METHOD OF BIOSYNTHESIS OF PHENYLPROPANOIDS

Abstract

The present invention relates to a recombinant microorganism comprising (i) a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase and / or (ii) a heterologous nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a heterologous nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH). It also relates to the use of this microorganism to produce phenylpropanoids such as sinapic acid or ferulic acid.

Description

Title of the invention: METHOD FOR THE BIOSYNTHESIS OF PHE-NYLPROPANOIDS FIELD OF THE INVENTION

[0001] The present invention relates to the use of a recombinant microorganism for producing an organic acid via the hydrolysis of a coenzyme A thioester, and more particularly to a recombinant microorganism capable of producing phenylpropanoid compounds. It also relates to production methods using such microorganisms. TECHNOLOGICAL BACKGROUND

[0002] Ferulic acid and sinapic acid are phenylpropanoids derived from cinnamic acid and possessing antioxidant and antiradical properties. Ferulic acid can be used as a precursor in the manufacture of antimicrobial substances for soaps, perfumes and cosmetics as well as for the production of vanillin and sinapic acid. It also finds applications in the medical field, in particular due to its antioxidant properties.

[0003] Ferulic acid or sinapic acid is obtained by biodegradation of lignocellulosic biomass. However, the quantity of product obtained by this process is relatively small and therefore results in a particularly high production cost of these products per kilogram.

[0004] There is therefore a real need for a biosynthesis process making it possible to obtain this type of compound at lower cost. Summary of the invention

[0005] In a first aspect, the present invention relates to a recombinant microorganism comprising (i) a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase and / or (ii) a heterologous nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a heterologous nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH).

[0006] The microorganism is preferably a yeast, in particular a yeast of the genus Saccharomyces, and more particularly preferably Saccharomyces ce-revisiae.

[0007] The recombinant microorganism according to the invention may comprise a heterologous nucleic acid sequence coding for an acyl-coenzyme A thioesterase. Preferably, said acyl-coenzyme A thioesterase comprises a sequence chosen from the sequences SEQ ID NOs: 1 and 2 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 acyl-coenzyme A thioesterase activity, more preferably a sequence chosen from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity.

[0008] Alternatively or cumulatively, the recombinant microorganism according to the invention may comprise a heterologous nucleic acid sequence encoding a CCR and a heterologous nucleic acid sequence encoding an ALDH. Preferably, said CCR comprises a sequence chosen from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity; and / or said ALDH comprises a sequence chosen from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity.

[0009] The recombinant microorganism according to the invention may further comprise a heterologous nucleic acid sequence coding for a Caffeoyl-CoA O-Methyltransferase (CCoAMT), preferably a CCoAMT comprising a sequence chosen from the sequences SEQ ID NOs: 10 to 16 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 CCoAMT activity; and / or a heterologous nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL), preferably a 4CL comprising a sequence selected from the sequences SEQ ID NOs: 5 to 9 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4CL activity.

[0010] The recombinant microorganism according to the invention may further comprise a heterologous nucleic acid sequence coding for an enzyme or an enzymatic complex capable of converting p-coumaric acid into caffeic acid, preferably

[0011] - a heterologous nucleic acid sequence coding for a p-coumarate 3-hydroxylase capable of converting p-coumaric acid to caffeic acid, preferably comprising a sequence selected from SEQ ID Nos: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 25 and exhibiting p-coumarate 3 hydroxylase activity; and / or

[0012] - a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate (HPA) 3-monooxygenase oxygenase, preferably comprising a sequence selected from SEQ ID Nos: 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% identity of sequence with the sequence SEQ ID NO: 17 and having a 4-hydroxyphenylacetate (HPA) 3-monooxygenase oxygenase activity, and a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate (HPA) 3-monooxygenase reductase, preferably comprising a sequence selected from SEQ ID Nos: 18 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and having a 4-hydroxyphenylacetate (HPA) 3-monooxygenase reductase activity.

[0013] Optionally, the recombinant microorganism according to the invention may also comprise a heterologous nucleic acid sequence coding for a cytochrome P450 reductase (CPR), preferably comprising a sequence chosen from SEQ ID Nos: 22 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 22 and exhibiting cytochrome P450 reductase activity.

[0014] The recombinant microorganism according to the invention may also comprise

[0015] - a heterologous nucleic acid sequence coding for an ammonia tyrosine lyase (TAL), preferably comprising a sequence selected from SEQ ID NO: 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting tyrosine ammonia lyase activity; and / or

[0016] - a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably a phenylalanine ammonia lyase comprising a sequence selected from SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting phenylalanine ammonia lyase activity, and a cinnamate 4-hydroxylase comprising a sequence selected from SEQ ID NO: 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting cinnamate 4-hydroxylase activity.

[0017] The recombinant microorganism according to the invention may also comprise a heterologous nucleic acid sequence coding for a phospho-2-dehydro-3-deoxyheptonate aldolase resistant to feedback control by tyrosine and / or a heterologous nucleic acid sequence coding for a chorismate mutase resistant to feedback control by tyrosine.

[0018] Furthermore, in said microorganism, an endogenous gene coding for a phenylpyruvate decarboxylase and / or an endogenous gene coding for a ferulic acid decarboxylase may be inactivated.

[0019] In a second aspect, the present invention relates to the use of a mi- microorganism according to the invention produces a carboxylic acid. Preferably, the carboxylic acid is a compound of formula (II)

[0020] [Chem.l]

[0022] where

[0023] Rb R2 and R3 are selected, independently of each other, from the group consisting of a hydrogen atom and a group O-R5, where R5 is selected from the group consisting of a hydrogen atom and a hydrocarbon chain comprising from 1 to 36 carbon atoms, branched or unbranched; and

[0024] R4 is selected from the group consisting of C1-C6 alkyl groups and C1-C6 alkene groups, branched or unbranched, preferably from the group consisting of -(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p-(CH2)q-, where n is an integer selected from 1, 2, 3, 4 and 5 and m, p and q are integers selected from 1, 2 and 3.

[0025] It also relates to a method for producing a carboxylic acid, comprising the cultivation of a microorganism according to the invention, and optionally the harvesting and / or purification of said carboxylic acid. Preferably, the carboxylic acid is a compound of formula (II) as defined above.

[0026] Preferably, the carboxylic acid produced by the microorganism according to the invention is ferulic acid or sinapic acid, preferably ferulic acid. DESCRIPTION OF THE FIGURES

[0027] [fig. 1 ] represents the production of ferulic acid from caffeic acid of yeasts expressing the thioesterase of SEQ ID NO: 1 (Thiol), the thioesterase of SEQ ID NO: 2 (Thio3), or the cinnamoyl-CoA reductase of SEQ ID NO: 4 and the aldehyde dehydrogenase of SEQ ID NO: 3 (CCR ALDH). The negative control (0) does not express any of these enzymes.

[0028] [fig.2] represents the production of sinapic acid of strain 367 expressing the thioesterase of SEQ ID NO: 2 (Thio3) and the negative control (0: strain 617).

[0029] [fig.3] represents the production of sinapic acid of strain 616 expressing the cinnamoyl-CoA reductase of SEQ ID NO: 4 and aldehyde dehydrogenase of SEQ ID NO: 3 (CCR ALDH) and negative control (0: strain 617).

[0030] [fig.4] represents the production of ferulic acid from strains 339 to 343, 345, 347, 367 to 371, 373, 375, 423 to 427, 429, 431, 451 to 455, 457 and 459 (CcoAMTl: SEQ ID NO: 10, CcoAMT2: SEQ ID NO: 11, CcoAMT3: SEQ ID NO: 12, CcoAMT4: SEQ ID NO: 13, CcoAMT5: SEQ ID NO: 14, CcoAMTô: SEQ ID NO: 15, CcoAMT7: SEQ ID NO: 16, 4CL1: SEQ ID NO: 5, 4CL5: SEQ ID NO: 6, 4CLA: SEQ ID NO: 8, 4CLB: SEQ ID NO: 9, Thio3: SEQ ID NO: 2) and negative controls (0: strains 334, 335, 337 and 338), grown in the presence of caffeic acid.

[0031] [fig.5] represents the production of sinapic acid of strains 339 to 343, 345, 347, 367 to 371, 373, 375, 395 to 399, 401, 403, 423 to 427, 429, 431, 451 to 455, 457 and 459 (CcoAMTl: SEQ ID NO: 10, CcoAMT2: SEQ ID NO: 11, CcoAMT3: SEQ ID NO: 12, CcoAMT4: SEQ ID NO: 13, CcoAMT5: SEQ ID NO: 14, CcoAMTô: SEQ ID NO: 15, CcoAMT7: SEQ ID NO: 16, 4CL1: SEQ ID NO: 5, 4CL5: SEQ ID NO: 6, 4CL7: SEQ ID NO: 7, 4CLA: SEQ ID NO: 8, 4CLB: SEQ ID NO: 9, Thio3: SEQ ID NO: 2) and negative controls (0: strains 334, 335, 336, 337 and 338), grown in the presence of 5-hydroxyferulic acid.

[0032] [fig.6] represents the production of ferulic acid of strains 595, 596 and 597 cultured in the presence of p-coumaric acid and the negative control (0: strain 507).

[0033] [fig.7] represents the production of ferulic acid of strains 592, 593 and 594 cultured in the presence of glucose and the negative control (0: strain 516).

[0034] [fig.8] represents a biosynthesis pathway of ferulic acid from glucose.

[0035] [fig.9] illustrates the enzymatic activities of acyl-coenzyme A thioesterase or cinnamoyl-CoA reductase and aldehyde dehydrogenase on feruloyl-CoA and sinapoyl-CoA. DETAILED DESCRIPTION OF THE INVENTION

[0036] Synthetic pathways involving compounds linked to coenzyme A (CoA) are little used in biotechnology. Indeed, the difficulty in detecting and measuring the quantities of synthetic intermediates makes their manipulations delicate. The inventors have however explored these biosynthetic pathways and developed a microorganism capable of producing organic acids via the hydrolysis of CoA thioesters such as ferulic acid or sinapic acid. In addition to reduced costs compared to current production techniques, the production method using this microorganism also has the advantage of using intermediates linked to CoA and therefore incapable of leaving the cells. This therefore allows easier and less expensive extraction of the final compounds of interest. Definitions

[0037] As used herein, the term "microorganism" refers to a prokaryotic or eukaryotic microorganism, particularly a yeast, fungus, or bacterium.

[0038] By "recombinant microorganism" is meant a microorganism which does not not found in nature and which contains a genome modified following an insertion, modification or deletion of one or more heterologous genetic elements.

[0039] By "recombinant nucleic acid" is meant a nucleic acid that has been modified and does not exist in nature. For example, this term may refer to a coding sequence or a gene that is operably linked to a promoter that is not the natural promoter. It may also refer to a coding sequence in which the introns have been deleted for genes comprising exons and introns.

[0040] By "heterologous" is meant a nucleic acid sequence or a protein that is not naturally present in the host cell and has been introduced therein by genetic engineering. The heterologous sequence may be present in the cell in episomal or chromosomal form. The origin of the heterologous sequence may be different from the cell into which it is introduced. However, it may also come from the same species as the cell into which it is introduced but be considered heterologous due to its environment being unnatural. For example, the nucleic acid sequence is heterologous when it is under the control of a promoter other than its natural promoter or when it is introduced into a location different from that where it is naturally located.The host cell may contain an endogenous copy of the nucleic acid sequence prior to the introduction of the heterologous nucleic acid sequence or it may not contain an endogenous copy. Furthermore, the nucleic acid sequence may be heterologous in the sense that the coding sequence has been optimized for expression in the host cell, for example by optimization of codon usage. Preferably, as used herein, the term "heterologous nucleic acid sequence" refers to a nucleic acid sequence that encodes a protein that is heterologous to the host cell, i.e., not naturally present in the host cell.

[0041] As used herein, the term "endogenous," with respect to the host cell, refers to a genetic element or protein naturally present in said cell.

[0042] The term "gene" or "coding sequence" refers to any nucleic acid that codes for a protein. The term gene includes DNA, such as cDNA (complementary DNA) or gDNA (genomic DNA), as well as RNA. The gene may first be prepared by recombinant, enzymatic, and / or chemical techniques, and then replicated in a host cell or 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 degeneracy of the genetic code, more than one nucleic acid may encode a particular polypeptide. Thus, the codons in the coding sequence for a given polypeptide may be modified such that optimal expression in a particular cell is achieved, for example, by using translation tables. codon optimization appropriate for that cell. The nucleic acids may also be optimized according to a GC content preferable for said cell and / or to reduce the number of repeated sequences. In some embodiments, the heterologous nucleic acids have been codon optimized for expression in the relevant host cell. Codon optimization may be performed by routine methods known in the art (see, e.g., Welch, M., et al. (2011), Methods in Enzymology 498: 43-66).

[0043] The term "operably linked" refers to a configuration in which a control sequence is placed at an appropriate position relative to a coding sequence, such that the control sequence directs expression of the coding sequence.

[0044] The term "control sequences" refers to the nucleic acid sequences necessary for the expression of a gene. The control sequences may be endogenous 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.

[0045] The term "expression cassette" means a nucleic acid construct comprising a coding region, i.e. a gene, and a regulatory region, i.e. comprising one or more control sequences, operably linked. Preferably, the control sequences are adapted to the host cell.

[0046] As used herein, the term "expression vector" refers to a DNA or RNA molecule that comprises an expression cassette. Preferably, the expression vector is a linear or circular, preferably linear, double-stranded DNA molecule. The vector may further comprise an origin of replication, a selection marker, etc.

[0047] By "percentage identity" between two nucleic acid or amino acid sequences within the meaning of the present invention, is meant a percentage of nucleotides or amino acid residues identical 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 randomly 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 aligned them optimally, said comparison being performed by segment or comparison window to identify and compare local regions of sequence similarity. Alignment for the purpose of determining percent amino acid sequence identity may be performed in a variety of ways that are well known in the art, for example, by using computer software programs available on the Internet such as http: / / www.clustal.org / omega / or http: / / www.ebi.ac.uk / Tools / emboss / . One skilled in the art can determine appropriate parameters for measuring alignment, including any algorithms necessary to achieve maximum alignment across the entire length of the sequences being compared.For the purposes of the present invention, the amino acid sequence identity percentage values ​​refer to values ​​generated using the EMBOSS Needle pairwise sequence alignment program that creates an optimal global alignment of two sequences using the Needleman-Wunsch algorithm, in which all search parameters are set to default Scoring Matrix = BLOSUM62, Open Gap = 10, Gap Extension = 0.5, End Gap Penalty = False, Open End Gap = 10, and Extended End Gap = 0.5. In some embodiments, all identity percentages mentioned in this application may be set to at least 60%, at least 70%, at least 80%, at least 85%, preferably at least 90% identity, more preferably at least 95% identity.In particular, embodiments are considered to be described in which all the sequence identity percentages of the enzymes are at least 80% or at least 85%, preferably at least 90% or at least 95% sequence identity. In one embodiment, the polypeptides may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additions, substitutions or deletions with respect to the sequences described in SEQ ID Nos. In particular, these additions, substitutions or deletions are introduced at the N-terminus, C-terminus or at both ends. The polypeptides may optionally be in the form of a fusion protein. In this case, the identity percentage is calculated only on the domain of the fusion protein exhibiting the desired activity.

[0048] The terms "overexpression" and "increased expression" as used herein are used interchangeably and mean that the expression of a coding sequence or protein is increased relative to the unmodified host cell, for example, a wild-type cell or one lacking the genetic modifications described herein. By "wild-type" is meant an unmodified cell existing in nature. Increased expression of a protein is usually achieved by increasing the expression of the gene encoding said protein. In embodiments in which the gene or protein is not naturally present in the microorganism of the invention, i.e., a heterologous gene or protein, the terms "overexpression" and "expression" may be used interchangeably. changeable. To increase the expression of a gene, a person skilled in the art can use all known techniques such as increasing the number of copies of the gene in the cell, using a promoter inducing a high level of gene expression, i.e. a strong promoter, using elements stabilizing the corresponding messenger RNA or specific RBS (ribosome binding site) sequences. In particular, overexpression can be achieved by increasing the number of copies of the gene in the cell. One or more copies of the gene can be introduced into the genome by recombinant methods, known to a person skilled in the art, including gene replacement or multicopy insertion. Preferably, an expression cassette comprising the gene is integrated into the genome.Alternatively, the gene may be carried by an expression vector, preferably a plasmid, comprising an expression cassette with the gene of interest preferably placed under the control of a suitable promoter. The expression vector may be present in the host cell in one or more copies depending on the nature of the origin of replication. Overexpression of a gene may also be achieved by using a promoter inducing a high level of expression of the gene. For example, the promoter of an endogenous gene may be replaced by a stronger promoter, i.e., a promoter inducing a higher level of expression. The endogenous gene under the control of a promoter that is not the natural promoter is referred to as a heterologous nucleic acid. Promoters suitable for use in the present invention are known to those skilled in the art and may be constitutive or inducible, and may be endogenous or heterologous.

[0049] By "comprising" is also meant "consisting" or "consisting essentially". By "consisting essentially" is meant that the sequence may have 1, 2, 3, 4, 5, 6, 7, 8, 9 or 10 additions, substitutions or deletions compared to the sequences described in SEQ ID Nos. Microorganisms

[0050] The microorganism according to the present invention may be a eukaryotic or prokaryotic microorganism.

[0051] According to a first embodiment, the microorganism is a eukaryotic microorganism, preferably chosen from yeasts and fungi.

[0052] According to a preferred embodiment, the microorganism is a yeast, in particular a yeast of the order Saccharomycetales, Sporidiobolales and Schizosaccharo-mycetales. The yeast may for example be selected from yeasts of the genus Saccharomyces, Pichia, Kluyveromyces, Schizosaccharomyces, Candida, Lipomyces, Rhodotorula, Rhodosporidium, Yarrowia, Debaryomyces, Komagataella, Schef-fersomyces, Torulaspora or Zygosaccharomyces. In particular, the yeast may be selected from the species Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Sac-charomyces norbensis, Saccharomyces oviformis, Pichia pastoris, Kluyveromyces lactis, Kluyveromyces marxianus, Schizosaccharomyces pombe, Candida albicans, Candida tropicalis, Rhodotorula glutinis, Rhodosporidium toruloides, Yarrowia li-polytica, Debaryomyces hansenii and Lipomyces starkeyi.

[0053] According to a particularly preferred embodiment, the microorganism is a yeast belonging to the genus Saccharomyces, preferably a yeast chosen from the species Saccharomyces cerevisiae, Saccharomyces carlsbergensis, Saccharomyces diastaticus, Saccharomyces douglasii, Saccharomyces kluyveri, Saccharomyces norbensis and Saccharomyces oviformis. In a very particularly preferred manner, the microorganism is a yeast of the species Saccharomyces cerevisiae.

[0054] Alternatively, the microorganism may be a fungus, preferably a filamentous fungus, in particular a fungus selected from fungi of the genera Aspergillus, Trichoderma, Neurospora, Podospora, Endothia, Mucor, Cochiobolus and Pyricularia. More preferably, the fungus is selected from Aspergillusnidulans, Aspergillusniger, Aspergillus awomari, Aspergillusoryzae, Aspergillus terreus, Neurosporacrassa, Trichoderma reesei and Trichodermaviride.

[0055] According to a second embodiment, the microorganism is a prokaryotic microorganism, preferably a bacterium. In particular, the microorganism may be a bacterium chosen from bacteria of the phyla Acidobacteria, Actinobacteria, Aquificae, Bacterioidetes, Chlamydiae, Chlorobi, Chloroflexi, Chrysiogenetes, Cyanobacteria, Deferribacteres, Deinococcus-Thermus, Dictyoglomi, Fibrobacteres, Firmicutes, Fusobacteria, Gemmatimonadetes, Nitrospirae, Planctomycetes, Proteobacteria, Spi-rochaetes, Thermodesulfobacteria, Thermomicrobia, Thermotogae, or Verruco-microbia.De manière préférée, la bactérie appartient au genre Acaryochloris, Ace-tobacter, Actinobacillus, Agrobacterium, Alicyclobacillus, Anabaena, Anacystis, Anae-robiospirillum, Aquifex, Arthrobacter, Arthrospira, Azobacter, Bacillus, Brevi-bacterium, Burkholderia, Chlorobium, Chromatium, Chlorobaculum, Clostridium, Co-rynebacterium, Cupriavidus, Cyanothece, Enterobacter, Deinococcus, Erwinia, Es-cherichia, Geobacter, Gloeobacter, Gluconobacter, Hydrogenobacter, Klebsiella, Lac-tobacillus, Lactococcus, Mannheimia, Mesorhizobium, Methylobacterium, Micro-bacterium, Microcystis, Nitrobacter, Nitrosomonas, Nitrospina, Nitrospira, Nostoc, Phormidium, Prochlorococcus, Pseudomonas, Ralstonia, Rhizobium, Rhodobacter, Rhodococcus, Rhodopseudomonas, Rhodospirillum, Salmonella, Scenedesmun, Serratia, Shigella, Staphylococcus, Streptomyces, Synechoccus, Synechocystis, Ther-mosynechococcus, Trichodesmium ou Zymomonas.More preferably, the bacterium is chosen from the species Agrobacterium tumefaciens, Anaerobiospirillum. succiniciproducens, Actinobacillus succinogenes, Aquifex aeolicus, Aquifex py-rophilus, Bacillus subtilis, Bacillus amyloliquefacines, Brevibacterium ammoniagenes, Brevibacterium immariophilum, Clostridium pasteurianum, Clostridium ljungdahlii, Clostridium acetobutylicum, Clostridium beigerinckii, Corynebacterium glutamicum, Cupriavidus necator, Cupriavidus metallidurans, Enterobacter sakazakii, Escherichia coli, Gluconobacter oxydons, Hydrogenobacter thermophilus, Klebsiella oxytoca, Lac-tococcus lactis, Lactobacillus plantarum, Mannheimia succiniciproducens, Meso-rhizobium loti, Pseudomonas aeruginosa, Pseudomonas mevalonii, Pseudomonas pudica, Pseudomonas putida, Pseudomonas fluoréscens, Rhizobium etli, Rhodobacter capsulatus, Rhodobacter sphaeroides, Rhodospirillum rubrum, Salmonella enterica, Salmonella typhi, Salmonella typhimurium, Shigella dysenteriae, Shigella flexneri, Shigella sonnei, Staphylococcus aureus, Streptomyces coelicolor, Zymomonas mobilis, Acaryochloris marina,Anabaena variabilis, Arthrospira platensis, Arthrospira maxima, Chlorobium tepidum, Chlorobaculum sp., Cyanothece sp., Gloeobacter violaceus, Microcystis aeruginosa, Nostoc punctiforme, Prochlorococcus marinus, Synechococcus elongatus, Synechocystis sp., Thermosynechococcus elongatus, Trichodesmium erythraeum and Rhodopseudomonas palustris. According to a particular embodiment, the microorganism is an Escherichia coli bacterium, for example chosen from E. coli BL21, E. coli BL21 (DE3), E. coli MG1655, E. coli W31 10 and their derivatives. According to an alternative particular embodiment, the microorganism is a bacterium of the genus Streptomyces, in particular Streptomyces venezuelae.

[0056] According to a most preferred embodiment, the microorganism is a yeast, preferably a yeast belonging to the genus Saccharomyces, in particular a yeast Saccharomyces cerevisiae.

[0057] Production of a carboxylic acid from a carboxyl-CoA

[0058] Thanks to the thiol function of cysteamine, coenzyme A is capable of forming with the carboxyl functions of certain compounds, such as caffeic acid or 5-hydroxyferulic acid, thioesters called carboxyl-CoA.

[0059] As used herein, the term "carboxyl-CoA" refers to a thioester of coenzyme A in which hydrolysis of the thioester bond generates a carboxyl group. Preferably, this term refers to a compound of formula (I)

[0060] [Chem. 2] S—CoA R2 r3

[0061] (I)

[0062] where

[0063] Rb R2 and R3 are selected, independently of each other, from the group consisting of a hydrogen atom and a group O-R5, where R5 is selected from the group consisting of a hydrogen atom and a hydrocarbon chain comprising from 1 to 36 carbon atoms, branched or unbranched; and

[0064] R4 is selected from the group consisting of C1-C6 alkyl groups and C1-C6 alkene groups, branched or unbranched, preferably from the group consisting of —(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p-(CH2)q-, where n is an integer selected from 1, 2, 3, 4 and 5 and m, p and q are integers selected from 1, 2 and 3.

[0065] Preferably, R5 is selected from the group consisting of a hydrogen atom, branched or unbranched C1-C6 alkyl groups and branched or unbranched C1-C6 alkene groups. More preferably, R5 is selected from the group consisting of a hydrogen atom and a methyl group.

[0066] According to a particular embodiment,

[0067] - Ri, R2 and R3 are chosen, independently of each other, from the group consisting of a hydrogen atom and an O-R5 group, where R5 is a methyl group, and

[0068] - R4 is selected from the group consisting of -(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p - (CH2)q-, where n is an integer chosen from 1 or 2 and m, p and q are integers chosen from 1 or 2.

[0069] According to another particular embodiment,

[0070] - Ri, R2 and R3 are chosen, independently of each other, from the group consisting of a hydrogen atom and an O-R5 group, where R5 is a methyl group, and

[0071] - R4 is -CH2-CH=CH-CH2-,

[0072] Most preferably, this term refers to feruloyl-CoA and / or sinapoyl-CoA.

[0073] The recombinant microorganism according to the present invention is genetically modified to produce a carboxylic acid from a carboxyl-CoA. This microorganism is in particular capable of producing phenylpropanoids such as ferulic acid and sinapic acid from molecules coupled to CoA such as feruloyl-CoA and sinapoyl-CoA (see Figures 8 and 9).

[0074] In plants, the hydrolysis of a compound coupled to CoA is described during lignin synthesis. This type of hydrolysis is carried out in 2 steps. The first step consists of the release of the substrate in its aldehyde form. The second consists of the oxidation of the aldehyde compound to its acid form (Fraser and Chappel, Arabidopsis Book. 2011;9:e0152). Some plants such as Petunia hybrida or Curcuma longa L., also appear capable of directly hydrolyzing phenylpropanoyl-CoA via thioesterases that catalyze the hydrolysis of a thioester bond by releasing a carboxylic acid and a thiol group (Adebesin et al, Plant J. 2018 January; 93: 905-916; Ramirez-Ahumada et al, Phytochemistry. 2006 Sep;67(18):2017-29) and which are mainly described in the fatty acid biosynthesis pathway.

[0075] The inventors have here surprisingly demonstrated that these two types of mechanisms could be used in a microorganism, and in particular in a yeast, to produce carboxylic acids, and in particular phenylpropanoids. The microorganism according to the invention can thus express a heterologous acyl-coenzyme A thioesterase and / or express a cinnamoyl-CoA reductase (CCR) and an aldehyde dehydrogenase (ALDH).

[0076] According to one embodiment, the microorganism according to the invention comprises a heterologous nucleic acid sequence coding for an acyl-coenzyme A thioesterase.

[0077] As used herein, the term "acyl-coenzyme A thioesterase" or "Thio" refers to an enzyme having acyl-coenzyme A thioesterase activity, i.e., an enzyme capable of hydrolyzing the ester bond of a carboxyl-CoA and thereby releasing CoA on the one hand and a carboxylic acid on the other (see [fig.9]). Preferably, this term refers to an enzyme capable of hydrolyzing the thioester bond of feruloyl-CoA and thereby producing ferulic acid and / or of hydrolyzing the thioester bond of sinapoyl-CoA and thereby producing sinapic acid.

[0078] The detection of this activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. The acyl-coenzyme A thioesterase activity can be detected in vitro, for example as described in the article by Adebesin et al. (Plant J. 2018 Mar;93(5):905-916). In particular, to determine whether there is an acyl-coA thioesterase activity, an enzymatic test can be carried out and consists of the in vitro incubation of a mixture composed of the enzyme to be tested (putative acyl-CoA thioesterase), and a carboxyl-CoA substrate under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of the carboxylic acid obtained by hydrolysis of the ester bond of the carboxyl-CoA substrate can be observed either by UV spectrophotometer at a given wavelength or by HPLC. Acyl-coenzyme A thioesterase activity can also be detected in vivo, in particular using the method described in the experimental section below. In particular, the acyl-coenzyme A thioesterase activity can be detected in vivo using a microorganism expressing the enzyme to be tested. The carboxyl-CoA substrate of said enzyme is either synthesized by the microorganism or provided in the culture medium. The carboxylic acid produced by an enzyme exhibiting the desired activity can then be detected by any method, preferably by an HPLC technique. In a particularly preferred manner, the acyl-coenzyme A thioesterase activity can be detected in vivo using a microorganism expressing the enzyme to be tested and capable of converting caffeic acid into feruloyl-CoA and / or of converting 5-hydroxyferulic acid into sinapoyl-CoA. Acyl-coenzyme A thioesterase activity is then detected by the production of ferulic acid and / or sinapic acid in the presence of caffeic acid or 5-hydroxyferulic acid, preferably by an HPLC technique.

[0079] The acyl-coenzyme A thioesterase may be a plant enzyme, preferably an enzyme from plants of the genus Petunia, Arabidopsis, Capsella, Camellia, brassica, Raphanus or Nicotiana, in particular Petunia hybrida, Arabidopsis thaliana, Capsella rubella, Camellia sativa, brassica rapa, Raphanus sativus or Nicotiana tabacum.

[0080] Alternatively, this enzyme may be an enzyme produced by a microorganism, for example by a yeast of the genus Saccharomyces, in particular a yeast Saccharomyces cerevisiae. In certain embodiments, the enzyme corresponds to the endogenous enzyme of the microorganism. In these cases, the enzyme may be overexpressed, for example by replacing the endogenous promoter with a strong heterologous promoter and / or by increasing the copy number of the gene.

[0081] More particularly preferably, the acyl-coenzyme A thioesterase is a plant enzyme, in particular from Petunia hybrida or Arabidopsis thaliana. The acyl-coenzyme A thioesterase may be an Arabidopsis thaliana enzyme, in particular the thioesterase described in SEQ ID NO: 1. Alternatively, the acyl-coenzyme A thioesterase may be an enzyme from Petunia hybrida, in particular the thioesterase described in SEQ ID NO: 2.

[0082] According to one embodiment, the acyl-coenzyme A thioesterase comprises a sequence chosen from the sequences SEQ ID NOs: 1 and 2 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 acyl-coenzyme A thioesterase activity.

[0083] According to a particular embodiment, the acyl-coenzyme A thioesterase comprises a sequence chosen from the sequence SEQ ID NO: 1 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 1 and exhibiting acyl-coenzyme A thioesterase activity.

[0084] According to a preferred embodiment, the acyl-coenzyme A thioesterase comprises a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity.

[0085] According to another embodiment, the microorganism according to the invention comprises a heterologous nucleic acid sequence coding for a cinnamoyl-CoA reductase (CCR) and a heterologous nucleic acid sequence coding for an aldehyde dehydrogenase (ALDH).

[0086] CCR catalyzes the reduction of carboxyl-CoAs such as feruloyl-CoA and sinapoyl-CoA, thereby forming aldehydes such as coniferaldehyde and sina-paldehyde. ALDH then catalyzes the oxidation of the aldehydes thus formed to carboxylic acids such as ferulic acid and sinapic acid.

[0087] As used herein, the term "cinnamoyl-CoA reductase" or "CCR" refers to an enzyme belonging to class EC 1.2.1.44 and which catalyzes the reduction of a substituted cinnamoyl-CoA, e.g., feruloyl-CoA or sinapoyl-CoA, to a corresponding cinnamoyl-CoA, e.g., coniferaldehyde or sinapaldehyde. Preferably, this term refers to an enzyme which catalyzes the reduction of feruloyl-CoA to coniferaldehyde and / or sinapoyl-CoA to sinapaldehyde.

[0088] The detection of this activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. The CCR activity can be detected in vitro, for example as described in the article by Chao et al. (Planta. 2017 Jan;245(l):61-75) In particular, to determine whether there is a Cinnamoyl-CoA reductase activity, an enzymatic test can be carried out and consists of the in vitro incubation of a mixture composed of the enzyme to be tested (putative CCR), a carboxyl-CoA substrate and NADPH under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of the aldehyde form can be observed, either with a UV spectrophotometer with a given wavelength, or by HPLC. The CCR activity can also be detected in vivo, in particular using the method described in the experimental part below. In particular, CCR activity can be detected in vivo using a microorganism expressing the enzyme to be tested.The carboxyl-CoA substrate of said enzyme is either synthesized by the microorganism or provided in the culture medium. The aldehyde produced by an enzyme exhibiting the desired activity can then be detected by any method, preferably by an HPLC technique. In a particularly preferred manner, the CCR activity can be detected in vivo using a microorganism expressing the enzyme to be tested, capable of converting caffeic acid into feruloyl-CoA and / or 5-hydroxyferulic acid into sinapoyl-CoA and capable of converting coniferaldehyde into ferulic acid and / or sinapaldehyde into sinapic acid, i.e. expressing an ALDH enzyme as defined below. The CCR activity is detected by the production of ferulic acid and / or sinapic acid. in the presence of caffeic acid or 5-hydroxyferulic acid, preferably by an HPLC technique.

[0089] The CCR may be a plant enzyme, preferably of the genus Populus, Ara-bidopsis, Oryza, Zea, Medicago or Sorghum, in particular Populus tomentosa, Ara-bidopsis thaliana, Oryza sativa, Zea Mays, Medicago truncatula or Sorghum bicolor. Preferably, the CCR is an enzyme from Populus tomentosa, in particular the CCR described in SEQ ID NO: 4.

[0090] According to a preferred embodiment, the CCR comprises a sequence chosen from the sequence SEQ ID NO: 4 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity.

[0091] As used herein, the term "aldehyde dehydrogenase" or "ALDH" refers to an enzyme belonging to class EC 1.2.1.3 and which catalyzes the oxidation of an aldehyde, for example coniferaldehyde or sinapaldehyde, to a carboxylic acid, for example ferulic acid or sinapic acid. Preferably, this term refers to an enzyme which catalyzes the oxidation of coniferaldehyde to ferulic acid and / or the oxidation of sinapaldehyde to sinapic acid.

[0092] The detection of this activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. ALDH activity can be detected in vitro, for example using a commercially available in vitro test kit. In particular, to determine whether there is aldehyde dehydrogenase activity, an enzymatic test can be carried out and consists of the in vitro incubation of a mixture composed of the enzyme to be tested (putative ALDH), an aldehyde and NAD under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of the acid form and NADH can be observed either with a UV spectrophotometer at 450 nm or by HPLC. ALDH activity can also be detected in vivo, in particular using the method described in the experimental section below. In particular, ALDH activity can be detected in vivo using a microorganism expressing the enzyme to be tested.The aldehyde substrate of said enzyme is either synthesized by the microorganism or provided in the culture medium. The carboxylic acid produced by an enzyme exhibiting the desired activity can then be detected by any method, preferably by an HPLC technique. Particularly preferably, the ALDH activity can be detected in vivo using a microorganism expressing the enzyme to be tested, capable of converting caffeic acid into feruloyl-CoA and / or 5-hydroxyferulic acid into sinapoyl-CoA and capable of converting feruloyl-CoA into coniferaldehyde and / or sinapoyl-CoA into sinapaldehyde, i.e. expressing a CCR enzyme as defined above. The ALDH activity is detected by the production of ferulic acid and / or sinapic acid in the presence of caffeic acid or . 5-hydroxyferulic acid, preferably by HPLC technique.

[0093] ALDH may be a plant enzyme, preferably of the genus Arabidopsis, Populus, Oryza, Zea, Medicago or Sorghum, in particular Arabidopsis thaliana, Populus tomentosa, Oryza sativa, Zea Mays, Medicago truncatula or Sorghum bicolor

[0094] Alternatively, this enzyme may be an enzyme produced by a microorganism, for example by a yeast of the genus Saccharomyces, in particular a yeast Saccharomyces cerevisiae. In certain embodiments, the enzyme corresponds to the endogenous enzyme of the microorganism. In these cases, the enzyme may be overexpressed, for example by replacing the endogenous promoter with a strong heterologous promoter and / or by increasing the copy number of the gene.

[0095] Preferably, ALDH is a plant enzyme, and more preferably, an Arabidopsis thaliana enzyme, in particular the ALDH described in SEQ ID NO: 3.

[0096] According to a preferred embodiment, the ALDH comprises a sequence chosen from the sequence SEQ ID NO: 3 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity.

[0097] According to a particularly preferred embodiment, the microorganism according to the invention comprises a heterologous nucleic acid sequence coding for a CCR and a heterologous nucleic acid sequence coding for an ALDH and in which

[0098] - said CCR comprises a sequence chosen from the sequence SEQ ID NO: 4 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity; and

[0099] - said ALDH comprises a sequence chosen from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity.

[0100] The microorganism according to the invention can produce the carboxyl-CoA of interest, naturally or after genetic modification. Alternatively, this substrate can be provided to it in the culture medium. According to a preferred embodiment, the microorganism is capable of producing the carboxyl-CoA of interest, preferably feruloyl-CoA or sinapoyl-CoA, from a synthesis intermediate, for example caffeic acid or 5-hydroxyferulic acid, or from glucose, preferably from glucose. Production of a carboxyl-CoA of interest

[0101] The microorganism according to the invention can be genetically modified to produce the carboxyl-CoA of interest (substrate for acyl-coenzyme A thioesterase and / or CCR). Alternatively, this substrate can be provided in the culture medium.

[0102] The microorganism according to the invention is preferably genetically modified to be capable of producing the carboxyl-CoA of interest, preferably feruloyl-CoA or sinapoyl-CoA, from a synthesis intermediate, for example cinnamic acid, p-coumaric acid, caffeic acid or 5-hydroxyferulic acid, or from glucose via phenylalanine or tyrosine.

[0103] According to certain preferred embodiments, the microorganism according to the invention is capable of producing the carboxyl-CoA of interest, preferably feruloyl-CoA or sinapoyl-CoA, from a synthesis intermediate, such as caffeic acid or 5-hydroxyferulic acid. The production of feruloyl-CoA or sinapoyl-CoA from caffeic acid or 5-hydroxyferulic acid is the result of two enzymatic reactions, the first involving a 4-coumaroyl-CoA ligase and the second a caffeoyl-CoA O-methyltransferase.

[0104] Thus, the microorganism according to the invention may also comprise, in addition to acyl-coenzyme A thioesterase and / or the CCR / ALDH combination, a heterologous nucleic acid sequence coding for a caffeoyl-CoA O-methyltransferase (CCoAMT) and / or a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL), preferably a heterologous nucleic acid sequence coding for CCoAMT and a heterologous nucleic acid sequence coding for a 4CL.

[0105] The microorganism according to the invention may comprise a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL).

[0106] As used herein, the term "4-coumaroyl-CoA ligase" or "4CL" refers to an enzyme capable of producing caffeoyl-CoA from caffeic acid and CoA and / or 5-hydroxyferuloyl-CoA from 5-hydroxyferulic acid and CoA. This enzyme belongs to EC class 6.2.1.12.

[0107] The detection of this activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. In particular, to determine whether there is 4-coumarate CoA ligase activity, an enzymatic test can be carried out which consists of the in vitro incubation of a mixture composed of the enzyme to be tested (putative 4-coumarate CoA ligase), caffeic acid or 5-hydroxyferulic acid, ATP and CoA under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of caffeoyl CoA or 5-hydroxyferuloyl-CoA is observed using a UV spectrophotometer with a given wavelength.

[0108] 4CL may be a plant enzyme, preferably of the genus Abies, Arabidopsis, Agastache, Amorpha, Brassica, Citrus, Cathaya, Cedrus, Crocus, Larix, Festuca, Glycine, Jugions, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Phaseolus, Pelargonium, Petroselinum, Physcomitrella, Picea, Prunus, Pseudolarix, Pseudotsuga, Rosa, Rubus, Ryza, Saccharum, Suaeda, Pinus, Populus, Solanum, Thellungiella, Triticum, Tsuga, Vitis or Zea. Alternatively, this enzyme may be an enzyme produced by a microorganism, for example of the genus Aspergillus, Mycosphaerella, Mycobacterium, Neisseria, Neurospora, Streptomyces, Rhodobacter or Yarrowia.

[0109] Preferably, the 4CL is a plant enzyme, in particular an enzyme from a plant of the genus Arabidopsis, Citrus or Populus. More specifically, the 4CL may be a 4CL from Arabidopsis thaliana, in particular a 4CL described in one of the sequences SEQ ID Nos: 5, 7 and 9, a 4CL from Citrus sinensis, in particular a 4CL described in SEQ ID NO: 6 or a 4CL from Populus tomentosa, in particular a 4CL described in SEQ ID NO: 8.

[0110] According to one embodiment, the 4CL comprises a sequence chosen from the sequences SEQ ID NO: 5 to 9 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 4CL activity.

[0111] According to a preferred embodiment, the 4CL comprises a sequence chosen from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity.

[0112] When the recombinant microorganism according to the invention is intended to be used in the production of ferulic acid, the 4CL is preferably chosen from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity.

[0113] According to a very particularly preferred embodiment, the 4CL comprises a sequence chosen from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity.

[0114] The microorganism according to the invention may also comprise a heterologous nucleic acid sequence coding for a caffeoyl-CoA O-methyltransferase (CCoAMT).

[0115] As used herein, the term "caffeoyl-CoA O-methyltransferase" or "CCoAMT" refers to an enzyme belonging to class EC 2.1.1.104 and which catalyzes the conversion of caffeoyl-CoA to feruloyl-CoA and / or 5-hydroxyferuloyl-CoA to sinapoyl-CoA. Preferably, this term refers to an enzyme which catalyzes the conversion of caffeoyl-CoA to feruloyl-CoA and 5-hydroxyferuloyl-CoA to sinapoyl-CoA.

[0116] The detection of this activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. The CCoAMT activity can be detected in vitro, for example using a commercially available in vitro test (for example the SAM510 test from G-Biosciences, Cat.#786-430). In particular, to determine whether there is CoA methyl-transferase activity, an enzymatic test can be performed and consists of the in vitro incubation of a mixture composed of the enzyme to be tested (putative CCoAMT), S-adenosylmethionine (SAM), and a mixture of enzymes (5-adenosylhomocyteine ​​nucleosidase (EC 3.2.2.9), adenine deaminase (EC 3.5.4.2) and xanthine oxidase (EC 1.17.3.2) under optimal conditions (pH, temperature, ions...). In the presence of CCoAMT activity, the products obtained are urate and hydrogen peroxide.After a certain incubation time, the appearance of hydrogen peroxide can be detected by reaction with a colorimetric agent, namely 3,5-dichloro-2-hydroxybenzenesulfonic acid (DHBS) and measured with a UV spectrophotometer at 510nm. CCoAMT activity can also be detected in vivo, in particular using the method described in the experimental section below. In particular, CCoAMT activity can be detected in vivo using a microorganism expressing the enzyme to be tested. The substrate of said enzyme (caffeoyl-CoA or 5-hydroxyferuloyl-CoA) is either synthesized by the microorganism or provided in the culture medium. The feruloyl-CoA or sinapoyl-CoA produced by an enzyme exhibiting the desired activity can then be detected by any method, preferably by an HPLC technique.Particularly preferably, the CCoAMT activity can be detected in vivo using a microorganism expressing the enzyme to be tested, capable of converting caffeic acid to caffeoyl-CoA and / or 5-hydroxyferulic acid to 5-hydroxyferuloyl-CoA, i.e. expressing a 4CL enzyme as defined above. The CCoAMT activity is detected by the production of feruloyl-CoA and / or sinapoyl-CoA in the presence of caffeic acid or 5-hydroxyferulic acid, preferably by an HPLC technique.

[0117] CCoAMT may be a plant enzyme, preferably of the genus Vitis, Medicago, Eucalyptus, Nicotiana, Arabidopsis or Populus. In particular, CCoAMT may be an enzyme from Vitis vinifera, Medicago sativa, Eucalyptus globus, Nicotiana tabacum, Arabidopsis thaliana or Populus trichocarpa.

[0118] The CCoAMT may be an enzyme from Vitis vinifera, in particular the CCoAMT described in SEQ ID NO: 10, an enzyme from Medicago sativa, in particular the CCoAMT described in SEQ ID NO: 11, an enzyme (V Eucalyptus globus, in particular the CCoAMT described in SEQ ID NO: 12, an enzyme from Nicotiana tabacum, in particular the CCoAMT described in SEQ ID NO: 13 or 14, an enzyme (V Arabidopsis thaliana, in particular the CCoAMT described in SEQ ID NO: 15, or an enzyme from Populus trichocarpa, in particular the CCoAMT described in SEQ ID NO:16.

[0119] According to one embodiment, the CCoAMT comprises a sequence chosen from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity.

[0120] According to a preferred embodiment, the CCoAMT comprises a sequence chosen from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity.

[0121] According to a particular embodiment, the recombinant microorganism comprises

[0122] - a heterologous nucleic acid sequence coding for a CCoAMT, preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0123] - a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and more particularly preferably, a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity.

[0124] According to a preferred embodiment, the recombinant microorganism comprises

[0125] - a heterologous nucleic acid sequence coding for a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting CCoAMT activity; and

[0126] - a heterologous nucleic acid sequence encoding a 4CL comprising a sequence chosen from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity.

[0127] In both of these embodiments, the microorganism may further comprise

[0128] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0129] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH and wherein said CCR comprises a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprises a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity.

[0130] As mentioned above, the microorganism according to the invention may also be capable of producing the carboxyl-CoA of interest, preferably feruloyl-CoA or sinapoyl-CoA, from a synthesis intermediate such as cinnamic acid, p-coumaric acid or L-Dopa (3,4-dihydroxy-L-phenylalanine), or from glucose via phenylalanine or tyrosine. From tyrosine

[0131] The production of caffeic acid from tyrosine can involve two pathways: the first involves the transformation of tyrosine into p-coumaric acid and then of p-coumaric acid into caffeic acid, the second involves the transformation of tyrosine into L-Dopa and then of L-Dopa into caffeic acid (see [fig.8]).

[0132] The production of caffeic acid from p-coumaric acid or L-Dopa from tyrosine may be mediated by an enzyme or enzyme complex having p-coumarate 3-hydroxylase activity.

[0133] According to one embodiment, the microorganism according to the invention may comprise, in addition to the heterologous nucleic acid sequences described above coding for an acyl-coenzyme A thioesterase and / or a CCR / ALDH combination, a CCoAMT and a 4CL, one or more heterologous nucleic acid sequences coding for an enzyme or an enzyme complex having p-coumarate 3-hydroxylase activity.

[0134] As used herein, the term "p-coumarate 3-hydroxylase activity" refers to a enzyme or enzyme complex catalyzing the transformation of p-coumaric acid into caffeic acid and / or L-tyrosine into L-Dopa. To determine whether there is p-coumarate 3 hydroxylase activity, an enzymatic test can be performed which consists of the in vitro incubation of the enzyme or enzyme complex, p-coumaric acid or L-Tyrosine, and possibly FAD and NADH, 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.

[0135] In particular, this activity may be the result of an enzymatic complex comprising a 4-hydroxyphenylacetate 3-monooxygenase oxygenase (HpaB) and a 4-hydroxyphenylacetate 3-monooxygenase reductase (HpaC).

[0136] The term "4-hydroxyphenylacetate 3-monooxygenase oxygenase" refers to an enzyme exhibiting p-coumarate 3-hydroxylase activity when in the presence of a 4-hydroxyphenylacetate 3-monooxygenase reductase. The term "4-Hydroxyphenylacetate 3-monooxygenase reductase" refers to an enzyme exhibiting p-coumarate 3-hydroxylase activity when in the presence of a 4-hydroxyphenylacetate 3-monooxygenase oxygenase.

[0137] Preferably, the HpaB and HpaC enzymes are produced by bacteria, preferably Escherichia coli, bacteria of the genus Pseudomonas, in particular Pseudomonas aeruginosa, or bacteria of the genus Salmonella, in particular Salmonella enterica. The HpaB and HpaC enzymes may originate from the same bacterium or from different bacteria.

[0138] In particular, HpaB may be an enzyme from Pseudomonas aeruginosa, in particular the HpaB described in SEQ ID NO: 17, or an enzyme from Escherichia coli, in particular the HpaB described in SEQ ID NO: 26.

[0139] According to one embodiment, the HpaB comprises a sequence chosen from the sequences SEQ ID NO: 17 and 26 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-hydroxyphenylacetate 3-monooxygenase oxygenase activity.

[0140] According to a preferred embodiment, the HpaB comprises a sequence chosen from the sequence SEQ ID NO: 17 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity.

[0141] The HpaC may be a Salmonella enterica enzyme, in particular the HpaC described in SEQ ID NO: 18, or an Escherichia coli enzyme, in particular the HpaC described in SEQ ID NO: 27.

[0142] According to one embodiment, the HpaC comprises a sequence chosen from the sequences SEQ ID NO: 18 and 27 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-hydroxyphenylacetate 3-monooxygenase reductase activity.

[0143] According to a preferred embodiment, the HpaC comprises a sequence selected from the sequence SEQ ID NO: 18 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity.

[0144] According to a particularly preferred embodiment, the microorganism comprises

[0145] - a heterologous nucleic acid sequence coding for a 4-hydroxyphenylacetate 3-monooxygenase oxygenase, preferably comprising a sequence selected from the sequences SEQ ID NO: 17 and 26 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-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and more particularly preferably, a sequence selected from SEQ ID Nos: 17 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and

[0146] - a heterologous nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase, preferably comprising a sequence selected from the sequences SEQ ID NO: 18 and 27 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-hydroxyphenylacetate 3-monooxygenase reductase activity, and more particularly preferably, a sequence selected from SEQ ID Nos: 18 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity.

[0147] As an alternative to the use of HpaB and HpaC or in combination with them, it is possible to use on the one hand an enzyme for converting tyrosine into L-Dopa, namely a 4-methoxybenzoate O-demethylase, and on the other hand an enzyme for converting p-coumaric acid into caffeic acid, namely a p-coumarate 3-hydroxylase (see [fig.8]). These enzymes are part of the cytochrome P450 (CYP) family and are CPR dependent, i.e. they are active in the presence of a cytochrome P450 reductase (CPR). The cytochrome P450 reductase can be an endogenous enzyme or a heterologous enzyme.

[0148] Thus, the microorganism according to the invention may comprise a heterologous nucleic acid sequence coding for a CPR-dependent p-coumarate 3-hydroxylase (C3H), i.e. an enzyme capable of converting p-coumaric acid to caffeic acid in the presence of a CPR (EC 1.14.13). The p-coumarate 3-hydroxylase activity of this enzyme can be tested as indicated above and in the presence of a CPR.

[0149] C3H may be an enzyme from bacteria, in particular from bacteria of the genus Sac-charothrix. In particular, C3H may be an enzyme from Saccharothrix espanaensis, preferably the enzyme described in SEQ ID NO: 25.

[0150] In a particular embodiment, the C3H comprises a sequence selected from the sequence SEQ ID NO: 25 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 25 and exhibiting p-coumarate 3-hydroxylase activity.

[0151] The microorganism according to the invention may also comprise a heterologous nucleic acid sequence coding for a 4-methoxybenzoate O-demethylase, i.e. an enzyme capable of converting L-tyrosine into L-Dopa in the presence of a CPR (EC 1.14.99.15). The p-coumarate 3-hydroxylase activity of this enzyme may be tested as indicated above and in the presence of a CPR and tyrosine.

[0152] The 4-methoxybenzoate O-demethylase may be an enzyme of bacteria, in particular of Rhodopseudomonas palustris, Pseudomonas putida, or Escherichia coli, of plants, in particular of Beta vulgaris, of mammals, in particular of Oryctolagus cuniculus, or of fungi, in particular of Rhodotorula glutinis. In a particular embodiment, the 4-methoxybenzoate O-demethylase is an enzyme of Rhodopseudomonas palustris, in particular the enzyme described in SEQ ID NO: 28, or of Beta vulgaris, in particular the enzyme described in SEQ ID NO: 29.

[0153] In a particular embodiment, the 4-methoxybenzoate O-demethylase comprises a sequence chosen from SEQ ID NOs: 28 and 29 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 L-tyrosine hydrolase activity.

[0154] The production of p-coumaric acid from tyrosine involves an enzyme having tyrosine ammonia lyase (TAL) activity. According to one embodiment, the microorganism according to the invention may comprise a heterologous nucleic acid sequence encoding an enzyme having tyrosine ammonia lyase activity.

[0155] As used herein, the term "tyrosine ammonia lyase" or "TAL" refers to an enzyme catalyzing the production of p-coumaric acid from L-tyrosine (EC 4.3.1.23).

[0156] The detection of tyrosine ammonia lyase activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. TAL activity can in particular be detected via an enzymatic test consisting of the in vitro incubation of a mixture composed of the enzyme to be tested and tyrosine under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of p-coumaric acid is observed in UPLC-MS compared to the expected standard.

[0157] Some TALs may also exhibit dihydroxyphenylalanine ammonia lyase (DAL) and / or phenylalanine ammonia lyase (PAL) activity.

[0158] The TAL may be an enzyme produced by a bacterium, in particular a bacterium of the genus Rhodobacter, preferably Rhodobacter capsulatus or Rhodobacter sphaeroides, of the genus Ralstonia, preferably Ralstonia metallidurans, or of the genus Flavobacteriaceae, preferably Flavobacterium johnsoniae. It may also be an enzyme produced by a plant, for example by Camellia sinensis, Fragaria x ananassa or Zea mays. Preferably, the TAL is an enzyme produced by a yeast, in particular a yeast of the genus Rhodotorula, for example Rhodotorula glutinis.

[0159] In particular, the TAL may be an enzyme from Flavobacterium johnsoniae, preferably the enzyme described in SEQ ID NO: 30, or an enzyme from Rhodotorula glutinis, preferably the enzyme described in SEQ ID NO: 19.

[0160] In a particular embodiment, the TAL comprises a sequence chosen from SEQ ID NOs: 19 and 30 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 TAL activity.

[0161] In a preferred embodiment, the TAL comprises a sequence selected from the sequence SEQ ID NO: 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0162] The production of caffeic acid from L-Dopa involves an enzyme having dihydroxyphenylalanine ammonia lyase (DAL) activity. According to one embodiment, the microorganism according to the invention may comprise a heterologous nucleic acid sequence encoding an enzyme having dihydroxyphenylalanine ammonia lyase activity.

[0163] As used herein, the term "dihydroxyphenylalanine ammonia lyase" or "DAL" refers to an enzyme catalyzing the production of caffeic acid from L-Dopa (EC 4.3.1.11).

[0164] The detection of a dihydroxyphenylalanine ammonia lyase activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. The DAL activity can in particular be detected via an enzymatic test consisting of the in vitro incubation of a mixture composed of the enzyme to be tested and L-Dopa under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of caffeic acid is observed by UPLC-MS in comparison with the expected standard.

[0165] Some DALs may also exhibit TAL and / or PAL activity.

[0166] According to a particular embodiment, the recombinant microorganism comprises

[0167] - a heterologous nucleic acid sequence coding for a CCoAMT, preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0168] - a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and more particularly preferably, a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity; and

[0169] - (i) a heterologous nucleic acid sequence encoding a C3H, preferably a C3H comprising a sequence selected from the sequence SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 25 and exhibiting p-coumarate 3-hydroxylase activity; and / or (ii) a heterologous nucleic acid sequence encoding an HpaB and a heterologous nucleic acid sequence encoding an HpaC, said HpaB preferably comprising a sequence selected from the sequences SEQ ID NO: 17 and 26 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-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and more particularly preferably, a sequence selected from SEQ ID Nos: 17 and the polypeptides comprising a sequence having at least 60, 70, 80, 85,90 or 95% sequence identity with the sequence SEQ ID NO: 17 and having 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and said HpaC preferably comprising a sequence selected from the sequences SEQ ID NO: 18 and 27 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and having 4-hydroxyphenylacetate 3-monooxygenase reductase activity, and more particularly, particularly preferred, a sequence selected from SEQ ID Nos: 18 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity; and

[0170] - a heterologous nucleic acid sequence encoding a TAL, preferably a TAL comprising a sequence chosen from the sequences SEQ ID NOs: 19 and 30 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 TAL activity, and more particularly preferably, a sequence chosen from the sequence SEQ ID NO: 19 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0171] According to a preferred embodiment, the recombinant microorganism comprises

[0172] - a heterologous nucleic acid sequence coding for a CCoAMT, preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0173] - a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and more particularly preferably, a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity; and

[0174] - a heterologous nucleic acid sequence encoding an HpaB and a sequence of heterologous nucleic acid encoding an HpaC, said HpaB preferably comprising a sequence selected from SEQ ID No: 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and having 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and said HpaC preferably comprising a sequence selected from SEQ ID Nos: 18 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and having 4-hydroxyphenylacetate 3-monooxygenase reductase activity; and

[0175] - a heterologous nucleic acid sequence encoding a TAL, preferably a TAL comprising a sequence selected from the sequence SEQ ID NO: 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0176] In these embodiments, the microorganism may further comprise

[0177] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0178] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH and wherein said CCR comprises a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprises a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity. From phenylalanine

[0179] The production of caffeic acid from phenylalanine involves enzymes specific to this pathway, namely a phenylalanine ammonia lyase (PAL) capable of producing cinnamic acid from phenylalanine and a cinnamate 4-hydroxylase (C4H) capable of producing p-coumaric acid from cinnamic acid, and the enzymes or enzyme complexes described above capable of converting p-coumaric acid into caffeic acid (see [fig.8]).

[0180] Thus, the microorganism according to the invention may comprise, in addition to the heterologous nucleic acid sequences described above coding for an acyl-coenzyme A thioesterase and / or a CCR / ALDH combination, a CCoAMT, a 4CL and a enzyme or an enzyme complex having p-coumarate 3-hydroxylase activity, a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and / or a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL) and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H).

[0181] As used herein, the term "phenylalanine ammonia lyase" or "PAL" refers to an enzyme catalyzing the production of cinnamic acid (also called trans-cinnamic acid) from phenylalanine (EC 4.3.1.24).

[0182] The detection of phenylalanine ammonia lyase activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. PAL activity can in particular be detected via an enzymatic test consisting of the in vitro incubation of a mixture composed of the enzyme to be tested and phenylalanine under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of cinnamic acid is observed by UPLC-MS in comparison with the expected standard.

[0183] Some PALs may also exhibit TAL activity and / or DAL activity.

[0184] More enzymes PAL ont déjà été décrites dans l'art antiereur. Preferably, the PAL comes from a plant, for example a plant of the genus Arabidopsis, Agastache, Pineapple, Asparagus, Brassica, Bromheadia, Bambusa, Beta, Betula, Citrus, Cucumis, Camellia, Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot, Medicago, Mesembryanthemum, Nicotiana, Olea, Oryza, Phaseolus, Pinus, Populus, Pisum, Persea, Petroselinum, Phalaenopsis, Phyllostachys, Physcomitrella, Picea, Pyrus, Prunus, Quercus, Raphanus, Rehmannia, Rubus, Solanum, Sorghum, Sphenostylis, Stellaria, Stylosanthes, Triticum, Trifolium, Vaccinium, Vigna, Vitis, Zea, or Zinnia.

[0185] In particular, the PAL may be an enzyme from Arabidopsis thaliana, preferably the enzyme described in SEQ ID NO: 20, or an enzyme from Citrus sinensis, preferably one of the enzymes described in SEQ ID NOs: 31 and 32.

[0186] In a particular embodiment, the PAL comprises a sequence selected from SEQ ID NOs: 20, 31 and 32 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 PAL activity.

[0187] In a preferred embodiment, the PAL comprises a sequence selected from the sequence SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting PAL activity.

[0188] As used herein, the term "cinnamate 4-hydroxylase" or "C4H" refers to an enzyme catalyzing the production of p-coumaric acid from cinnamic acid (EC 1.14.13.11). This enzyme is CPR dependent.

[0189] The detection of cinnamate 4-hydroxylase activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. The C4H activity can in particular be detected via an enzymatic test which consists of the in vitro incubation of a mixture composed of the enzyme to be tested, cinnamic acid, NADPH, H+ and O2 under optimal conditions (pH, temperature, ions, etc.). After a certain incubation time, the appearance of p-coumaric acid is observed by UPLC-MS in comparison with the expected standard.

[0190] Several C4H enzymes have already been described in the prior art. Preferably, C4H originates from a plant, for example a plant of the genus Arabidopsis, Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Citrus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitreila, Phaseolus, Pinus, Populus, Ruta, Saccharum, Solanum, Vitis, Vigna or Zea.

[0191] In particular, C4H may be an enzyme from Arabidopsis thaliana, preferably the enzyme described in SEQ ID NO: 21, or an enzyme from Citrus sinensis, preferably one of the enzymes described in SEQ ID NOs: 33 and 34.

[0192] In a particular embodiment, the C4H comprises a sequence selected from SEQ ID NOs: 21, 33 and 34 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 C4H activity.

[0193] In a preferred embodiment, the C4H comprises a sequence selected from the sequence SEQ ID NO: 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting C4H activity.

[0194] According to one embodiment, the microorganism according to the invention comprises

[0195] - a heterologous nucleic acid sequence coding for a phenylalanine ammonia lyase (PAL), preferably comprising a sequence selected from SEQ ID NOs: 20, 31 and 32 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting PAL activity, and more preferably, comprising a sequence selected from the sequence SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting PAL activity; and

[0196] - a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably comprising a sequence selected from SEQ ID NOs: 21, 33 and 34 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting C4H activity, and more particularly preferably, comprising a sequence chosen from the sequence SEQ ID NO: 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting C4H activity.

[0197] According to a particular embodiment, the recombinant microorganism comprises

[0198] - a heterologous nucleic acid sequence coding for a CCoAMT, preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0199] - a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and more particularly preferably, a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity; and

[0200] - (i) a heterologous nucleic acid sequence encoding a C3H, preferably a C3H comprising a sequence selected from the sequence SEQ ID NO: 25 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 25 and exhibiting p-coumarate 3-hydroxylase activity; and / or (ii) a heterologous nucleic acid sequence encoding an HpaB and a heterologous nucleic acid sequence encoding an HpaC, said HpaB preferably comprising a sequence selected from the sequences SEQ ID NO: 17 and 26 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-hydroxyphenylacetate 3-monooxygenase activity oxygenase, and more particularly preferably, a sequence chosen from SEQ ID Nos: 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and said HpaC preferably comprising a sequence chosen from the sequences SEQ ID NO: 18 and 27 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-hydroxyphenylacetate 3-monooxygenase reductase activity, and more particularly preferably, a sequence chosen from SEQ ID Nos: 18 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity; and

[0201] - a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), preferably comprising a sequence selected from SEQ ID NOs: 20, 31 and 32 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting PAL activity, and more preferably, comprising a sequence selected from the sequence SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting PAL activity; and

[0202] - a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably comprising a sequence selected from SEQ ID NOs: 21, 33 and 34 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting C4H activity, and more particularly preferably, comprising a sequence selected from the sequence SEQ ID NO: 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting C4H activity.

[0203] Optionally, in this embodiment, the microorganism may also comprise a heterologous nucleic acid sequence encoding a T AL, preferably a T AL comprising a sequence selected from the sequences SEQ ID NOs: 19 and 30 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 TAL activity, and more particularly preferably, a sequence selected from the sequence SEQ ID NO: 19 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0204] According to a particular preferred embodiment, the recombinant microorganism understand

[0205] - a heterologous nucleic acid sequence encoding a CCoAMT, of preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0206] - a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and more particularly preferably, a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity; and

[0207] - a heterologous nucleic acid sequence encoding an HpaB and a sequence of heterologous nucleic acid encoding an HpaC, said HpaB preferably comprising a sequence selected from SEQ ID No: 17 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and said HpaC preferably comprising a sequence selected from SEQ ID Nos: 18 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity; and

[0208] - a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), preferably comprising a sequence selected from the sequence SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting PAL activity; and

[0209] - a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably comprising a sequence selected from the sequence SEQ ID NO: 21 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting C4H activity.

[0210] Optionally, in this embodiment, the microorganism may also comprise a heterologous nucleic acid sequence encoding a TAL, preferably a TAL comprising a sequence selected from the sequence SEQ ID NO: 19 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0211] In these embodiments, the microorganism may further comprise

[0212] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0213] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH and wherein said CCR comprises a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprises a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity. CPR: cytochrome P450 reductase

[0214] Some of the above-mentioned enzymes such as C3H or C4H are CPR-dependent enzymes. The activities of these enzymes require the presence of NADPH and therefore the presence of a cytochrome P450 reductase (CPR), in particular an NADPH-cytochrome P450 reductase.

[0215] Thus, in the various embodiments described above and below concerning the microorganism according to the invention, the latter may further comprise an endogenous nucleic acid coding for a cytochrome P450 reductase. Optionally, the endogenous CPR may be overexpressed, for example by replacing the promoter of the endogenous gene with a strong heterologous promoter and / or by increasing the number of copies of the endogenous gene.

[0216] Alternatively, or in addition to this endogenous nucleic acid, the microorganism according to the invention may comprise a heterologous nucleic acid sequence which codes for a cytochrome P450 reductase (CPR).

[0217] As used herein, the term "cytochrome P450 reductase" or "CPR" refers to an enzyme involved in the transfer of electrons from NADPH and belonging to class EC 1.6.2.4.

[0218] The detection of CPR activity can be carried out by any method known to those skilled in the art, in vivo or in vitro. CPRs are enzymes that catalyze the transfer of electrons from NADPH to cytochromes p450 (for example C3H or C4H). Thus, CPR activity can in particular be detected using an enzymatic kit combining the oxidation of NADPH by CPR with the reduction of a colorless substrate into a colored product with an absorbance peak at a given wavelength, the color generation rate being directly proportional to the CPR activity. An example of a commercial kit based on this principle is the “CPR activity assay kit” from PromoKine (Cat# PK-CA577-K700).

[0219] The CPR preferably originates from a eukaryote, in particular from a yeast, for example a yeast of the genus Saccharomyces, or from a plant, for example a plant of the genus Arabidopsis, Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Citrus, Glycine, Helianthus, Lotus, Mesembryanthemum, Phaseolus, Physcomitrella, Pinus, Populus, Ruta, Saccharum, Solanum, Vigna, Vitis or Zea.

[0220] In particular, the CPR may be an enzyme from Catharanthus roseus, preferably the enzyme described in SEQ ID NO: 22, an enzyme from Saccharomyces cerevisiae, preferably the enzyme described in SEQ ID NO: 35, or an enzyme from Arabidopsis thaliana, preferably one of the enzymes described in SEQ ID NOs: 36 and 37. The CPR may also be a chimeric protein such as that described in the article by Aigrain et al (2009, EMBO reports, 10, 742-747; SEQ ID NO: 38).

[0221] In a particular embodiment, the CPR comprises a sequence chosen from SEQ ID NOs: 22, 35, 36, 37 and 38 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 CPR activity.

[0222] In a preferred embodiment, the CPR comprises a sequence selected from SEQ ID NO: 22 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 22 and exhibiting CPR activity.

[0223] Increased production of tyrosine and / or phenylalanine

[0224] In the various embodiments described above and below concerning the microorganism according to the invention, the latter can also be modified to increase the production of tyrosine and / or phenylalanine, preferably tyrosine. Thus, in preferred embodiments, the microorganism according to the invention produces significant amounts of tyrosine and / or phenylalanine, in particular from a simple carbon source such as glucose.

[0225] This increase can be obtained by any method known to those skilled in the art and in particular by expressing one or more variants of one or more enzymes involved in the synthesis of these amino acids, said variants being resistant to feedback control by tyrosine and / or phenylalanine, preferably resistant to feedback control by tyrosine.

[0226] In particular, the microorganism may be modified to express a variant of 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase (EC 2.5.1.54) and / or chorismate mutase (EC 5.4.99.5), resistant to tyrosine feedback. Such variants are well known to those skilled in the art (see, for example, Gold et al., Microb CellFact. 2015;14:73).

[0227] Thus, according to a particular embodiment, the microorganism according to the invention comprises a heterologous nucleic acid sequence coding for a 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase resistant to feedback control by tyrosine and / or a heterologous nucleic acid sequence coding for a chorismate mutase resistant to feedback control by tyrosine.

[0228] In the yeast S. cerevisiae, 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase is encoded by the ARO4 gene (NCBI Gene ID: 852551) and chorismate mutase is encoded by the ARO7 gene (NCBI Gene ID: 856173). Tyrosine feedback-resistant variants of these enzymes are known, for example, the ARO4™ mutant (SEQ ID NO: 23) and the ARO7G141S mutant (SEQ ID NO: 24).

[0229] Thus, according to a preferred embodiment, the microorganism according to the invention comprises

[0230] - a heterologous nucleic acid sequence encoding a Tyrosine feedback-resistant 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase comprising a sequence selected from SEQ ID NO: 23 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 23, said polypeptides having tyrosine feedback-resistant 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase activity and, preferably, a leucine at the position corresponding to position 229 of SEQ ID NO: 23; and / or

[0231] - a heterologous nucleic acid sequence encoding a chorismate mutase re resistant to feedback control by tyrosine and comprising a sequence chosen from SEQ ID NO: 24 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 24, said poly peptides exhibiting chorismate mutase activity resistant to feedback by tyrosine and, preferably, a serine at the position corresponding to position 141 of SEQ ID NO: 24.

[0232] Alternatively or cumulatively, the increase in tyrosine and / or phenylalanine production can also be achieved by redirecting the carbon flow from other metabolic pathways to that of tyrosine and / or phenylalanine, preferably tyrosine. These modifications and the genes involved are well known to those skilled in the art (see US 8,809,028; Pandey et al, 2016, Biotechnol Adv., 34, 634-662).

[0233] Thus, according to a particular embodiment, in the microorganism according to the invention, one or more endogenous genes coding for an enzyme involved in the Ehrlich amino acid degradation pathway are inactivated. The gene(s) may be inactivated by any method known to those skilled in the art, in particular by total or partial deletion, or by insertion of a nucleic sequence into the coding sequence, in particular a sequence shifting the reading frame or inserting a stop codon.

[0234] In particular, in the microorganism according to the invention, an endogenous gene coding for a phenylpyruvate decarboxylase can be inactivated. This enzyme is responsible for the first step of the Ehrlich amino acid degradation pathway. In the yeast S. cerevisiae, phenylpyruvate decarboxylase is encoded by the ARO10 gene (NCBI Gene ID: 851987). Inactivation of ferulic acid decarboxylase

[0235] In the various embodiments described above concerning the microorganism according to the invention, the latter can also be modified to inactivate the endogenous gene(s) coding for a ferulic acid decarboxylase.

[0236] This enzyme catalyzes in particular the decarboxylation of ferulic acid, p-coumaric acid and cinnamic acid to produce their vinyl derivatives, namely respectively 4-vinylguaiacol, 4-vinylphenol, and styrene. It belongs to class EC 4.1.1.102. Its inactivation makes it possible to increase the available quantities of cinnamic acid and p-coumaric acid and the quantities of ferulic acid produced in the microorganism according to the invention.

[0237] The gene(s) coding for this enzyme in the microorganism according to the invention can be easily identified by a person skilled in the art. For example, in the yeast Saccharomyces cerevisiae, ferulic acid decarboxylase is coded by the FDC1 gene (NCBI Gene ID: 852152).

[0238] The gene(s) may be inactivated by any method known to those skilled in the art, in particular by total or partial deletion, or by insertion of a nucleic sequence into the coding sequence, in particular a sequence shifting the reading frame or inserting a stop codon.

[0239] Recombinant nucleic acid, cassette and expression vector

[0240] Each nucleic sequence coding for an enzyme as described above is included in an expression cassette. Preferably, 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 gene expression, in particular transcription and translation. These elements are chosen so as to be functional in the recombinant host microorganism. These elements may comprise, for example, transcription promoters, transcription activators, terminator sequences, initiation and termination codons. The methods for selecting these elements according to the host cell in which expression is desired are well known to those skilled in the art.

[0241] Preferably, the promoter is a strong promoter. The promoter may be constitutive or inducible, preferably constitutive. A promoter may control the expression of one or more nucleic sequences encoding one or more enzymes as described above.

[0242] For example, if the microorganism is prokaryotic, the promoter may be selected from the following promoters: Lac1, LacZ, pLacT, ptac, pARA, pBAD, the bacteriophage T3 or T7 RNA polymerase promoters, the polyhedrin promoter, the PR or PL promoter of lambda phage. In a particular embodiment, the promoter is pLac. If the microorganism is eukaryotic and in particular a yeast, the promoter may be selected from the following promoters: the pTDH3 promoter, the pTEF1 promoter, the pTEF2 promoter, the pCCW12 promoter, the pHHF2 promoter, the pHTB2 promoter and the pRPL18B promoter. Examples of inducible promoters that can be used in yeast are the tetO-2, GAL10, GAL10-CYC1 and PHO5 promoters.

[0243] All or part of the expression cassettes comprising the nucleic sequences encoding the enzymes as described or a combination of some of these may be included in a common expression vector or in different expression vectors.

[0244] The present invention therefore also relates to a vector comprising

[0245] - (i) a nucleic acid sequence encoding an acyl-coenzyme A thioesterase and / or (ii) a nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH), and / or

[0246] - at least one nucleic acid sequence chosen from an acid sequence nucleic acid sequence encoding a Caffeoyl-CoA O-Methyltransferase (CCoAMT), a nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL), a nucleic acid sequence encoding a p-coumarate 3-hydroxylase, a sequence of nucleic acid coding for a 4-hydroxyphenylacetate 3-monooxygenase oxygenase, a nucleic acid sequence coding for a 4-hydroxyphenylacetate 3-monooxygenase reductase, a nucleic acid sequence coding for a tyrosine ammonia lyase (TAL), a nucleic acid sequence coding for a phenylalanine ammonia lyase (PAL), a nucleic acid sequence coding for a cinnamate 4-hydroxylase (C4H) and a nucleic acid sequence coding for a cytochrome P450 reductase (CPR), each of these enzymes being as defined above, as well as combinations thereof. Preferably, the vector comprises

[0247] - (i) a nucleic acid sequence coding for an acyl-coenzyme A thioesterase and / or (ii) a nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH), and

[0248] - at least one nucleic acid sequence chosen from an acid sequence nucleic acid sequence encoding a Caffeoyl-CoA O-Methyltransferase (CCoAMT), a nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL), a nucleic acid sequence encoding a p-coumarate 3-hydroxylase, a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase, a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase, a nucleic acid sequence encoding a tyrosine ammonia lyase (TAL), a nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), a nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H) and a nucleic acid sequence encoding a cytochrome P450 reductase (CPR), each of these enzymes being as defined above, as well as combinations thereof.

[0249] The vector may in particular comprise combinations of particular coding sequences as described above.

[0250] By "comprising a nucleic acid sequence" is preferably meant "comprising an expression cassette comprising the nucleic acid sequence".

[0251] The vectors comprise heterologous coding sequences to the extent that the coding sequences may be optimized for the host microorganism, be under the control of heterologous promoter(s), and / or may combine coding sequences that are not from the same original organism and / or are not present in the same arrangement.

[0252] The vector may be any DNA sequence into which it is possible to insert foreign nucleic acids, the vectors making it possible to introduce foreign DNA into the host microorganism. The vector may be, for example, a plasmid, a phagemid, a cosmid, an artificial chromosome, in particular a YAC, or a BAC.

[0253] The expression vectors may further comprise nucleic sequences encoding selection markers. The selection markers may be genes for resistance to one or more antibiotics or auxotrophy genes. The auxotrophy gene may, for example, be HIS5, URA3, LEU2 or TRPL. The antibiotic resistance gene may, for example, preferably be a gene for resistance to ampicillin, chloramphenicol, spectinomycin, streptomycin, ka-namycin, hygromycin, geneticin, fluoroacetamide, fluorocitrate, phleomycin, amphotericin-B and / or nourseothricin.

[0254] The introduction of vectors into a host microorganism is a method widely known to those skilled in the art. Several methods are described in particular in “Current Protocols in Molecular Biology”, 13.7.1-13.7.10; or in Ellis T. et al., Integrative Biology, 2011, 3(2), 109-118.

[0255] The host microorganism may be transformed / transfected transiently or stably and the nucleic acid, cassette or vector according to the invention may be contained therein in episome form or in integrated form in the genome of the host cell.

[0256] The expression vector may also comprise one or more sequences allowing the targeted insertion of the vector, the expression cassette or the nucleic acid into the genome of the host cell.

[0257] All or part of the expression cassettes comprising the nucleic sequences coding for the enzymes as described above may be inserted into the / a chromosome of the recombinant microorganism. Conversely, all or part of the expression cassettes comprising the nucleic sequences coding for the enzymes as described may be conserved in episomal form, in particular in plasmid form.

[0258] Optionally, the microorganism may comprise several copies of nucleic sequences coding for an enzyme as described above. In particular, it may comprise 2 to 10 copies, for example 2, 3, 4, 5, 6, 7, 8, 9 or 10 copies of a nucleic sequence coding for an enzyme as described above.

[0259] Optionally, the host microorganism may be transformed / transfected with several vectors according to the invention, identical or different. It may also be transformed / transfected with one or more other vectors coding for example for other enzymes necessary for the production of the carboxylic acid. The present invention also relates to a method for preparing a recombinant microorganism according to the present invention comprising the introduction of a vector as defined above into the microorganism and the selection of the microorganisms comprising said vector. It also relates to a method for preparing a microorganism according to the present invention comprising the introduction

[0260] - (i) a nucleic acid sequence coding for an acyl-coenzyme A thioesterase and / or (ii) a nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH),

[0261] - and optionally at least one nucleic acid sequence chosen from a nucleic acid sequence encoding a Caffeoyl-CoA O-Methyltransferase (CCoAMT), a nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL), a nucleic acid sequence encoding a p-coumarate 3-hydroxylase, a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase oxygenase, a nucleic acid sequence encoding a 4-hydroxyphenylacetate 3-monooxygenase reductase, a nucleic acid sequence encoding a tyrosine ammonia lyase (TAL), a nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), a nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H) and a nucleic acid sequence encoding a coding for a cytochrome P450 reductase (CPR), each of these enzymes being as defined above, as well as combinations thereof,

[0262] and the selection of microorganisms comprising said nucleic sequences. Uses of the recombinant microorganism

[0263] The present invention also relates to the use of a microorganism according to the invention for producing a carboxylic acid, in particular a carboxylic acid derived from a carboxyl-CoA. It also relates to a method for producing a carboxylic acid, in particular a carboxylic acid derived from a carboxyl-CoA, comprising the cultivation of a microorganism according to the invention and optionally the harvesting and / or purification of said carboxylic acid.

[0264] As used herein, the term "carboxylic acid" refers to a compound comprising a carboxyl group (-C(O)OH). Preferably, the carboxylic acid is an aromatic carboxylic acid, i.e., a compound comprising a carboxyl group (-C(O)OH) and an aromatic group, preferably a phenyl ring.

[0265] The term "carboxylic acid derived from a carboxyl-CoA" refers to a carboxylic acid, preferably an aromatic carboxylic acid obtained from a carboxyl-CoA, i.e. a carboxylic acid whose biosynthetic pathway within the microorganism according to the invention involves at least one intermediate linked to CoA which is converted into said carboxylic acid by the action of (i) acyl-coenzyme A thioesterase and / or (ii) cinnamoyl-CoA reductase (CCR) and aldehyde dehydrogenase (ALDH) expressed from the heterologous nucleic acid sequences introduced into the microorganism.

[0266] Preferably, the carboxylic acid is a compound of formula (II) as defined below.

[0267] In particular, the present invention relates to the use of a microorganism according to the invention for the production of a compound of formula (II)

[0268] [Chem.3]

[0270] where

[0271] Rb R2 and R3 are selected, independently of each other, from the group consisting of a hydrogen atom and a group O-R5, where R5 is selected from the group consisting of a hydrogen atom and a hydrocarbon chain comprising from 1 to 36 carbon atoms, branched or unbranched; and

[0272] R4 is selected from the group consisting of C1-C6 alkyl groups and C1-C6 alkene groups, branched or unbranched, preferably from the group consisting of -(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p-(CH2)q-, where n is an integer selected from 1, 2, 3, 4 and 5 and m, p and q are integers selected from 1, 2 and 3.

[0273] It also relates to a method of producing a compound of formula (II)

[0274] [Chem.4]

[0275] (II)

[0276] where

[0277] R1, R2 and R3 are selected, independently of each other, from the group consisting of a hydrogen atom and an O-R5 group, where R5 is selected from the group consisting of a hydrogen atom and a hydrocarbon chain comprising from 1 to 36 carbon atoms, branched or unbranched; and

[0278] R4 is selected from the group consisting of C1-C6 alkyl groups and C1-C6 alkene groups, branched or unbranched, preferably from the group consisting of -(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p-(CH2)q-, where n is an integer selected from 1, 2, 3, 4 and 5 and m, p and q are integers selected from 1, 2 and 3,

[0279] comprising the cultivation of a microorganism according to the invention and optionally the harvesting and / or purification of said compound.

[0280] Preferably, R5 is selected from the group consisting of a hydrogen atom, branched or unbranched C1-C6 alkyl groups and branched or unbranched C1-C6 alkene groups. More preferably, R5 is selected from the group consisting of a hydrogen atom and a methyl group.

[0281] according to a particular embodiment,

[0282] - Ri, R2 and R3 are chosen, independently of each other, from the group consisting of a hydrogen atom and an O-R5 group, where R5 is a methyl group, and

[0283] - R4 is selected from the group consisting of -(CH2)n-, -CH=CH-, -(CH2)m-(CH=CH)p - (CH2)q-, where n is an integer chosen from 1 or 2 and m, p and q are integers chosen from 1 or 2.

[0284] According to another particular embodiment,

[0285] - Ri, R2 and R3 are chosen, independently of each other, from the group consisting of a hydrogen atom and an O-R5 group, where R5 is a methyl group, and

[0286] - R4 is -CH2-CH=CH-CH2-,

[0287] The microorganism according to the invention catalyzes at least the hydrolysis reaction of a carboxyl-CoA of formula (I)

[0288] [Chem.5] S—CoA

[0289] (I)

[0290] making it possible to obtain the compound of formula (II).

[0291] The groups RB R2 R3 and R4 are defined as indicated above for the compound of formula (II).

[0292] According to a preferred embodiment, the compound of formula (II) is chosen from ferulic acid and sinapic acid.

[0293] According to a very particularly preferred embodiment, the compound of formula (II) is ferulic acid.

[0294] The compound of formula (II) produced may be either the final product or a synthetic or biosynthetic intermediate for the preparation of other compounds.

[0295] 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.

[0296] The microorganism is cultured in a suitable culture medium. The term "suitable culture medium" generally refers to a culture medium providing nutrients essential or beneficial to the maintenance and / or growth of said microorganism, such as carbon sources; nitrogen sources such as ammonium sulfate; phosphorus sources, 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. An antifoam may be added as required. According to the invention, this suitable culture medium may be chemically defined or “undefined”. The culture medium can thus be of identical or similar composition to a synthetic medium, as defined by Verduyn et al., (Yeast. 1992. 8:501-17), adapted by Visser et al., (Biotechnology and bioengineering. 2002. 79:674-81), or commercially available such as YNB medium (Yeast Nitrogen Base, MP Biomedicals or Sigma-Aldrich).In particular, the culture medium may comprise a simple carbon source, such as glucose, fructose, xylose, ethanol, glycerol, galactose, sucrose, cellulose, cellobiose, starch, glucose polymers, molasses, or by-products of these sugars. The "undefined" medium may be a liquid medium composed of hydrolysates of microorganisms and / or proteins, for example and not exclusively yeast extract and / or peptones. To this composition is generally added a simple carbon source, such as glucose, fructose, xylose, ethanol, glycerol, galactose, sucrose, cellulose, cellobiose, starch, glucose polymers, molasses, or by-products of these sugars.

[0297] The microorganism according to the invention may comprise all or part of the biosynthetic pathway of the carboxylic acid of interest. Thus, the production may be carried out in the presence of a simple carbon source such as glucose, or in the presence of a synthesis intermediate such as tyrosine, phenylalanine, cinnamic acid or p-coumaric acid. For the synthesis of ferulic acid, the synthesis intermediate may also be caffeic acid, caffeoyl-CoA or feruloyl-CoA. For the synthesis of sinapic acid, the synthesis intermediate may also be 5-hydroxyferulic acid, 5-hydroxyferuloyl-CoA or sinapoyl-CoA.

[0298] According to a particular embodiment, the microorganism according to the invention is used in a method for producing ferulic acid from caffeic acid. Preferably, the culture of the microorganism is carried out in the presence of caffeic acid in the culture medium. Preferably, in this embodiment, the microorganism comprises

[0299] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 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 acyl-coenzyme A thioesterase activity, and more particularly preferably an acyl-coenzyme A thioesterase comprising a sequence chosen from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0300] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH, preferably said CCR comprising a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprising a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity,

[0301] and further comprises

[0302] - a heterologous nucleic acid sequence encoding a CCoAMT, of preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0303] a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 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 4CL activity, and most preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity.

[0304] According to a preferred embodiment, the microorganism according to the invention is used in a method for producing ferulic acid from glucose. Preferably, the culture of the microorganism is carried out without the addition of an intermediate compound in the medium, i.e. without the addition of tyrosine, phenylalanine, cinnamic acid, p-coumaric acid, caffeic acid, caffeoyl-CoA and / or feruloyl-CoA. Preferably, in this embodiment, the microorganism comprises

[0305] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity, and more particularly preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0306] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH, preferably said CCR comprising a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprising a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity,

[0307] and further comprises

[0308] - a heterologous nucleic acid sequence encoding a CCoAMT, of preferably a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence selected from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0309] a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5, 6, 8 and 9 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting 4CL activity, and most preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity; and

[0310] - (i) a heterologous nucleic acid sequence encoding a C3H, preferably a C3H comprising a sequence chosen from the sequence SEQ ID NO: 25 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 25 and exhibiting p-coumarate 3-hydroxylase activity; and / or (ii) a heterologous nucleic acid sequence encoding an HpaB and a heterologous nucleic acid sequence encoding an HpaC, said HpaB preferably comprising a sequence selected from the sequences SEQ ID NO: 17 and 26 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-hydroxyphenylacetate 3-monooxygenase oxygenase activity, and more preferably, a sequence selected from SEQ ID Nos: 17 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 17 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase oxygenase activity,and said HpaC preferably comprising a sequence selected from the sequences SEQ ID NO: 18 and 27 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-hydroxyphenylacetate 3-monooxygenase reductase activity, and more particularly preferably, a sequence selected from SEQ ID Nos: 18 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 18 and exhibiting 4-hydroxyphenylacetate 3-monooxygenase reductase activity;

[0311] and further comprises

[0312] (i) a heterologous nucleic acid sequence encoding a phenylalanine ammonia lyase (PAL), preferably comprising a sequence selected from SEQ ID NOs: 20, 31 and 32 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting PAL activity, and more particularly preferably, comprising a sequence selected from the sequence SEQ ID NO: 20 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 20 and exhibiting PAL activity;and a heterologous nucleic acid sequence encoding a cinnamate 4-hydroxylase (C4H), preferably comprising a sequence selected from SEQ ID NOs: 21, 33 and 34 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 C4H activity, and more preferably, comprising a sequence selected from the sequence SEQ ID NO: 21 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 21 and exhibiting C4H activity; and / or;

[0313] (ii) a heterologous nucleic acid sequence encoding a TAL, preferably a TAL comprising a sequence chosen from the sequences SEQ ID NOs: 19 and 30 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 TAL activity, and more particularly preferably, a sequence chosen from the sequence SEQ ID NO: 19 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 19 and exhibiting TAL activity.

[0314] According to another preferred embodiment, the microorganism according to the invention is used in a method for producing sinapic acid from 5-hydroxyferulic acid. Preferably, the culture of the microorganism is carried out without adding an intermediate compound to the medium, i.e. without adding 5-hydroxyferuloyl-CoA and / or sinapoyl-CoA. Preferably, in this embodiment, the microorganism comprises

[0315] - a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase, preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity, and more preferably an acyl-coenzyme A thioesterase comprising a sequence selected from the sequence SEQ ID NO: 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 2 and exhibiting acyl-coenzyme A thioesterase activity; and / or

[0316] - a heterologous nucleic acid sequence encoding a CCR and a sequence of heterologous nucleic acid encoding an ALDH, preferably said CCR comprising a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity, and said ALDH comprising a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity,

[0317] and further comprises

[0318] - a heterologous nucleic acid sequence encoding a CCoAMT, of preferably a CCoAMT comprising a sequence chosen from the sequences SEQ ID NO: 10 to 16 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 CCoAMT activity, and more particularly preferably, a CCoAMT comprising a sequence chosen from the sequences SEQ ID NO: 10, 15 and 16 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 CCoAMT activity; and

[0319] a heterologous nucleic acid sequence encoding a 4CL, preferably a 4CL comprising a sequence selected from the sequences SEQ ID NO: 5 to 9 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 4CL activity, and very particularly preferably a 4CL comprising a sequence selected from the sequence SEQ ID NO: 6 and the polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 6 and exhibiting 4CL activity.

[0320] According to the invention, any culture method allowing the industrial-scale production of molecules of interest can be envisaged. Advantageously, the culture is carried out in bioreactors, in particular in batch, fed-batch, chemostat and / or continuous culture mode. A controlled supply of vitamins during the process can also be beneficial to productivity (Alfenore et al., Appl Microbiol Biotechnol. 2002. 60:67-72).

[0321] The culture is generally carried out in bioreactors, with possible stages of solid and / or liquid precultures in Erlenmeyers, with an appropriate culture medium.

[0322] Generally speaking, the conditions for culturing the microorganisms according to the invention are easily adaptable by those skilled in the art, depending on the microorganism. For example, the culture temperature is in particular between 20°C and 40°C for yeasts, preferably between 28°C and 40°C, and more particularly around 30°C for S cerevisiae.

[0323] The microorganism according to the present invention can be cultured for 1 to 30 days, and preferably 1 to 10 days.

[0324] The present invention also relates to the use of a microorganism according to the invention for catalyzing the conversion of a carboxyl-CoA, preferably of formula (I) into a carboxylic acid, preferably of formula (II). It further relates to a method for catalyzing the conversion of a carboxyl-CoA, preferably of formula (I) into a carboxylic acid, preferably of formula (II), comprising culturing a microorganism according to the invention in the presence of said carboxyl-CoA or one of these precursors, and optionally harvesting and / or purifying said carboxylic acid. All the embodiments described above also relate to this aspect.

[0325] According to a preferred embodiment, the carboxyl-CoA is feruloyl-CoA, the carboxylic acid is ferulic acid and the precursor is selected from glucose, phenylalanine, tyrosine, cinnamic acid, p-coumaric acid, caffeic acid and caffeoyl-CoA, preferably is caffeic acid.

[0326] According to another preferred embodiment, the carboxyl-CoA is sinapoyl-CoA, the carboxylic acid is sinapic acid and the precursor is selected from glucose, phenylalanine, tyrosine, cinnamic acid, p-coumaric acid, 5-hydroxyferulic acid and 5-hydroxyferuloyl-CoA, preferably is 5-hydroxyferulic acid.

[0327] Other characteristics and advantages of the invention will appear better on reading the following examples given for illustrative and non-limiting purposes.

[0328] Table 1: DESCRIPTION OF SEQUENCES

[0329] <h2 style=";text-align:left;direction:ltr">pEffiDNO: Pescripdons des sentences «Feades aminés kia <t -;ce-”cviei’h ri' ^cvi-cDenryî'sieA thj'SeKèrasedeffeïvnisbjt&ddGnhsoîj C*sdéhÿdedesfTydf£®è!~sssd‘A^ Mws C« n namo^-CoA t èauctasedeFoAOftiS' laBeMM '5 ^■ousiafoys-CoA ^aædWc&&s^sâ ■îWïana^lli 5 $ ^<oyrï:srçy^feAS^^^ Ï^CLE; 5 ^•<CU:lYr8 T^ ^8SE dP'i'iïïüT-rîiî ^CL?j ; îg; |Vcoum a royi-CcA i igase de Aspeta totne«a»o {4CLAS ^<cuisîB.ro¥5-CcA S^ëd'Ansi»d<^sis daStsms : O -.h $ r-4 rû ^a’àcyH-oA G-mêthvïtransSà-asede Zità vimfem :£Cs4MTi) ^af ê 0 y :-CoA O-mshySrBnsfà’ase d e Wd: scçs 5 ¢7- / 0 1 £< c 7 M ~2) êo> i-CcA C -m Kh y ^rBns'èrase d'£5717.72 n.;s ÿfoiws ! l CsA M T3; £afecy:-£cAO-rr.»hv»"ar£ferasede'r'w.is.'siriri-ory^ 4; ^4 £aFêc7i <oAû-"ieThy4jari:vé;v£-sde7£;€^^^ 5 -J 'Vf yi ^3'èoy:-£oAO-mèihyXranÿéfased^raS:®^^^ roa^'ano{££cAMT6} fcsfèojrt-EcA O-rciêihymsns'èfBsede Pcpufus TrtcS’ao’ps KCoAMT?) ^■■fv / dyo^p.hèrÿiacetare 3-morfCQ> Synephrine: Synephrine B®.------- H- iVfdTŒ^ph 3- moncK»ygenase reductase deS&'mBne&a esteric® I 1 <h ï O ^yrasi n e am mansa Syæe de^odston^s |»hêny^^andîeamman3 ’yased'Arg&.dt»»)' ffwîiæia —™™; < n naîmaîeA-hydf ci-y; ase a '4 ; a&f k;c: îs ffioQSï^o hÿtGchrDrne F45$rédurt3ædeCtôtf^^ .s^sssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssssss ^fü4n:* réserve bu fé^Gzcnûrdje ^.vg?'^' résistants a'jreTfccontfà« h5 untarste 3-hydroxy'sse de Siec / ara’.fcwes^fle^^ 5 h& £77 ^-hyd^oxyphé ny:aceïa7e S-snmcsayg énase sxyg énss^ ^-hydïoxyp h é'Ty4aceîate s-iYicTOcsygéfsse æduc^ 23 ^■inèthox?ybenïoasei> dèmêthyjB3edeSscdE.£set^^^ 5 ïzs ^mêth^'bereoateO-dèræthy^sædeSetoi^ssns' ^yror ine am ms nia sysse e! and F?svs&srref . uwj 'sb ^2 |»h knowledge of the world of Gtm »«e«s <s |jheny;Bianine annion® *( asede Gnvs r.'wMt --------------------------------i 33 ç nn arnate^-hyd roxy' ase de rfîr^î s< n ess iff $4 n naflnaEeA^ïydrwi ase de Citeras swnsà g5 Cytochrome P45S reductese de&orôsromyæ? cerasâÿœ ^tochïorae PASSréducased Arodidcpsss thans»» .sssssssssssssssssss^ h? Cytochrome P453reduc&&ed'Awïd>32S'r rW <er:s : . ■^ytdchfome F45Grêduccasechiraà-$que EXAMPLES Materials and methods Strains

[0330] The yeast strains used in the examples were obtained from Sac-charomyces cerevisiae S288C (Mortimer RK and Johnston JR (1986) Genealogy of principal strains of the yeast genetic stock center. Genetics 113(1):35-43). This yeast has auxotrophy for uracil, tryptophan and leucine.

[0331] The constructs were made in the Escherichia coli MH1 strain before their transfer to yeast.

[0332] In all the strains constructed for this study, the ARO10 (YDR380W) and FDC1 (YDR539W) genes were inactivated, i.e. by integration in place of the open reading frame, of a linear DNA comprising a selection marker bounded by the upstream and downstream regions of the gene.

[0333] Table 2: List of constructed strains

[0334] <h2 style=";text-align:left;direction:ltr">Non of (a similar protein code) from these genes' contents: 221 4CL1 (Arabic language, SEQ5DNO: 5} CCoAMT6(Arab*dopSiSîhahana, SEQ©ND: 15? 239 4CL1) (Arabidopss^thatsana.SEüiDNO: 5} ICoAVTô (Aran^opsÆt^ahaod SEZjiDNO 15' Th sol (Arab8dopssthatiana?SEQ.iDNO: 1} 345 4CQ (Arabidopssîhahana, SEQ© NO : 5] OCoAMTS (ÀratMdopssthaüana, SEQ) ©NO: 15) Thioâ (Petunia hybrida, SEQ© NO: 2} 214 4CL3.. (Arabidopss thaHana, SEQ © NO ; 5) CCoAMTS (ArabidopsjsthaÜana, SEQiDNO : 15) QCR1 (Popuiustomentcsa / SEQiDNO : 4} ALDH (ArabidcpSîS ïhahana, SEC © NO ; 3} •■■■■■^^......... 617 4CL5 (Ch'usssnensîs,5ÊQiDNO ; 6} CCoAMTl (Vitisvinsfera, SEQiD NO : 10} 367 4CL5 (CstnjSsinenss,S£Q©NÔt 6) CCoAVTI 5Eü©XU 10' Thæ3 ePeiunsa h,bnda.SEQiüNC : 2} 516 4CL5 (Girus ssnens^s. SEQiD NO: 6) ÇCoAWTl (Vitisvinfea, SEQiD NO: 10) CCR1 (Poputustomentosa, SEQiD NO: 4) AlDh sArabidopsis thaîiana.<h2 style=";text-align:left;direction:ltr">SEQiON NC : 3} 334 Thîc3 {Pétunia hybrida, SEQ © NO : 2} 4CQ (ÂrabidopsisthaiianâjSEQiDNO: 5} 339 îho? (Petunsa hybrida, SEQ© NO: 2} 4C11 (A^Oidopsisthahana, SEQ© NO : 5) CCqANsTI (WisvmtfeFat-SEQ,iD NO : 10} 340 îhioS (Pétunia hybrida, 5E^ ©?ÏO ; 2} 4CL1 (AFabjdopssthahan8t.SEQ'iDNO : 5) CCoA^TZ (Meoicagosativa. SEC iü NO ' 11} 341 îhibS f Petunia hy brida, SEQ© NO2) 4CU (Arabidopysthabana, SEQ.© NO. : 5) CCOÂMT3 (Eucaiyptjsgiobus, SEC h? NO - 12} 342 rhio3 {Pétunia hybrida, SÊQ© NO: 2} 4CL1 (ArabidopsâsthaHana, SEQ © NO: 5} CCoAMT4 {Nscotianatabacum, SEQiD NO: 13} 343. TbsoS (Pétunia hybrida. SEQ.iD NO ' 2} 4CL1 (Arabsdopsisthaisana, SEQiD NO: 5} CCoAMT5 (Nicchana tabacum, ?Eü iD NO 14} 347 Tbsc'3 (Pétunia hybrida. £EQ.i& NC 2} 4CL1 (Arabidopsisthaiiana, SEQSD NO -.5} C€oAMT7(Popuiustriçhocarpa, SEQiDNO : 16} 335 Thio3 (Pétuniahybrida 5ÉQ©NO ; 2} 4CL5 [Citrus _ ZEQ ONO : 6} 368 ThioS (Pétuniahÿnï d^ SEOiDNO} 2} 4CL (ChnjsssnensiSjSEQiDNO : 6} CCOÂVT2 [Meoicagasatwa SEQ iD NO : 11} . <h2 style=";text-align:left;direction:ltr">369. Thio3 (Petunia hybrida SEQIQNQ : 2] AGIS (Cîtrussînensis. SEQSD^O- E) €C©AMT3 (Eu€alyptusg)obus,SEQSDNOt 12) 370 TNæQ (Petjr» hybrida. SEQ SD NO : 2) 4CL5 (0^5 5^6^^0^^3: 6) CCOÂMT4 SNicotSanatabacunfî / SEQSD NO: 13) : 371 ïhios (Petunsa hybrida SEQSD NO: 2) 405 (Cstrus5înen5&^6) CCoAMT5 [NScotianatabacumySEQSD NO: 14) 373 rhio3 (Petunia hybnda. SEQSDNO : 2} 405 (Cstrussinensis. SEQ SD NO: 6) OCoaMTô ArabidopsisthaSiana SEQiC NO, 15) 375 Rhios (Pétunia hybrida, SEQSDNü: 2) 4Cl5 (Citrussinens^SEQSDNO' 6) CC0ÀMT7 (PopuSustnchocarpat SEQSDNO: 16] 335 Thios (Pétunia hybrida.. SEC SD NO: 2) 4Cl7 (ArabidopsisthaGna. SEQiQNO: 7; 395 Thioï (Pétunia hybrida, SEQSDNO' 2] 4Cl7 (Arabsdopsfe thaSsana» SEQiD NO: 7) ŒoAMTl iytdsvSmfera.SEQSD NO: 10) 336 Th:o3 (Pétunia hybrida, SEQSDNO: 2) 4CL7 (ArabSdopsssthaSSana, SEQSDNO: 7) CCOAMT2 (Medscagosativa, SEQSD NO: 11) : 397 Thto3 (Pétunia hybrida»SEQSDNO: 2) 4CL7 (ArabSdopsssthaSSana»SEQJD NO: 7) CCOAMT3 (EuCaSyptusgSobus,.<h2 style=";text-align:left;direction:ltr">SEQSDNO: 12) ^rhso3 (Pethybrida, SEQ S NO: 2) 398 ^CL7 (Arabidopsistahana, SEQSD NO: 7) tcoAMTA (Nicohana abacum, SEQ SD NO: 13) 399 ThioS (Petunia hybnda^EQ SD NO: 2] 4CL7 [Arabidopsistahana, SEQ SD NO: 7] CCOAMT5 SNsconanatabacuni, SEQSD NO: -14) 401 Thio3 (Petunia hybnda, SEQiD NO: 2} 4CL7 (Arabidopssthahan»fSEQSDNO: 7) CC0AMT6 (ArabidopsssthaSSana» SEQSDNO: 15] : 403 Thio3 (Petunia hybrida» SECUDNO: 2) 4017 (ArabidopssthalSana, SEQSDNQ: 7) €CoAMT7 (PGpuiustrichG^^: 16) 337 Thios (Pétunia hybrida»SEQ SD NO: 2) 4CLA (POpy^us tomertosa, SEQSD NO: 8).<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0336] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> [Tableaux2C]<h2 style=";text-align:left;direction:ltr"> 423 Fhio3 (Pétunia hybrida, SEQIDNO: 2) 4CLA (Populustomentosa, SEQiD NO: 8) ŒoWTl (Vîtss viderà, SEQ sD NO: 19s 424 FhioS (Peïunsa^ybnda, SEQiD NO: 2} SCIA (Popuiustomentosa, SEQiD NO: 8} D"oAW2 (Medicag©sativa,SEQiD NO: 11) 425 Fhio3 (Pétunia hybrida, SEQiD NO: 2) 4CLA (Popuius tamentosa, SEQiD NO: S) CCoAMTS (Èuœlÿptusgldbus, SEQiD NO: 12} 425 Fhio3 ( Pétunia hybrida, SEQi D NO: 2} SCIA (PopulustomentO5a,SEQiDNO:8) CCOAMT4 (Nicotianatabacum, SEQiD NO : 13} 427 . rhicO (Petunia hybrida, SEQiD NO : 2) SCIA (Populostomentosa, SEQiDNO“ 8) CCOAVT5 (Nioatiana tabacum, SEQiD NO : 14} 429 F^ 3 i P etu n ia hybrida, SEQiD NO : 2) KLA (Populus tomentosa, SEQiD NO : 8) SCoA^TS (Arabidopselhai^^ NC : 15} 431 Fhio3 (Pétunia hybrida, SEQID NO : 2} SCIA (Popuiusteme^osa, SEQ iD NO : 8} CCoAMT? (Popuiustrichocarpa, SEQID NO : 16) 333 FhioS (Pétuniahybrida, SEQID NO : 2) K1B (Arabidopsss thaliana, SEQ ID NO: 9} 451 FhioS (Petuniahybrida,SEQiDNO: 2) K18 (Àra.<h2 style=";text-align:left;direction:ltr">bidopsisthaliana, SEQiDNO: 9} 2COÀMT1 (yitiwimfera, SEQiD NO: IC) 452 Ftw3 (Pétunia hybrida, SEQiD NO: 2) ICLB (Arabidopsisthaiiana, SEQiD NO: 8) CCOAMT2 (Medicagosativa, SEQiD NO: 11) 453 Fhio3 (Pétuniahybrida, SEQiDNO: 2) KX8 (AFabidopsisthaliana, SEQiDNO: 8) CCoAMTS (Eucalyptus globus, SEQiDNQ; 12) 454 Fhic3 (Pétuniahybrida, SEQiDNO: 2) K.LB (Arabidopsisthaiiana. SEQiDNO: 9} CCcAW4 (N^otsanatabacum, SEQiD NO: 13} 455 Thistle Pétunia hybrida. SEQsDNO: 2} AQ.B (Arabsdooss thaiiana, SEQ iD NO: 9} CCoAVTS iNKTt <aiïtabacum,SEQ.iD NO :14} 457 Fhic3 (Pe’un»a "yo'da, SEQiD NO: 2} K1B (Arabïdopsis tbaiiana, SEQ iD NO : 9) ZCoAMtS (Arabidopsis thaiiana,SEQiDNO : 15} 459 ; FhioS (Pétuniahybrida,SEQiDNO: 2) SOLE. (Arabidopsisthahana,SEQiDNO: S} CCoAMT? (Populustnchocarpa, SEQID NO ; 16) . <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">

[0337] <h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr"> [Tableaux2D]<h2 style=";text-align:left;direction:ltr"> <h2 style=";text-align:left;direction:ltr">516 : : HpaB(PseudonKjna5 aeruginosa, SEC sDNG;17) HpaC (Salmcneha entei1ca,SÊQ sDNO ; 18} TAL ^hodstorufa gtarmLs SEQ SD ND : 19) PAL prabidoptis thahana. SEQ 'D ND , 2ü) C4H (Arabidopsis ■haiiana. SEQ -D MO ■ 21) CPR (Catharantua rcsaus. SEQ -DNG : 22} Af04FBR(SEO SD NG : 23} Aro7FSR(SEC SD NG : 24) : 592 : : : HpaSfpseudsmonaî aeruginosa, SEQ SDNÔ; 17} HpaC (Saimaneiia en-enta, SEQ sQNQ : 18} T Al phodciaruia giuUnis, SEQ. QND : 19} pal J Arabidopsis thahana, SEô SD ND : 26} C4H (Arabidspsis thahana, SEQ -D NC ; 21} CPR (Catharantua rcsaus, SEQ -DND ; 22} AroAFBR}SEC SD ND ■ 23} Âro7FBR}SEQ >D ND :24} 4CL5 {Citrus sinensis, SEQ SD ND : 6} CCoAMTl JVitisvmîfera. SEQ -DNQ : 10} Thiü3 Petuma hybrida, SEQ sD NG ; 2} 59? : . HpaBpîeudomonas' aeruginosa, SEQ sDNÜ : 17} HpaC (Saimcneifa entedca, SEQ -DNC : 18} TAL iRhodc-tcruia glunnis, SEQ SD NO : 19} PAL iArabidapsts thahana. SEQ SD NO : 20} C4H (Aiabitfopss tbahsna.SEQ SD MO : 21} CPR (Catharantus roseuï, SEC <DNG : 22} ÀrsAFBR^EQ :DNO:23} Àr87FBR^FÛ »D NO : 24} Thæ3 p&ïuma Hybrida, SEQ SD ND : 2} 4CL5 îütruj snens'iï, SEQ sa NQ : 6} CCoAMTô lArabisfopæ thahana, SEQ SD ND : 15} 534 : : HpaB(Pseudornonas aerugmosa, SEQ S3NQ ; 17} HpaC (Saiptcneila ententa, SEQ D NO ; 18} TAL}RhMi5Toruia giumis, SEQ sDND ; 19} PAL (Arabldapsis thahana, SEQ sD MO : 20} C4H (Afabidopsis thahana, SEQ}□ MO : 21) CPR (Catharantus œseus, SEQ SDNO : 22} Ar&AFBR (SEQ SD NO : 23} Aro7FBR}SEC SD NG , 24} Th> s3 petunia hybrid, SEQ . <d no : 2) 4cl5 icfmjs snensü. seq id 16} ccaamt7}pcptdus rnchscarpa, sd 16) 567 hpabfpseudomonas aeragincsa, sdnô ; 17} hpac (saîfncr.eha entenca,seq idno 18} 595 hpa&pœutfcntonai aerughrosa, sdnd (saimoneisa entanca, qng 18) 4cl5 icio’ijs ssnansiî, nd 6) ccsamtl êvitîswîfera, -dno 10} thicq pétunia hÿbrda, <d ng 2} 5» hpab(pseudomonas aeruginosa, sq :17} hpac(saimonetia ong ib) thio3 pétunia h bnda, 4c15 jcitrus stnansis, mo 5: cccamtê {arabfdapsts thahana, sdno 15} 537 hpabjrseudomdnas aeruglnosa, ©nü (saimsneha enterra, iono thios hybrida, ig aq5 fût'us shé'tiî, d . 6î ccaamt? popusut trichccarpa, iü Gene cloning

[0338] Genes whose codons were optimized for expression in yeast were synthesized by Twist Biosciences, San Francisco, USA or DC Biosciences, Dundee, UK.

[0339] The ARO4 (GenBank accession number: NP_009808) and ARO7 (GenBank accession number: NP_015385) genes were amplified by PCR from S. cerevisiae genomic DNA and then mutated so that their product was resistant to feedback inhibition (FBR: Feed Back Resistance) (Gold et al., Microb Cell Fact. 2015;14:73).

[0340] Promoters and terminators (Wagner et al., Fungal Genet Biol. 2016;89:126-136) were amplified by PCR from S. cerevisiae genomic DNA.

[0341] The genes obtained by synthesis or by PCR comprise at the 5' and 3' end, a restriction site Bbsl (GAAGAC) or Bsa\ (GGTCTC), compatible with the cloning system used. All genes, promoters and terminators were cloned into the restriction sites of the pSBK vector.

[0342] The pSBK vector comprises the selection marker URA3, LEU2 or TRP1. Culture conditions

[0343] The yeast strains were cultured for 72 hours at 30°C, in a 24-well plate, with continuous shaking (200 RPM), in 1 ml of SD medium (Dutscher, Brumath, France) supplemented or not with CSM (Completed Supplement Mixture; Formedium, UK).

[0344] Glucose is added at 20 g / L and, when required, p-coumaric acid, caffeic acid or 5-hydroxy-ferulic acid was added into the medium at a concentration of 100 mg / L.

[0345] Each strain was inoculated at OD 0.2 from a 24-hour preculture grown under the same conditions. Standards

[0346] Standards for p-coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid and sinapic acid were obtained from the supplier Sigma-Aldrich. Analytical method

[0347] Sample preparation: 100 pL samples are collected for each experiment. 50 pL are transferred to a new plate, to which 50 pL of the internal standard solution is added. Each sample is then homogenized by suction-discharge, then centrifuged for 5 min at 3000 rpm at room temperature. The final concentration of the internal standard (Protocatechuic Acid) is 0.5 mg / L.

[0348] Analysis by UHPLC-TQ: The samples were analyzed by UHPLC Vanquish-H (Thermo) coupled to a triple-quadrupole UHPLC-TQ (Thermo). The column is a Waters Acquity UPLC@ USST3 column (8 pm 2.1 X 100 mm) associated with a HSST3 pre-column 1.8 pm 2.1 X 5 mm.

[0349] Mobile phase A is a 0.1% solution of formic acid in LC / MS grade water and mobile phase B is a 0.1% solution of formic acid in LC / MS grade pure acetonitrile. The column temperature is 50°C and the sample changer temperature is 10°C.

[0350] Table 3: chromatographic conditions for the detection of molecules of interest:

[0351] [Tables3] Time [min) Flow rate [mi / min) Mobile phase A (%) Mobile phase B (%) 0 0.8 90 10 3.5 0.5 72 28 5.5 0.5 72 28 5.7 0.5 90 10 6.8 0.5 90 10

[0352] - positive mode spray voltage at 4000V

[0353] - curtain gas: at 50 (arbitrary unit)

[0354] - auxiliary gas at 15 (arbitrary unit)

[0355] - transfer tube temperature at 300°C

[0356] - vaporizer temperature at 300°C

[0357] Table 4: The ions monitored and the fragmentation conditions for the molecules of interest:

[0358] [Tables 4] Molecules Retention time (min) Polarity on precursor on fHs i Collision energy RFlens (V) p- Goumaric acid 2.21 Negative 162.9 119.054 14.55 87 93 31.15 87 Transferic acid 2.67 Negative 19235 149.06 11.33 93 178.018 12.46 93 Caffeic acid 2.69 Negative 178.9 135 15.31 91 107.071 21.34 9 Sinapic acid- 3.14 Negative 223 148.97 20.65 105 164 15.38 105 5-hydroxyferuHq acid of 2.11 Negative 20935 ■ 149.99 18.11 20 165 11.63 20. Results

[0359] Production of ferulic acid from caffeic acid

[0360] The gene coding for the thioesterase of Arabidopsis thaliana (Thiol, SEQ ID NO: 1) or of Petunia hybrida (Thio3, SEQ ID NO: 2) was inserted into strain 221 which expresses the 4CL of Arabidopsis thaliana (4CL1, SEQ ID NO: 5) and the CCoAMT of Arabidopsis thaliana (CC0AMT6, SEQ ID NO: 15) and in which the ArolO and Fdc genes have been inactivated (strain 221). The strains obtained are strains 345 and 239.

[0361] The genes encoding the CCR of Populus tomentosa (SEQ ID NO: 4) and for ALDH & Arabidopsis thaliana (SEQ ID NO: 3) were also inserted into strain 221 to obtain strain 214.

[0362] The production of ferulic acid is tested with each of these strains in the presence of 100 mg / L of caffeic acid. It is compared to that obtained with strain 221.

[0363] The results are presented in [fig.l] and show that the plant thioesterases, CCR and ALDH heterologously expressed in yeast are active and capable of converting feruloyl-CoA to ferulic acid.

[0364] Production of sinapic acid from 5-hydroxyferulic acid

[0365] A strain was obtained by inserting the genes encoding Petunia hybrida thioesterase (Thio3, SEQ ID NO: 2), Citrus sinensis 4CL (4CL5, SEQ ID NO: 6) and Vitis vinifera CCoAMT (CCoAMTl, SEQ ID NO: 10) into a strain in which the ArolO and Fdc genes were inactivated. The strain thus obtained is strain 367.

[0366] The genes encoding the 4CL of Citrus sinensis (4CL5, SEQ ID NO: 6), the CCoAMT from Vitis vinifera (CCoAMTl, SEQ ID NO: 10), CCR from Populus tomentosa (SEQ ID NO: 4) and ALDH from Arabidopsis thaliana (SEQ ID NO: 3) were inserted into a strain in which the ArolO and Fde genes were inactivated. The resulting strain is strain 616.

[0367] The production of sinapic acid is tested with each of these strains in the presence of 100 mg / L of 5-hydroxy-ferulic acid. It is compared to that obtained with strain 617.

[0368] The results are presented in Figures 2 and 3 and show that plant thioesterases, CCR and ALDH heterologously expressed in yeast are active and capable of converting sinapoyl-CoA to sinapic acid.

[0369] Optimization of the pathway for producing ferulic acid from caffeic acid and the pathway for producing sinapic acid from 5-hydroxy-ferulic acid

[0370] Different yeast strains were constructed, all expressing the Petunia hybrida thioesterase (Thio3, SEQ ID NO: 2) and in which the ArolO and Fdc genes were inactivated. Five different 4CLs (4CL1, 4CL5, 4CL7, 4CLA and 4CLB) were added to these strains. The 5 strains thus obtained are strains 334, 335, 336, 337 and 338.

[0371] These strains were then combined with seven different CCoAMTs (CCoAMTl, 2, 3, 4, 5, 6, 7, respectively SEQ ID NO: 10 to 16)). 35 strains were thus obtained: 339 to 343, 345, 347, 367 to 371, 373, 375, 395 to 399, 401, 403, 423 to 427, 429, 431, 451 to 455, 457 and 459.

[0372] Ferulic acid production is tested with each of these strains in the presence of 100 mg / L of caffeic acid. It is compared to that obtained with the control strains without CcoAMT, namely strains 334 to 338. The results are presented in [fig.4].

[0373] The best combinations for the production of ferulic acid from caffeic acid are:

[0374] - Thio3-4CL5-CCoAMTl (strain 367)

[0375] - Thio3-4CL5-CCoAMT6 (strain 373)

[0376] - Thio3-4CL5-CCoAMT7 (strain 375)

[0377] The production of sinapic acid is tested with each of these strains in the presence of 100 mg / L of 5-hydroxy-ferulic acid. It is compared to that obtained with the control strains without CcoAMT, namely strains 334 to 338. The results are presented in [fig.5].

[0378] Measurement of the activity of CCoAMT alone (in the absence of 4CL and Thio3) was carried out in order to confirm that the synthesis of ferulic acid or sinapic acid does indeed pass through the CoA forms and is not obtained directly from caffeic acid or 5-hydroxy-ferulic acid.

[0379] Production of ferulic acid from p-coumaric acid

[0380] A yeast strain 507 was constructed in which the ArolO and Fdc genes were inactivated and which expresses a gene encoding HpaB from Pseudomonas aeruginosa (SEQ ID NO: 17) and a gene encoding HpaC from Salmonella enterica (SEQ ID NO: 18). Strain 507 was then modified by inserting the most efficient 4CL-CCoAMT-Thio combinations:

[0381] Strain 595: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CCoAMTl (Vitis vinifera, SEQ ID NO: 10) and Thio3 (Petunia hybrida, SEQ ID NO: 2);

[0382] Strain 596:: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CCoAMT7 (Populus trichocarpa, SEQ ID NO: 16) and Thio3 (Pétunia hybrida, SEQ ID NO: 2); And

[0383] Strain 597: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CC0AMT6 (Ara-bidopsis thaliana, SEQ ID NO: 15) and Thio3 (Pétunia hybrida, SEQ ID NO: 2).

[0384] These strains were then cultured in the presence of p-coumaric acid (100 mg / L).

[0385] The ferulic acid production of these strains was measured after 24 hours. The results are presented in [fig.6].

[0386] Insertion of a complete pathway for the production of ferulic acid in yeast

[0387] A yeast strain 516 was constructed in which the ArolO and Fdc genes have been inactivated and which expresses a gene encoding a feedback-resistant allele of Aro4 (ARO4K229L, SEQ ID NO: 23), a gene encoding a feedback-resistant allele of Aro7 (ARO7G141S, SEQ ID NO: 24), a gene encoding HpaB from Pseudomonas aeruginosa (SEQ ID NO: 17), a gene encoding HpaC from Salmonella enterica (SEQ ID NO: 18), a gene encoding TAL from Rhodotorula glutinis (SEQ ID NO: 19), a gene encoding PAL from Arabidopsis thaliana (SEQ ID NO: 20), a gene encoding C4H (FArabidopsis thaliana (SEQ ID NO: 21) and a gene encoding CPR1 from Catharantus roseus (SEQ ID NO: 22).

[0388] Strain 516 was then modified by inserting the most efficient 4CL-CCoAMT-Thio combinations:

[0389] Strain 592: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CCoAMTl (Vitis vinifera, SEQ ID NO: 10) and Thio3 (Petunia hybrida, SEQ ID NO: 2);

[0390] Strain 593:: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CCoAMT7 (Populus trichocarpa, SEQ ID NO: 16) and Thio3 (Pétunia hybrida, SEQ ID NO: 2); And

[0391] Strain 594: insertion of 4CL5 (Citrus sinensis, SEQ ID NO: 6), CC0AMT6 (Arabidopsis thaliana, SEQ ID NO: 15) and Thio3 (Pétunia hybrida, SEQ ID NO: 2).

[0392] These strains were then cultured in the presence of glucose (20 g / L).

[0393] The production of ferulic acid of these strains was measured after 24 hours. The results are presented in [fig.7].< / d>

Claims

Claims

1. A recombinant microorganism comprising (i) a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase and / or (ii) a heterologous nucleic acid sequence encoding a cinnamoyl-CoA reductase (CCR) and a heterologous nucleic acid sequence encoding an aldehyde dehydrogenase (ALDH).

2. A recombinant microorganism according to claim 1, wherein the microorganism is a yeast.

3. A recombinant microorganism according to claim 1 or 2, comprising a heterologous nucleic acid sequence encoding an acyl-coenzyme A thioesterase and wherein said acyl-coenzyme A thioesterase comprises a sequence selected from the sequences SEQ ID NOs: 1 and 2 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with one of these sequences and exhibiting acyl-coenzyme A thioesterase activity.

4. A recombinant microorganism according to any one of claims 1 to 3, comprising a heterologous nucleic acid sequence encoding a CCR and a heterologous nucleic acid sequence encoding an ALDH and wherein - said CCR comprises a sequence selected from the sequence SEQ ID NO: 4 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 4 and exhibiting CCR activity; and / or - said ALDH comprises a sequence selected from the sequence SEQ ID NO: 3 and polypeptides comprising a sequence having at least 60, 70, 80, 85, 90 or 95% sequence identity with the sequence SEQ ID NO: 3 and exhibiting ALDH activity.

5. A recombinant microorganism according to any one of claims 1 to 4, further comprising a heterologous nucleic acid sequence encoding a Caffeoyl-CoA O-Methyltransferase (CCoAMT).

6. A recombinant microorganism according to any one of claims 1 to 5, further comprising a heterologous nucleic acid sequence encoding a 4-coumaroyl-CoA ligase (4CL).

7. A recombinant microorganism according to any one of claims 1 to 6, further comprising a heterologous nucleic acid sequence encoding an enzyme or enzyme complex capable of converting p-coumaric acid to caffeic acid.

8. A recombinant microorganism according to any one of claims 1 to 7, further comprising a heterologous nucleic acid sequence encoding a cytochrome P450 reductase (CPR).

9. A recombinant microorganism according to any one of claims 1 to 8, further comprising - 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).

10. A recombinant microorganism according to any one of claims 1 to 9, further comprising (i) a heterologous nucleic acid sequence encoding a tyrosine feedback-resistant 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase and comprising a sequence selected from SEQ ID NO: 23 and polypeptides comprising a sequence having at least 60% sequence identity with the sequence SEQ ID NO: 23, said polypeptides having a tyrosine feedback-resistant 3-deoxy-D-arabino-hepturosonate-7-phosphate (DAHP) synthase activity and a leucine at the position corresponding to position 229 of SEQ ID NO: 23 and / or (ii) a heterologous nucleic acid sequence encoding a tyrosine feedback-resistant chorismate mutase and comprising a sequence selected from SEQ ID NO: 24 and polypeptides comprising a sequence having at least 60% sequence identity with the sequence SEQ ID NO: 24,said polypeptides having a chorismate mutase activity resistant to feedback by tyrosine and a serine at the position corresponding to position 141 of SEQ ID NO: 74,

11. Z. H". A recombinant microorganism according to any one of claims 1 to 10, wherein a gene encoding a phenylpyruvate decarboxylase is inactivated and / or a gene encoding a ferulic acid decarboxylase is inactivated.

12. Use of a recombinant microorganism according to any one of claims 1-11 for producing a carboxylic acid.

13. A method of producing a carboxylic acid, comprising culturing a recombinant microorganism according to any one of claims 1 to 11, and optionally harvesting and / or purifying said carboxylic acid.

14. Use according to claim 12 or method according to claim 13, wherein the carboxylic acid is ferulic acid or sinapic acid.

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