121 + FL 554 + FL 602 + FL 808.
  • Strain SC1568 comprises the constructs FL 121 + FL 556 + FL 602 + FL 808.
  • the strain SC2410 comprises the constructs FL 121 + FL 1100 + FL 602 + FL 808.
  • the strain SC1579 comprises the constructs FL 401 + FL 547 + FL 602 + FL 828.
  • the strain SC1584 comprises the constructs FL 401 + FL 554 + FL 602 + FL 828.
  • the strain SC1621 comprises the constructs FL 401 + FL 556 + FL 602 + FL 828.
  • the strain SC1626 comprises the constructs FL 401 + FL 603 + FL 602 + FL 828.
  • the control strain (without the genes) possessing the constructions TT LEU, TT URA, TT TRP and TT HIS is called CF237.
  • Tables 20 and 21 below show the production of erodictyol (Table 20) and luteolin (Table 21) obtained by cultivating the strains comprising the F3′H listed in Table 6 and the constructions of Table 7, in the presence respectively naringenin and apigenin.
  • strains are well capable of producing erodictyol from naringenin, in different concentrations depending on the F3′H and the CPR used (see FIG. 2).
  • strains are well capable of producing luteolin from apigenin, in different concentrations depending on the F3′H and the CPR used (see FIG. 3).
  • Tables 22 and 23 below show the production of apigenin (Table 22) and luteolin (Table 23) obtained by culturing the strains comprising the FNSII listed in Table 8 and the constructions of Table 9, in the presence respectively of naringenin and erodictyol.
  • Apigenin concentration in mg.l 1 )
  • strains are well capable of producing apigenin and luteolin from naringenin and erodictyol, in different concentrations depending on the SNSF used ( Figures 4 and 5).
  • All strains are capable of producing diosmetin from naringenin.
  • the production of diosmetin is greatly increased by the addition of a CPR.
  • SC2424, SC2425, SC2426, SC2427, SC1500 and SC2428 have all the enzymes from the pathway to erodyctiol and luteolin and are capable of producing luteolin and erodictyol at from glucose.
  • strain SC1500 corresponds to Figure 6, where the peaks of erodictyol and luteolin are observed. Similar results are obtained for the strains SC2424, SC2425, SC2426, SC2427, and SC2428. The production of erodictyol and luteolin for each of the strains SC2424, SC2425, SC2426, SC2427, SC1500 and SC2428 is shown in Figure 19.
  • PAL and C4H enzymes to the biosynthetic pathway makes it possible to obtain significantly higher concentrations of erodictyol and luteolin.These can be up to 6 times greater than the concentrations obtained with the strains containing the same enzymes with the exception of PAL and C4H (cf. figure 19, for example by comparing the strain SC2425 without PAL / C4H and the strain SC1500 with PAL / C4H or the strain SC2426 without PAL / C4H and the strain SC2428 with PAL / C4H).
  • the results for the production of hesperetin and diosmetin from erodictyol and luteolin by the strains SC1612, SC1614, SC2147 and SC2151 are presented respectively in Figures 7, 8, 20 and 21.
  • the yeast strains SC1612, SC1614, SC2147 and SC2151 are well capable of producing hesperetin and / or diosmetin.
  • strains SC2147, SC2151 and SC1612 are capable of specifically producing hesperetin, that is to say of specifically methylating the hydroxyl in position 4 'of erodictyol (FIG. 20).
  • the strain SC1614 for its part produces a mixture of hesperetin and homoeriodictyol.
  • strain SC2151 is moreover capable of producing around 40 mg / L of hesperetin (FIG. 20).
  • the strains SC2147, SC1612 and SC1614 are for their part capable of producing diosmetin from luteolin ( Figure 21).
  • the yeast strain SC744 is well capable of producing diosmetin from hesperetin.
  • the results for the production of hesperetin from erodictyol by the strain EC26, EC41 and EC43 are shown in Figures 10, 22 and 23 and the production of diosmetin from luteolin by the strains EC26 and EC43 are shown in Figures 11 and 24.
  • the E. coli strains EC26, EC41 and EC43 are well capable of producing hesperetin and / or diosmetin.
  • the results for the production of diosmetin from hesperetin by strain EC30 are shown in Figure 12.
  • the E. coli EC30 strain is well capable of producing diosmetin from hesperetin.
  • strain SC1508, SC2408 and SC2409 possessing all the enzymes of the pathway up to hesperetin and diosmetin are capable of producing hesperetin and / or diosmetin from glucose ( Figure 27).
  • strain SC2408 produces on the order of 25 mg / L of hesperetin and approximately 5 mg / L of diosmetin.
  • the different strains are well capable of producing resperidin and / or diosmin from hesperetin and diosmetin, in different concentrations depending on the GTs used.
  • strains are well capable of producing rhesperidin and diosmin from hesperetin and diosmetin, in different concentrations depending on the RHMs used.
  • the different strains are well capable of producing hesperidin and diosmin from hesperetin and diosmetin, in different concentrations depending on the GTs, RHMs and RHATs used.
  • the yeasts tested with the different constructs are well capable of producing hesperidin and diosmin.
  • strains are well capable of producing hesperidin from hesperetin.
  • strains are well capable of producing diosmin from diosmetin.
  • strains possessing all the enzymes of the pathway are capable of producing hesperidin and / or diosmin.

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