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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
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  • C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
  • C12N1/14—Fungi; Culture media therefor
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  • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
  • C12N15/09—Recombinant DNA-technology
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
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  • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
  • C12N9/14—Hydrolases (3)
  • C12N9/16—Hydrolases (3) acting on ester bonds (3.1)
  • C12N9/18—Carboxylic ester hydrolases (3.1.1)
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  • C12P7/00—Preparation of oxygen-containing organic compounds
  • C12P7/40—Preparation of oxygen-containing organic compounds containing a carboxyl group including Peroxycarboxylic acids
  • C12P7/42—Hydroxy-carboxylic acids
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  • C12Y301/00—Hydrolases acting on ester bonds (3.1)
  • C12Y301/01—Carboxylic ester hydrolases (3.1.1)
  • C12Y301/01042—Chlorogenate hydrolase (3.1.1.42)

Definitions

• the present invention relates to a recombinant microorganism, preferably a recombinant yeast, capable of producing caffeic acid comprising a heterologous gene encoding an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl bond. -shikimate to produce caffeic acid from caféoyl-shikimate. Said microorganism, preferably said recombinant yeast, may also be capable of producing ferulic acid from the resulting caffeic acid.
• the present invention also relates to a method of producing caffeic acid and a method of producing caffeic acid and / or ferulic acid, using microorganisms, preferably yeasts, according to the invention.
• the invention also relates to the use of the microorganisms, preferably yeasts, according to the invention to produce caffeic acid and / or ferulic acid.
• Ferulic acid is an organic hydroxycinnamic acid found in many plants and involved in the synthesis of lignin. This molecule is used in many applications. In cosmetics and therapy, ferulic acid is known for its antioxidant properties because it reacts with free radicals such as reactive oxygen derivatives. Research is also being carried out on applications in Alzheimer's disease, in cardiovascular disease, in diabetes, in atherosclerosis, in coronary heart disease in inflammatory pathologies or in cancer. Apart from these applications in human health, ferulic acid is also used in food for its preservative antimicrobial properties, and as a precursor in the manufacture of vanillin, a flavoring often used in place of the natural extract of vanilla. The production of ferulic acid is thus an ever-increasing need, as its properties are characterized.
• Caffeic acid is a precursor of ferulic acid, which is synthesized in plants as an intermediary in the synthesis of lignin. This molecule is mainly used for its anti-inflammatory, antiviral, anti-cancer and antioxidant properties.
• ferulic acid is mainly recovered by chemical hydrolysis of lignocellulosic biomasses. The extraction yield of this process is however relatively low and consequently entails a particularly high production cost of the ferulic acid per kilogram.
• Ferulic acid production has indeed been considered in Escherichia coli, but although production rates of pathway intermediates are high, the final ferulic acid production yield remains limited (Kang S. -Y., Choi O., Lee KJ, Hwang BY, Uhm T. -B., Hong YS 2012. Artificial biosynthesis of phenylpropanoic acids in a tyrosine overproducing Escherichia coli strain. Microbial Cell Factories 11: 153.).
• Extraction of caffeic acid is also carried out from plant materials, which similar to ferulic acid results in low yield and high production costs.
• the general inventive concept common to this invention is a new method of synthesizing these molecules via an alternative metabolic pathway having as main intermediate caffeoyl-shikimate in the same recombinant microorganism, preferably the same yeast.
• a recombinant microorganism preferably a recombinant yeast, comprising genes originating from plants, and more particularly originating from the lignin biosynthetic pathway, made it possible to produce caffeic acid. and ferulic acid, in particular from glucose, making it possible to increase the production yields of these compounds and to reduce their cost.
• the invention consists of a recombinant microorganism, preferably a recombinant yeast, capable of producing caffeic acid, comprising
• microorganism a living organism of microscopic size, in particular bacteria or unicellular fungi such as yeasts, or any prokaryotic or eukaryotic cells.
• recombinant microorganism a microorganism which has been genetically modified by the introduction and optionally the modulation of the expression and / or the blocking and / or the activation of genes.
• Yeasts are unicellular eukaryotic microorganisms of the fungal kingdom.
• yeast By “recombinant yeast” is understood according to the invention a yeast which has been genetically modified by the introduction and optionally the modulation of the expression and / or the blocking and / or the activation of genes.
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a caféoyl-shikimate esterase (CSE).
• CSE caféoyl-shikimate esterase
• Caffeic acid is a phenylpropanoid and an acid-phenol present in plants, and acting as an intermediate in the biosynthesis of lignin.
• the crude chemical formula of caffeic acid is C9H8O4 and its molar mass is 180.16g / mol.
• the chemical structure of caffeic acid is:
• heterologous gene is understood that the gene has been introduced by genetic engineering into the cell. It can be present there in episomal or chromosomal form. The origin of the gene may be different from the cell into which it is introduced. However, the gene can also come from the same species as the cell into which it is introduced but it is considered heterologous due to its unnatural environment. For example, the gene is heterologous because it is under the control of a promoter other than its natural promoter, it is introduced in a place different from this one where it is naturally located. The host cell may contain a copy of the endogenous gene prior to the introduction of the heterologous gene, or it may not contain an endogenous copy. Additionally, the nucleic acid sequence may be heterologous in that the coding sequence has been optimized for expression in the host microorganism.
• producing caffeic acid is meant the obtaining of this compound, including its synthesis, by the recombinant microorganism, preferably the recombinant yeast, according to the invention.
• hydrolase is meant an enzyme capable of breaking a covalent bond, preferably by hydrolysis.
• breaking, preferably hydrolyzing, the caffeoyl-shikimate bond is understood the chemical and enzymatic decomposition breaking a covalent bond of this compound by enzymatically, preferably by hydrolysis, to allow the production of shikimate and caffeic acid.
• a caffeoyl-shikimate esterase is an enzyme capable of hydrolyzing the caffeoyl-shikimate bond. It exists, for example, in the lignin biosynthesis pathway (Ha CM, Escamilla-Trevino L, Yarce JCS, Kim H., Ralph J., Chen F., Dixon RA 2016. An essential role of caffeoyl shikimate esterase in monolignol biosynthesis in Medicago truncatula. Plant J.
• CSE Caffeoyl shikimate esterase
• the heterologous gene encoding a CSE is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding a CSE is the CSE gene from Medicago truncatula (MtCSE) (XM_003609990.3, Genbank, SEQ ID NO. 9) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Medicago truncatula (MtCSE) and exhibiting cafeoyl-shikimate esterase activity.
• MtCSE Medicago truncatula
• Medicago truncatula is a species of the Fabaceae family, of the Faboideae subfamily.
• the heterologous gene encoding a CSE is the CSE gene of Arabidopsis thaliana (AtCSE) (At1g52760, GenBank, SEQ ID NO. 3) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Arabidopsis thaliana (AtCSE) and exhibiting caffeoylshikimate esterase activity.
• AtCSE Arabidopsis thaliana
• Arabidopsis thaliana or Arabette des dames is a species of the Brassicaceae family.
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a chlorogenic acid esterase (ChlE).
• Chlorogenic acid esterase (ChlE) is an enzyme conventionally having chlorogenic acid hydrolysis activity, and used herein to hydrolyze the caffeoyl-shikimate bond and thereby produce caffeic acid from caffeoyl-shikimate.
• the heterologous gene for a ChlE is the ChlE gene of Bifidobacterium animalis subsp. Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• Lactis is a species of lactic acid bacteria isolated from the feces of chickens, rabbits and humans, as well as fermented milk.
• the heterologous gene for a ChlE is the ChlE gene from Ustilago maydis (UmChlE) (HG970190.1, GenBank, SEQ ID NO. 15) or a gene encoding a sequence having at least 55, 60 , 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Ustilago maydis (UmChlE) and exhibiting chlorogenic acid esterase activity.
• Ustilago maydis is a pathogenic fungus causing smut in corn in particular.
• the heterologous gene for a ChlE is the ChlE gene of Lactobacillus johnsonii (LaChlE) (SPPI01000004.1: 37780-38526, GenBank, SEQ ID NO. 8) or a gene encoding a sequence having at at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Lactobacillus johnsonii (LaChlE) and exhibiting chlorogenic acid esterase activity.
• LaChlE Lactobacillus johnsonii
• Lactobacillus johnsonii is a lactic acid bacteria that is part of the healthy vaginal microbiota.
• the heterologous gene for a ChlE is the ChlE gene of Salinibacter ruber (SrChlE) (CP030369.1: 2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having at at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Salinibacter ruber ChlE (SrChlE) and exhibiting chlorogenic acid esterase activity.
• Salinibacter ruber is a halophilic red bacterium that thrives in an environment with a high salt content.
• percentage identity between two gene sequences within the meaning of the present invention is intended to denote a percentage of nucleotides or of identical amino acid residues between the two sequences to be compared, obtained after the best alignment, this percentage being purely statistical and the differences between the two sequences being distributed at random and over their entire length.
• the best alignment or optimal alignment is the alignment for which the percentage identity between the two sequences to be compared is the highest.
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• a heterologous gene encoding an enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A.
• the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
• the enzyme is capable of producing coumaroyl-CoA.
• This enzymatic reaction involves ATR and can be carried out in one or more steps.
• a 4-coumarate-CoA ligase (4CL) is an enzyme that catalyzes the formation of coumaroyl-CoA from coumaric acid and coenzyme A.
• the heterologous gene encoding a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding said 4CL is mutated in order to decrease the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid relative to the parent gene. encoding an unmutated 4CL.
• 4CL is 4CL from Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
• Populus tomentosa or Chinese white poplar is a species of the Salicaceae family.
• the amino acid in the case where the 4CL is the 4CL of Populus tomentosa, the amino acid:
• - at position 236 is an Alanine (Y236A) or a Phenylalanine (Y236F); and or
• - at position 240 is an Alanine (S240A); and or
• - at position 305 is an Alanine (G305A); and or
• the 4CL is the 4CL of Arabidopsis thaliana (At1g51680, GenBank, SEQ ID NO. 1).
• the amino acid in the case where the 4CL is the 4CL of Arabidopsis thaliana, the amino acid:
• - at position 264 is an Alanine (S264A); and or
• - at position 329 is an Alanine (G329A); and or
• the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQ ID NO. 12).
• Streptomyces coelicolor is a Gram positive soil bacterium.
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• HCT Hydroxycinnamoyl-Transferase
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• CPR1 Cytochrome P450 reductase
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• HCT Hydroxycinnamoyl-Transferase
• CPR1 Cytochrome P450 reductase
• HCT Hydroxycinnamoyl-Transferase
• the heterologous gene encoding an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
• Coumarate 3 Hydroxylase (C3H) is an enzyme involved in the synthesis of lignin, and which catalyzes the production of caféoyl shikimate from coumaroyl shikimate.
• the heterologous gene encoding a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
• Cytochrome P450 reductase is a membrane oxidoreductase which allows the transfer of electrons from NADPH to cytochrome P450.
• the heterologous gene encoding a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Catharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
• Tyrosine Amonia Lyase is an enzyme that converts tyrosine to p-coumaric acid with the release of ammonia.
• the heterologous gene encoding a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rhodotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
• Rhodotorula glutinis is a pink yeast of the genus Rhodotorula.
• a Phenylalanine Amonia-Lyase is an enzyme that catalyzes the transformation of phenylalanine into cinnamic acid, with the release of ammonia.
• Cinnamate 4-Hydroxylase or T rans-cinnamate 4-monooxygenase, is an enzyme that converts trans-cinnamic acid (CA) into p-coumaric acid.
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• said recombinant microorganism preferably said recombinant yeast, further comprises the invalidation for:
• said recombinant microorganism preferably said recombinant yeast, further comprises:
• a 3-deoxy-7-phosphoheptulonate synthase (AR04), or DAHP synthase is a transferase which occurs at the first step of the shikimate pathway, and which catalyzes the reaction: phosphoenolpyruvate + D-erythrose-4-phosphate + H20 ⁇ 3 -deoxy-D- arabinoheptulosonate-7-phosphate + phosphate.
• a chorismate mutase (AR07) is an isomerase which is involved in the shikimate pathway, and which is known to catalyze a pericyclic reaction. This enzyme catalyzes the reaction: chorismate ⁇ prephenate.
• AR04 and AR07 are two genes involved in the de novo synthesis of aromatic amino acids such as phenylalanine and tyrosine in yeast.
• the AR04 gene (NP_009808, GenBank) is mutated so that the amino acid at position 229 is Leucine (K229L).
• the AR07 gene (NP_015385, GenBank) is mutated so that the amino acid at position 141 is a Serine (G141S).
• Phenylpyruvate decarboxylase (AR010) is an enzyme involved in particular in the degradation of phenylalanine and tyrosine in yeast.
• mutations of AR04 and AR07 and invalidation of AR010 are performed to optimize increased production of aromatic amino acids such as phenylalanine and tyrosine.
• said recombinant microorganism preferably said recombinant yeast, is capable of producing ferulic acid.
• said recombinant microorganism preferably said recombinant yeast, is capable of producing ferulic acid from caffeic acid. obtained, and further includes:
• COMPT caffeoyl-O-methyl transferase
• said recombinant microorganism preferably said recombinant yeast, capable of producing ferulic acid from the caffeic acid obtained further comprises:
• SAM2 S-Adenosylmethyltransferase
• Ferulic acid is an organic hydroxycinnamic acid found in many plants and involved in the synthesis of lignin.
• the crude chemical formula of ferulic acid is C 10 H 10 O 4 and this compound has a molar mass of 194.18 g / mol.
• SAM2 S-adenosylmethionine synthase 2
• the SAM2 gene is the SAM2 gene of S. accharomyces cerevisiae and is a copy of that understood naturally (YDR502C, SGD Database, SEQ ID NO. 11).
• a caffeoyl-O-methyl transferase is an enzyme which catalyzes the reaction: S-adenosyl-L-methionine + caffeic acid ⁇ S-adenosyl-L-homocysteine + ferulic acid.
• the heterologous gene encoding COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g54160, GenBank, SEQ ID NO. 2).
• said yeast according to the invention capable of producing ferulic acid further comprises the invalidation of the gene encoding a ferulic acid decarboxylase 1 (FDC1).
• FDC1 ferulic acid decarboxylase 1
• a ferulic acid decarboxylase is an enzyme which catalyzes the decarboxylation of aromatic carboxylic acids such as ferulic acid, p-coumaric acid or cinnamic acid, producing the corresponding vinyl derivatives 4-vinylphenol, 4-vinylguaiacol and styrene, respectively, which play the role of aromatic metabolites.
• said recombinant microorganism preferably said recombinant yeast
• said recombinant microorganism is a species of the branching of Ascomycota, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygosaccharomyces.
• said recombinant microorganism preferably said recombinant yeast, is of the species Saccharomyces cerevisiae.
• Saccharomyces cerevisiae is a unicellular eukaryotic yeast, occurring in ovoid to rounded form, about 6 to 12 ⁇ m in length and 6 to 8 ⁇ m in width.
• the invention relates to a process for modifying a microorganism, preferably a yeast, to produce caffeic acid, comprising the introduction of:
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a caféoyl-shikimate esterase (CSE).
• the heterologous gene encoding a CSE is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding a CSE is the CSE gene from Medicago truncatula (MtCSE) (XM_003609990.3, Genbank, SEQ ID NO. 9) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Medicago truncatula (MtCSE) and exhibiting cafeoyl-shikimate esterase activity.
• MtCSE Medicago truncatula
• the heterologous gene encoding a CSE is the CSE gene of Arabidopsis thaliana (AtCSE) (At1g52760, GenBank, SEQ ID NO. 3) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Arabidopsis thaliana (AtCSE) and exhibiting caffeoylshikimate esterase activity.
• AtCSE Arabidopsis thaliana
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a chlorogenic acid esterase (ChlE).
• the heterologous gene for a ChlE is the ChlE gene of Bifidobacterium animalis subsp. Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• the heterologous gene for a ChlE is the ChlE gene from Ustilago maydis (UmChlE) (HG970190.1, GenBank, SEQ ID NO. 15) or a gene encoding a sequence having at least 55, 60 , 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Ustilago maydis (UmChlE) and exhibiting chlorogenic acid esterase activity.
• the heterologous gene for a ChlE is the ChlE gene of Lactobacillus johnsonii (LaChlE) (SPPI01000004.1: 37780-38526, GenBank, SEQ ID NO.
• the heterologous gene for a ChlE is the ChlE gene of Salinibacter ruber (SrChlE) (CP030369.1: 2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having at at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Salinibacter ruber ChlE (SrChlE) and exhibiting chlorogenic acid esterase activity.
• Salinibacter ruber SrChlE
• GenBank GenBank
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• a heterologous gene encoding an enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A.
• the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
• the heterologous gene encoding a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding a 4CL is mutated in order to decrease the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid relative to the parent gene. encoding an unmutated 4CL.
• 4CL is 4CL from Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
• the amino acid in the case where the 4CL is the 4CL of Populus tomentosa, the amino acid:
• - at position 236 is an Alanine (Y236A) or a Phenylalanine (Y236F); and or - at position 240 is an Alanine (S240A); and or
• - at position 305 is an Alanine (G305A); and or
• the 4CL is the 4CL of Arabidopsis thaliana (At1g51680, GenBank, SEQ ID NO. 1).
• the amino acid in the case where the 4CL is the 4CL of Arabidopsis thaliana, the amino acid:
• - at position 264 is an Alanine (S264A); and or
• - at position 329 is an Alanine (G329A); and or
• the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQ ID NO. 12).
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• HCT Hydroxycinnamoyl-Transferase
• TAL Tyrosine Amonia Lyase
• PAL Phenylalanine Amonia-Lyase
• C4H Cinnamate 4-Hydroxylase
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• CPR1 Cytochrome P450 reductase
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• HCT Hydroxycinnamoyl-Transferase
• C3H Coumarate 3 Hydroxylase
• TAL Tyrosine Amonia Lyase
• PAL Phenylalanine Amonia-Lyase
• C4H Cinnamate 4-Hydroxylase
• CPR1 Cytochrome P450 reductase
• the heterologous gene encoding an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
• the heterologous gene encoding a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
• the heterologous gene encoding a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Catharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
• the heterologous gene encoding a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rhodotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the invalidation for:
• said process for modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of:
• the AR04 gene is mutated so that the amino acid at position 229 is Leucine (K229L).
• the AR07 gene is mutated so that the amino acid at position 141 is a Serine (G141S).
• said yeast is a species of the Ascomycota branch, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygosaccharomyces.
• said yeast is of the species Saccharomyces cerevisiae.
• the invention relates to a method of modifying a microorganism, preferably a yeast, to produce ferulic acid.
• the invention relates to a process for modifying a microorganism, preferably a yeast, to produce ferulic acid from the caffeic acid obtained, further comprising the introduction of:
• said method of modifying a microorganism, preferably a yeast, to produce ferulic acid from the caffeic acid obtained further comprises the introduction of:
• SAM2 S-Adenosylmethyltransferase
• SAM2 gene is the SAM2 gene of Saccharomyces cerevisiae and is a copy of that understood naturally (YDR502C, SGD Database, SEQ ID NO. 11).
• the heterologous gene encoding COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g54160, GenBank, SEQ ID NO. 2).
• said process for modifying a microorganism, preferably a yeast, to produce ferulic acid from the caffeic acid obtained according to the invention further comprises the invalidation of the gene encoding a ferulic acid decarboxylase 1 (FDC1).
• FDC1 ferulic acid decarboxylase 1
• the invention relates to a method for producing caffeic acid, comprising a step of: a. Culturing of the recombinant microorganisms, preferably the recombinant yeasts, as defined according to the invention in a culture medium.
• said method of producing caffeic acid further comprises a step of: b. Recovery of the caffeic acid obtained in step a.
• the caffeic acid is produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caféoyl-shikimate, added to the culture medium before or after step a.
• the caffeic acid is produced from glucose.
• the invention relates to a method for producing ferulic acid, comprising a step of: a. Culturing of the recombinant microorganisms, preferably the recombinant yeasts, as defined according to the invention in a culture medium.
• said method of producing ferulic acid further comprises a step of: b. Recovery of the ferulic acid obtained in step a.
• said method of producing caffeic acid and / or ferulic acid comprises a step of: a. Cultivation of recombinant microorganisms, preferably recombinant yeasts, capable of producing caffeic acid as defined according to the invention in a culture medium, or a ’. Cultivation of recombinant microorganisms, preferably recombinant yeasts, capable of producing ferulic acid from caffeic acid obtained according to the invention in a culture medium; step a or a ’preferably being followed by a step of: b. Recovery of caffeic acid and / or ferulic acid obtained in step a. or a ’.
• ferulic acid is produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caféoyl-shikimate, added to the culture medium before or after 'step a or a'.
• ferulic acid is produced from glucose.
• the invention relates to a method for producing caffeic acid and / or ferulic acid, comprising a step of: a. Cultivation of the recombinant microorganisms, preferably the recombinant yeasts, capable of producing ferulic acid from the caffeic acid obtained as defined according to the invention in a culture medium.
• said method of producing caffeic acid and / or ferulic acid further comprises a step of: b. Recovery of the caffeic acid and / or of the ferulic acid obtained in step a.
• caffeic acid and / or ferulic acid are produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caféoyl-shikimate, added in the culture medium before or in step a.
• caffeic acid and / or ferulic acid are produced from glucose.
• the invention relates to the use of the recombinant microorganism, preferably the recombinant yeast, capable of producing ferulic acid from the caffeic acid obtained according to the invention to produce acid caffeic and / or ferulic acid.
• the recombinant microorganism preferably the recombinant yeast, capable of producing ferulic acid from the caffeic acid obtained according to the invention to produce acid caffeic and / or ferulic acid.
• the invention relates to the use of the recombinant microorganism, preferably the recombinant yeast, producing caffeic acid according to the invention to produce caffeic acid.
• the invention relates to the use of the recombinant microorganism, preferably the recombinant yeast, producing ferulic acid according to the invention to produce ferulic acid.
• ferulic acid is produced from caffeic acid produced by said recombinant microorganism, preferably said recombinant yeast, according to the invention.
• the invention relates to at least one expression vector for the production of caffeic acid in a recombinant host microorganism, preferably a recombinant host yeast, comprising a coding sequence for a gene heterologous to the microorganism.
• recombinant host preferably to the recombinant yeast host, encoding an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate.
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a caféoyl-shikimate esterase (CSE).
• CSE caféoyl-shikimate esterase
• the heterologous gene encoding a CSE is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding a CSE is the CSE gene from Medicago truncatula (MtCSE) (XM_003609990.3, Genbank, SEQ ID NO. 9) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Medicago truncatula (MtCSE) and exhibiting cafeoyl-shikimate esterase activity.
• MtCSE Medicago truncatula
• the heterologous gene encoding a CSE is the CSE gene of Arabidopsis thaliana (AtCSE) (At1g52760, GenBank, SEQ ID NO. 3) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of the CSE of Arabidopsis thaliana (AtCSE) and exhibiting caffeoylshikimate esterase activity.
• AtCSE Arabidopsis thaliana
• the enzyme capable of breaking, preferably hydrolyzing, the caféoyl-shikimate bond to produce caffeic acid from caféoyl-shikimate is a chlorogenic acid esterase (ChlE).
• the heterologous gene for a ChlE is the ChlE gene of Bifidobacterium animalis subsp. Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• Lactis (BiChlE) (CP001606.1: 789353-790141, GenBank, SEQ ID NO. 4) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
• the heterologous gene for a ChlE is the ChlE gene from Ustilago maydis (UmChlE) (HG970190.1, GenBank, SEQ ID NO. 15) or a gene encoding a sequence having at least 55, 60 , 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Ustilago maydis (UmChlE) and exhibiting chlorogenic acid esterase activity.
• the heterologous gene for a ChlE is the ChlE gene of Lactobacillus johnsonii (LaChlE) (SPPI01000004.1: 37780-38526, GenBank, SEQ ID NO. 8) or a gene encoding a sequence having at at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of ChlE from Lactobacillus johnsonii (LaChlE) and exhibiting chlorogenic acid esterase activity.
• the heterologous gene for a ChlE is the ChlE gene of Salinibacter ruber (SrChlE) (CP030369.1: 2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having at at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Salinibacter ruber ChlE (SrChlE) and exhibiting chlorogenic acid esterase activity.
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for a heterologous gene coding for an enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A.
• the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
• the heterologous gene encoding a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
• the heterologous gene encoding a 4CL is mutated in order to decrease the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid relative to the parent gene. encoding an unmutated 4CL.
• 4CL is 4CL from Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
• the amino acid in the case where the 4CL is the 4CL of Populus tomentosa, the amino acid:
• - at position 236 is an Alanine (Y236A) or a Phenylalanine (Y236F); and or
• - at position 240 is an Alanine (S240A); and or
• - at position 305 is an Alanine (G305A); and or
• the 4CL is the 4CL of Arabidopsis thaliana (At1g51680, GenBank, SEQ ID NO. 1).
• the amino acid in the case where the 4CL is the 4CL of Arabidopsis thaliana, the amino acid:
• - at position 264 is an Alanine (S264A); and or
• - at position 329 is an Alanine (G329A); and or
• the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQ ID NO. 12).
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for:
• HCT Hydroxycinnamoyl-Transferase
• TAL Tyrosine Amonia Lyase
• PAL Phenylalanine Amonia-Lyase
• C4H Cinnamate 4-Hydroxylase
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for:
• CPR1 Cytochrome P450 reductase
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for:
• HCT Hydroxycinnamoyl-Transferase
• heterologous gene encoding a Cytochrome P450 reductase (CPR1).
• CPR1 Cytochrome P450 reductase
• the heterologous gene encoding an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
• the heterologous gene encoding a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
• the heterologous gene encoding a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Catharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
• the heterologous gene encoding a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rhodotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for:
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism, preferably a recombinant host yeast further comprises a sequence allowing the invalidation of:
• said at least one expression vector for the production of caffeic acid in a recombinant host microorganism preferably a recombinant host yeast, further comprises a coding sequence for:
• the AR04 gene is mutated so that the amino acid at position 229 is Leucine (K229L).
• the AR07 gene is mutated so that the amino acid at position 141 is a Serine (G141S).
• said at least one vector for expression in a recombinant host microorganism allows the production of ferulic acid from the caffeic acid obtained, and further comprises a sequence coding for:
• said at least one expression vector in a recombinant host microorganism preferably a recombinant host yeast, allowing the production of ferulic acid from the caffeic acid obtained further comprises a coding sequence for:
• SAM2 S-Adenosylmethyltransferase
• said yeast is a species of the Ascomycota branch, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygosaccharomyces.
• said yeast is of the species Saccharomyces Cerevisiae.
• the SAM2 gene is the SAM2 gene of Saccharomyces cerevisiae and is a copy of that understood naturally (YDR502C, SGD Database, SEQ ID NO. 11).
• the heterologous gene encoding COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis Thaliana (At5g54160, UniProtKB).
• said at least one vector for expression in a recombinant host microorganism, preferably a recombinant host yeast, allowing the production of ferulic acid from the caffeic acid obtained further comprises a sequence allowing the invalidation of the gene encoding a ferulic acid decarboxylase 1 (FDC1).
• FDC1 ferulic acid decarboxylase 1
• FIG. 1 illustrates the hydrolysis reaction of caféoyl-shikimate to caffeic acid and shikimate using the caféoyl-shikimate esterase (CSE) according to the invention.
• CSE caféoyl-shikimate esterase
• FIG. 2 illustrates the metabolic pathways for the production of caffeic acid and ferulic acid in yeast according to one embodiment of the invention.
• FIG. 3 illustrates the production of caffeic acid in a recombinant yeast according to one embodiment of the invention from p-coumaric or from glucose via CSE (UHPLC-TQ method), at 24h, 48h and 72h.
• FIG. 4 illustrates the production of the various intermediates produced by recombinant yeast according to one embodiment of the invention, analyzed by a qualitative method (UHPLC-HRMS method (high resolution mass spectrometry).
• FIG. 5 illustrates the analysis of the compounds present in the culture supernatant of recombinant yeasts according to one embodiment of the invention, the presence of the compounds being determined by the UHPLC-TQ method.
• the first peak with a retention time of 2.1 min corresponds to caffeic acid and the second at 3.25 min to ferulic acid.
• FIG. 6 is a chromatograph characterizing the production of ferulic acid in recombinant yeast according to an embodiment of the invention from glucose.
• the peak at 2.01 corresponds to caffeic acid and the peak at 3.15 corresponds to ferulic acid.
• FIG. 7 is a chromatogram characterizing the hydrolysis of caféoyl-shikimate to caffeic acid and shikimate using CSE (from Medicago truncatula, MtCSE) in recombinant yeast according to one embodiment of the invention.
• CSE from Medicago truncatula, MtCSE
• the peak with a retention time of 3.23 min corresponds to caffeic acid and the second at 3.78 min to caféoyl-shikimate.
• FIG. 8 is a chromatogram characterizing the hydrolysis of caféoyl-shikimate to caffeic acid and shikimate using ChlE (from Lactobacillus johnsonii, LaChlE) in recombinant yeast according to one embodiment of the invention.
• Fig. 9 is a chromatogram characterizing the presence of caféoyl-shikimate in the control samples.
• the peak with a retention time of 3.88 min corresponds to caffeoyl-shikimate.
• AR04 NP_009808, Genbank
• AR07 NP_015385, Genbank
• the genes obtained by synthesis or PCR include at the 5 ’and 3’ end, a Bbs ⁇ (GAAGAC) or Bsa ⁇ (GGTCTC) restriction site, compatible with the cloning system used. All genes, promoters and terminators were restriction cloned into the pSBK vector. Promoters and terminators (Wargner et al., 2015) were amplified by PCR from the genomic DNA of the yeast S. cerevisiae.
• the vector pSBK comprises a yeast selection marker: URA3, LEU2 or TRP1.
• Table 1 The different genes used to produce a recombinant yeast according to the invention. Mutation of the 4CL:
• P. tomentosa 4CL mutants Y236A and Y236F were constructed by PCR. Gene deletion:
• the AR010 (YDR380W) and FDC1 (YDR539W) genes were invalidated by deletion, i.e. by integration instead of the open reading frame, of a linear DNA comprising a selection marker bounded by the upstream regions and downstream of the gene.
• the yeast model used in this study is Saccharomyces cerevisiae strain FY1679-28A (Tettelin et al., 1995 - Table 1 page 85), auxotrophic for uracil, tryptophan, and leucine.
• the constructions were carried out in the strain of Escherichia coli MH1 before their transfer to yeast.
• the yeast strains were cultured for 72 hours at 30 ° C., in a 24-well plate, with continuous stirring (200 RPM), in 1 ml of SD medium (Dutscher, Brumath, Fr) supplemented or not with CSM (Completed Supplement Mixture; Formedium, UK). Glucose is added at 20 g / L. or p-coumaric acid was added to the medium at a concentration of 100 mg.l-1.
• UHPLC-TQ Analysis by UHPLC-TQ: The samples were analyzed by a UHPLC Vanquish-H (Thermo) coupled to a triple-quadrupole UHPLC-TQ (Thermo).
• the column is a Waters Acquity UPLC @ USST3 column (8 ⁇ m 2.1 X 100 mm) associated with a 1.8 ⁇ m 2.1 X 5 mm HSST3 pre-column.
• Mobile phase A is a solution of 0.1% formic acid in LC / MS grade water and mobile phase B is a solution of 0.1% formic acid in pure acetonitrile of LC / MS quality.
• the temperature of the column is 50 ° C and the temperature of the autosampler is 10 ° C.
• the parameters of the electrospray source are: - voltage of the positive mode spray at 4000V
• UHPLC-HRMS method High resolution mass spectrometry: Preparation of the samples: Samples of 100 ⁇ L are collected for each experiment. 50 ⁇ l are transferred to a new plate, to which are added 50 ⁇ l of acetonitrile. Each sample is then homogenized by suction-discharge, then centrifuged for 5 min at 3000 rpm at room temperature. Analysis by UHPLC-HRMS: The samples were analyzed by UHPLC Vanquish-H (Thermo) coupled to UHPLC-HRMS. The column is a Waters Acquity UPLC @ USST3 column (8 ⁇ m 2.1 X 100 mm) associated with a 1.8 ⁇ m 2.1 X 5 mm HSST3 pre-column.
• Mobile phase A is a solution of 0.1% formic acid in LC / MS grade water and mobile phase B is a solution of 0.1% formic acid in pure acetonitrile of LC / MS quality.
• the temperature of the column is 50 ° C and the temperature of the sample changer is 10 ° C.
• the parameters of the electrospray source are:
• Example 2 Production of caffeic acid from glucose or p-coumaric acid.
• caffeic acid from glucose or p-coumaric acid was tested in 4 strains, the differences of which relate to the choice of the mutated 4CL (either Y236A or Y236F) and of the CSEs used (A. thaliana or. truncatula).
• Figure 3 depicts the results of the production of caffeic acid from p-coumaric or glucose via CSE (UHPLC-TQ method) using the yeast according to the invention.
• the AR04K229L-AR07G141S-TAL-HCT-C3H-CPR1 genes were added and the AR010 gene was invalidated by deletion.
• These strains therefore diverge only by the 4CL and CSEs used (L16A3: 4CLY236A-AtCSE; L16B5: 4CLY236A-MtCSE; L16C2: 4CLY236F-AtCSE; L16D5: 4CLY236F-MtCSE).
• the accumulation of the various intermediates demonstrates the possibility of producing caffeic acid using the yeast according to the invention, as indicated by the presence of p-coumaroyl-shikimate and caféoyl-shikimate.
• Example 4 Production of ferulic acid from p-coumaric acid
• the yeast according to the invention capable of producing ferulic acid possesses the methytransferase of A. rabidobsis thaliana (COMT) and is invalidated for the FDC1 gene.
• This strain was incubated 72 hours in the presence of p-coumaric acid and the production of caffeic acid and ferulic acid was determined by the UHPLC-TQ method.
• the chromatogram shows the production of caffeic acid and ferulic acid ( Figure
• the first peak with a retention time of 2.1 min corresponds to caffeic acid and the second at 3.25 min to ferulic acid.
• the yeast according to the invention capable of producing ferulic acid possesses Arabidobsis thaliana methytransferase (COMT) and is invalidated for the FDC1 gene.
• This strain was incubated for 72 hours in the presence of glucose and the production of ferulic acid was determined by the UHPLC-TQ method.
• the chromatogram shows the production of caffeic acid and ferulic acid ( Figure
• the first peak with a retention time of 2.01 min corresponds to caffeic acid and the second at 3.15 min corresponds to ferulic acid.
• Example 6 Test for hydrolysis of caféoyl-shikimate to caffeic acid and shikimate using CSE:
• the caféoyl-shikimate sample was prepared from the culture supernatant of a producer strain.
• the release of caffeic acid from caffeoyl-shikimate was tested using a strain containing CSE from Medicago truncatula (MtCSE).
• Example 7 Test for the hydrolysis of caféoyl-shikimate to caffeic acid and shikimate using ChLE:
• the caféoyl-shikimate sample was prepared from the culture supernatant of a producer strain.
• the release of caffeic acid from caffeoyl-shikimate was tested using a strain containing Lactobacillus johnsonii ChlE.

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