Recombinant yeast capable of producing caffeic acid and / or ferulic acid.
Patent Information
- Application Number
- FR2019012573
- Authority / Receiving Office
- FR · FR
- Patent Type
- Patents
- Current Assignee / Owner
- Filing Date
- 2019-11-08
- Publication Date
- 2025-03-07
- Estimated Expiration
- 2039-11-08
Smart Images
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Abstract
Description
Title of the invention: Recombinant yeast capable of producing caffeic acid and / or ferulic acid. FIELD OF THE INVENTION
[0001] The present invention relates to a recombinant microorganism, preferably a recombinant yeast, capable of producing caffeic acid comprising a heterologous gene coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate. Said microorganism, preferably said recombinant yeast, may also be capable of producing ferulic acid from the caffeic acid obtained. The present invention also relates to a method for producing caffeic acid and a method for producing caffeic acid and / or ferulic acid, using the microorganisms, preferably yeasts, according to the invention. Finally, the invention also relates to the use of the microorganisms, preferably yeasts, according to the invention for producing caffeic acid and / or ferulic acid. STATE OF THE ART
[0002] Ferulic acid is an organic hydroxycinnamic acid present in many plants, and participating in the synthesis of lignin. This molecule is used in many applications. In the cosmetic and therapeutic fields, ferulic acid is known for its antioxidant properties because it reacts with free radicals such as reactive oxygen species. Research is also being carried out on applications in Alzheimer's disease, cardiovascular diseases, diabetes, atherosclerosis, coronary heart disease, inflammatory pathologies and even cancers. Apart from these applications in human health, ferulic acid is also used in food for its antimicrobial preservation properties, and as a precursor in the manufacture of vanillin, a flavoring often used instead of natural vanilla extract.The production of ferulic acid is thus a constantly growing need, as its properties are characterized.
[0003] Caffeic acid is a precursor of ferulic acid, which is synthesized in plants as an intermediate in lignin synthesis. This molecule is mainly used for its anti-inflammatory, antiviral, anticancer and antioxidant properties.
[0004] Today, ferulic acid is mainly recovered by chemical hydrolysis of lignocellulosic biomass. The extraction yield of this process is however relatively low and therefore results in a particularly high production cost of ferulic acid per kilogram.
[0005] Ferulic acid production has indeed been considered in Escherichia coli, but although the production rates of the pathway intermediates are high, the final yield of ferulic acid production remains limited (Kang S.-Y., Choi O., Lee KJ, Hwang BY, Uhm T.-B., Hong YS 2012. Artificial biosynthesis of phenyl-propanoic acids in a tyrosine overproducing Escherichia coli strain. Microbial Cell Factories 11:153.).
[0006] The extraction of caffeic acid is also carried out from plant materials, which, in the same way as for ferulic acid, results in low yield and high production costs.
[0007] A pathway for the production of caffeic acid has been described in yeast (Liu L., Liu H., Zhang W., Yao M., Li B., Liu D., Yuan Y. 2019. Engineering the biosynthesis of caffeic Acid in Saccharomyces cerevisiae with heterologous enzyme combinations. Engineering 5: 287-295). It consists of the direct conversion of p-coumaric acid into caffeic acid via a single hydroxylation step. However, the hydroxylases used in these studies, SAM5 (Zhang H., Stephanopoulos G. 2012. Engineering E. coli for caffeic acid biosynthesis from renewable sugars. Applied Mi-crobiology and Biotechnology 97:3333-3341), C0UM3H, HpaBC (Furuya T. & Kino K. 2014. Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives. Appl Microbiol Biotechnol 98:1145-1154.) and Cypl99A2 (Furuya T. & Kino K. 2014.Catalytic activity of the two-component flavin-dependent monooxygenase from Pseudomonas aeruginosa toward cinnamic acid derivatives. Appl Microbiol Biotechnol 98:1145-1154), are relatively inefficient in yeast, predicting low production yield.
[0008] Thus, there remains a need to improve the production yields of caffeic acid and ferulic acid, while lowering the costs of its production.
[0009] The general inventive concept common to this invention is a new method of synthesizing these molecules by passing through an alternative metabolic pathway having as its main intermediate caffeoyl-shikimate in the same microorganism, preferably the same recombinant yeast.
[0010] Surprisingly, the Applicant has discovered that a recombinant microorganism, preferably a recombinant yeast, comprising genes originating from plants, and more particularly from the lignin biosynthesis pathway, makes it possible to produce caffeic acid and ferulic acid from glucose in particular, making it possible to increase the production yields of these compounds and to reduce their cost. Statement of the invention
[0011] According to a first aspect, the invention consists of 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-shikimate bond to produce caffeic acid from caffeoyl-shikimate.
[0012] A microorganism is understood to mean a living organism of microscopic size, in particular bacteria or unicellular fungi such as yeasts, or any prokaryotic or eukaryotic cells.
[0013] According to the invention, the term “recombinant microorganism” means a microorganism which has been genetically modified by the introduction and optionally the modulation of the expression and / or the blocking and / or the inactivation of genes.
[0014] Yeasts are unicellular eukaryotic microorganisms of the fungal kingdom.
[0015] According to the invention, the term "recombinant yeast" means a yeast which has been genetically modified by the introduction and optionally the modulation of the expression and / or the blocking and / or the inactivation of genes.
[0016] Preferably, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a caffeoyl-shikimate esterase (CSE).
[0017] The reaction carried out is described in [fig.l].
[0018] Caffeic acid is a phenylpropanoid and phenolic acid found in plants and acting as an intermediate in lignin biosynthesis. The chemical formula of caffeic acid is C9H8O4 and its molar mass is 180.16g / mol.
[0019] The chemical structure of caffeic acid is:
[0020] [Chem.l]
[0021] By "heterologous gene" is meant that the gene has been introduced by genetic engineering into the cell. It may be present 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 is considered heterologous because of 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 into a different location from 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. Furthermore, the nucleic acid sequence may be heterologous in the sense that the coding sequence has been optimized for expression in the host microorganism.
[0022] By "producing caffeic acid" is meant the obtaining of this compound, including its synthesis, using the recombinant microorganism, preferably recombinant yeast, according to the invention.
[0023] By “hydrolase” is meant an enzyme capable of breaking a covalent bond, preferably by hydrolysis.
[0024] By "breaking, preferably hydrolyzing, the caffeoyl-shikimate bond" is meant the chemical and enzymatic decomposition breaking a covalent bond of this compound enzymatically, preferably by hydrolysis, in order to allow the production of shikimate and caffeic acid.
[0025] A caffeoyl-shikimate esterase (CSE) 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. 86950:363-75; and Vanholme R., Cesarino I., Rataj K., Xiao Y., Sundin L., Goeminne G., Kim H., Cross J., Morreel K., Araujo P., Welsh L., Haustraete J., McClellan C., Vanholme B., Ralph J., Simpson GG, Halpin C., Boerjan W. 2013. Caffeoyl shikimate esterase (CSE) is an enzyme in the lignin biosynthetic pathway in Arabidopsis Science. 341:1103-6.).
[0026] Preferably according to the invention, the heterologous gene coding for a CSE is a gene originating from a prokaryotic or eukaryotic organism.
[0027] According to one embodiment, the heterologous gene encoding a CSE is the Medicago truncatula CSE (MtCSE) gene (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 Medicago truncatula CSE (MtCSE) and exhibiting caffeoyl-shikimate esterase activity.
[0028] Medicago truncatula, or truncated alfalfa, is a species of the Fabaceae family, of the Faboideae subfamily.
[0029] According to one embodiment, the heterologous gene encoding a CSE is the Arabidopsis thaliana CSE gene (AtCSE) (Atlg52760, 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 caffeoyl-shikimate esterase activity.
[0030] Arabidopsis thaliana or Rockcress is a species of the Bras-sicaceae family.
[0031] According to one embodiment, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a chlorogenic acid esterase (ChlE).
[0032] A chlorogenic acid esterase (ChlE) is an enzyme classically having chlorogenic acid hydrolysis activity, and used herein to hydrolyze the caffeoyl-shikimate bond and thereby produce caffeic acid from caffeoyl-shikimate.
[0033] According to one embodiment, 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 of Bifidobacterium animalis subsp. Lactis (BiChlE) and having chlorogenic acid esterase activity.
[0034] Bifidobacterium animalis subsp. Lactis is a species of lactic acid bacteria isolated from chicken, rabbit and human feces, as well as from fermented milk.
[0035] According to one embodiment, the heterologous gene for a ChlE is the Ustilago maydis ChlE (UmChlE) gene (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 Ustilago maydis ChlE (UmChlE) and exhibiting chlorogenic acid esterase activity.
[0036] Ustilago maydis is a pathogenic fungus that causes corn smut in particular.
[0037] According to one embodiment, the heterologous gene for a ChlE is the Lactobacillus johnsonii ChlE (LaChlE) gene (SPPI01000004.1:37780-38526, GenBank, SEQ ID NO. 8) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Lactobacillus johnsonii ChlE (LaChlE) and exhibiting chlorogenic acid esterase activity.
[0038] Lactobacillus johnsonii is a lactic acid bacterium that is part of the vaginal microbiota healthy.
[0039] According to one embodiment, the heterologous gene for a ChlE is the Salinibacter ruber ChlE (SrChlE) gene (CP030369.1:2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having 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.
[0040] Salinibacter ruber is a halophilic red bacterium that thrives in a highly concentrated salt environment.
[0041] By "percentage identity" between two gene 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 is the highest.
[0042] Preferably according to the invention, 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.
[0043] Preferably, the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
[0044] By capable of catalyzing the formation of the bond between coumaric acid and coenzyme A, it is understood according to the invention that from these two compounds, the enzyme is capable of producing coumaroyl-CoA. This enzymatic reaction involves ATP and can be carried out in one or more stages.
[0045] A 4-coumarate-CoA ligase (4CL) is an enzyme that catalyzes the formation of coumaroyl-CoA from coumaric acid and coenzyme A.
[0046] Preferably according to the invention, the heterologous gene coding for a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
[0047] Preferably according to the invention, the heterologous gene coding for said 4CL is mutated in order to reduce the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid compared to the parent gene coding for a non-mutated 4CL.
[0048] According to one embodiment, the 4CL is the 4CL of Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
[0049] Populus tomentosa or Chinese white poplar is a species of the Salicaceae family.
[0050] According to one embodiment, 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 - at position 329 is an Alanine (G329A)
[0051] According to one embodiment, the 4CL is the 4CL of Arabidopsis thaliana (Atlg51680, GenBank, SEQ ID NO. 1).
[0052] According to one embodiment, 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 - at position 353 is an Alanine (G353A).
[0053] Since 4CL is not specific for p-coumaric acid and also accepts caffeic acid as a substrate, the latter released by the CSE is likely to be converted back into caffeoyl-CoA by 4CL. These mutations are made to overcome this problem, and reduce the affinity of 4CL for caffeic acid while improving its specificity for p-coumaric acid.
[0054] According to one embodiment, the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQ ID NO. 12).
[0055] Streptomyces coelicolor is a Gram-positive soil bacterium.
[0056] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises: - A heterologous gene encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene coding for a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene coding for a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene coding for a Cinnamate 4-Hydroxylase (C4H).
[0057] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises: - A heterologous gene encoding a Cytochrome P450 reductase (CPR1).
[0058] According to one embodiment of the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises: - A heterologous gene encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene encoding a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene encoding a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene encoding a Cinnamate 4-Hydroxylase (C4H); and - A heterologous gene encoding a Cytochrome P450 reductase (CPR1).
[0059] A Hydroxycinnamoyl-Transferase (HCT) is an enzyme of the transferase family, which catalyzes the reaction: 4-coumaroyl-CoA + shikimate CoA + 4-coumaroylshikimate.
[0060] Preferably according to the invention, the heterologous gene coding for an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
[0061] A Coumarate 3 Hydroxylase (C3H) is an enzyme involved in the synthesis of lignin, and which catalyzes the production of caffeoyl shikimate from coumaroyl shikimate.
[0062] Preferably according to the invention, the heterologous gene coding for a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
[0063] A Cytochrome P450 reductase (CPR1) is a membrane oxidoreductase that allows the transfer of electrons from NADPH to cytochrome P450.
[0064] Preferably according to the invention, the heterologous gene coding for a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Ca-tharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
[0065] Catharanthus roseus, or Madagascar periwinkle, is a plant of the Apocynaceae family native to Madagascar.
[0066] A Tyrosine Amonia Lyase (TAL) is an enzyme that converts tyrosine to p-coumaric acid with release of ammonia.
[0067] Preferably according to the invention, the heterologous gene coding for a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rho-dotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
[0068] Rhodotorula glutinis is a pink yeast of the genus Rhodotorula.
[0069] A Phenylalanine Amonia-Lyase (PAL) is an enzyme that catalyzes the transformation of phenylalanine into cinnamic acid, with the release of ammonia.
[0070] A Cinnamate 4-Hydroxylase (C4H), or Trans-cinnamate 4-monooxygenase, is an enzyme that converts trans-cinnamic acid (CA) into p-coumaric acid.
[0071] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises: - A gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition compared to the parent gene.
[0072] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises invalidation for: - a gene coding for a Phenylpyruvate decarboxylase (ARO10).
[0073] According to one embodiment of the invention, said recombinant microorganism, preferably said recombinant yeast, further comprises: - A gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a mutated chorismate mutase (ARO7) so that the product is resistant to feedback inhibition relative to the parent gene; and / or the invalidation of: - a gene coding for a Phenylpyruvate decarboxylase (AR01O).
[0074] By "invalidation" is meant the suppression or inhibition of the expression of the gene, and thus the suppression or inhibition of the activity of the enzyme.
[0075] A 3-deoxy-7-phosphoheptulonate synthase (ARO4), or DAHP synthase, is a transferase that occurs at the first step of the shikimate pathway, and catalyzes the reaction: phosphoenolpyruvate + D-erythrose-4-phosphate + H2O 3-deoxy-D-arabinoheptulosonate-7-phosphate + phosphate.
[0076] A chorismate mutase (ARO7) is an isomerase involved in the shikimate pathway, and is known to catalyze a pericyclic reaction. This enzyme catalyzes the reaction: chorismate prephenate.
[0077] ARO4 and ARO7 are two genes involved in the de novo synthesis of aromatic amino acids such as phenylalanine and tyrosine in yeast.
[0078] Preferably, the ARO4 gene (NP_009808, GenBank) is mutated so that the amino acid at position 229 is a Leucine (K229L).
[0079] Preferably, the ARO7 gene (NP_015385, GenBank) is mutated so that the amino acid at position 141 is a Serine (G141S).
[0080] These mutations of ARO4 and ARO7 have the effect of optimizing increased production of aromatic amino acids in yeast.
[0081] A Phenylpyruvate decarboxylase (ARO10) is an enzyme involved in particular in the degradation of phenylalanine and tyrosine in yeast.
[0082] Together, the mutations of ARO4 and ARO7 and the invalidation of ARO10 are carried out to optimize increased production of aromatic amino acids such as phenylalanine and tyrosine.
[0083] According to one embodiment, said recombinant microorganism, preferably said recombinant yeast, is capable of producing ferulic acid.
[0084] Preferably, said recombinant microorganism, preferably said recombinant yeast, is capable of producing ferulic acid from the caffeic acid obtained, and further comprises: - A heterologous gene encoding a caffeoyl-O-methyl transferase (COMT).
[0085] Even more preferably, said recombinant microorganism, preferably said recombinant yeast, capable of producing ferulic acid from the caffeic acid obtained further comprises: - A gene encoding S-Adenosylmethyltransferase (SAM2).
[0086] Ferulic acid is an organic hydroxycinnamic acid present in many plants, and participating in the synthesis of lignin.
[0087] The chemical formula of ferulic acid is CioHio04 and this compound has a molar mass of 194.18 g / mol.
[0088] The chemical structure of ferulic acid is:
[0089] [Chem.2] O H 3C0^Y%A 0H
[0090] An S-Adenosylmethyltransferase, or S-adenosylmethionine synthase 2 (SAM2) catalyzes the formation of S-adenosylmethionine from methionine and ATP. The reaction comprises two steps catalyzed by the same enzyme: the formation of S-adenosylmethionine and triphosphate and the subsequent hydrolysis of the triphosphate. In the context of the invention, it makes it possible to transform methionine into S-adenosylmethionine and S-adenosilhomocysteine, which allows the methylation of caffeic acid into ferulic acid.
[0091] Preferably, the SAM2 gene is the Saccharomyces cerevisiae SAM2 gene and is a copy of the naturally occurring one (YDR502C, SGD Database, SEQ ID NO. H).
[0092] A caffeoyl-O-methyl Transferase (COMT) is an enzyme that catalyzes the reaction: S-adenosyl-L-methionine + caffeic acid S-adenosyl-L-homocysteine + ferulic acid.
[0093] Preferably according to the invention, the heterologous gene coding for COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g54160, GenBank, SEQ ID NO. 2).
[0094] Preferably, said yeast according to the invention capable of producing ferulic acid further comprises the invalidation of the gene coding for a ferulic acid decarboxylase 1 (FDC1).
[0095] A ferulic acid decarboxylase is an enzyme that 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 act as the aromatic metabolites.
[0096] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, is a species of the As-comycota phylum, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygo-saccharomyces.
[0097] Preferably according to the invention, said recombinant microorganism, preferably said recombinant yeast, is of the species Saccharomyces cerevisiae.
[0098] Saccharomyces cerevisiae is a unicellular eukaryotic yeast, appearing in ovoid to rounded form, approximately 6 to 12 pm in length and 6 to 8 pm in width.
[0099] According to an embodiment where all of the genes discussed above are respectively introduced and / or invalidated, the metabolic pathways for the production of caffeic acid and ferulic acid in the yeast according to the invention are illustrated in [fig.2]
[0100] According to a second aspect, the invention relates to a method of modifying a microorganism, preferably a yeast, to produce caffeic acid, comprising the introduction of: - A heterologous gene encoding an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate.
[0101] Preferably, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a caffeoyl-shikimate esterase (CSE).
[0102] Preferably according to the invention, the heterologous gene coding for a CSE is a gene originating from a prokaryotic or eukaryotic organism.
[0103] According to one embodiment, the heterologous gene encoding a CSE is the Medicago truncatula CSE (MtCSE) gene (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 Medicago truncatula CSE (MtCSE) and exhibiting caffeoyl-shikimate esterase activity.
[0104] According to one embodiment, the heterologous gene encoding a CSE is the Arabidopsis thaliana CSE gene (AtCSE) (Atlg52760, 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 Arabidopsis thaliana CSE (AtCSE) and exhibiting caffeoyl-shikimate esterase activity.
[0105] According to one embodiment, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a chlorogenic acid esterase (ChlE).
[0106] According to one embodiment, 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 of Bifidobacterium animalis subsp. Lactis (BiChlE) and exhibiting chlorogenic acid esterase activity.
[0107] According to one embodiment, the heterologous gene for a ChlE is the Ustilago maydis ChlE (UmChlE) gene (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 Ustilago maydis ChlE (UmChlE) and exhibiting chlorogenic acid esterase activity.
[0108] According to one embodiment, the heterologous gene for a ChlE is the Lactobacillus johnsonii ChlE (LaChlE) gene (SPPI01000004.1:37780-38526, GenBank, SEQ ID NO. 8) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Lactobacillus johnsonii ChlE (LaChlE) and exhibiting chlorogenic acid esterase activity.
[0109] According to one embodiment, the heterologous gene for a ChlE is the Salinibacter ruber ChlE (SrChlE) gene (CP030369.1:2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having 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.
[0110] Preferably according to the invention, 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.
[0111] Preferably, the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
[0112] Preferably according to the invention, the heterologous gene coding for a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
[0113] Preferably according to the invention, the heterologous gene coding for a 4CL is mutated in order to reduce the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid compared to the parent gene coding for a non-mutated 4CL.
[0114] According to one embodiment, the 4CL is the 4CL of Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
[0115] According to one embodiment, 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 - at position 329 is an Alanine (G329A)
[0116] According to one embodiment, the 4CL is the 4CL of Arabidopsis thaliana (Atlg51680, GenBank, SEQ ID NO. 1).
[0117] According to one embodiment, 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 - at position 353 is an Alanine (G353A).
[0118] According to one embodiment, the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQID NO. 12).
[0119] Preferably according to the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of: - A heterologous gene encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene coding for a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene coding for a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene coding for a Cinnamate 4-Hydroxylase (C4H).
[0120] Preferably according to the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of: - A heterologous gene encoding a Cytochrome P450 reductase (CPR1).
[0121] According to one embodiment of the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of: - A heterologous gene encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene encoding a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene encoding a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene encoding a Cinnamate 4-Hydroxylase (C4H); and - A heterologous gene encoding a Cytochrome P450 reductase (CPR1).
[0122] Preferably according to the invention, the heterologous gene coding for an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
[0123] Preferably according to the invention, the heterologous gene coding for a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
[0124] Preferably according to the invention, the heterologous gene coding for a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Ca-tharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
[0125] Preferably according to the invention, the heterologous gene coding for a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rho-dotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
[0126] Preferably according to the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of: - A gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition compared to the parent gene.
[0127] Preferably according to the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises invalidation for: - a gene coding for a Phenylpyruvate decarboxylase (ARO10).
[0128] According to one embodiment of the invention, said method of modifying a microorganism, preferably a yeast, to produce caffeic acid further comprises the introduction of: - A gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or invalidation of: - a gene coding for a Phenylpyruvate decarboxylase (ARO10).
[0129] Preferably, the ARO4 gene is mutated so that the amino acid at position 229 is a Leucine (K229L).
[0130] Preferably, the ARO7 gene is mutated so that the amino acid at position 141 is a Serine (G141S).
[0131] Preferably according to the invention, said yeast is a species of the Ascomycota phylum, preferably chosen from the genera Schizosac-charomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, To-rulaspora and / or Zygosaccharomyces.
[0132] Preferably according to the invention, said yeast is of the species Saccharomyces cerevisiae.
[0133] According to one embodiment, the invention relates to a method of modifying a microorganism, preferably a yeast, to produce ferulic acid.
[0134] Preferably, the invention relates to a method for modifying a microorganism, preferably a yeast, to produce ferulic acid from the caffeic acid obtained, further comprising the introduction of: - A heterologous gene encoding caffeoyl-O-methyl Transferase (COMT).
[0135] Preferably, said method of modifying a microorganism, preferably a yeast, to produce ferulic acid from the caffeic acid obtained, further comprises the introduction of: - A gene encoding S-Adenosylmethyltransferase (SAM2).
[0136] Preferably, the SAM2 gene is the Saccharomyces cerevisiae SAM2 gene and is a copy of the naturally occurring one (YDR502C, SGD Database, SEQ ID NO. H).
[0137] Preferably according to the invention, the heterologous gene coding for COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g54160, GenBank, SEQ ID NO. 2).
[0138] Preferably, said method of 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 coding for a ferulic acid decarboxylase 1 (FDC1).
[0139] According to a third aspect, the invention relates to a method for producing caffeic acid, comprising a step of: a. Cultivation of recombinant microorganisms, preferably recombinant yeasts, as defined according to the invention in a culture medium.
[0140] Preferably, said method of producing caffeic acid further comprises a step of: b. Recovery of the caffeic acid obtained in step a.
[0141] According to the invention, recovery means isolating the target compound, here caffeic acid and / or ferulic acid, from the rest of the other compounds.
[0142] Examples of methods for recovering caffeic acid and ferulic acid are described in the literature, notably in Yin Y. et al., “Polydopamine-coated magnetic molecularly imprinted polymer for the selective solid-phase extraction of cinnamic acid, ferulic acid and caffeic acid from radix scrophulariae sample”, J Sep Sci. 2016 Apr;39(8): 1480-8, and in Ning Li. et al., “Separation and purification of the antioxidant compounds, caffeic acid phenethyl ester and caffeic acid from mushrooms by molecularly imprinted polymer”, Food Chem. 2013 Aug 15;139(1-4):1161-7.
[0143] Preferably according to the invention, the caffeic acid is produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caffeoyl-shikhnate, added to the culture medium before or at step a.
[0144] Even more preferably, the caffeic acid is produced from glucose.
[0145] According to a fourth aspect, the invention relates to a method for producing ferulic acid, comprising a step of: a. Cultivation of recombinant microorganisms, preferably recombinant yeasts, as defined according to the invention in a culture medium.
[0146] Preferably, said method of producing ferulic acid further comprises a step of: b. Recovery of the ferulic acid obtained in step a.
[0147] According to one embodiment, said method for 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 the 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'.
[0148] Preferably according to the invention, ferulic acid is produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caffeoyl-shikhnate, added to the culture medium before or at step a or a'.
[0149] Preferably, ferulic acid is produced from glucose.
[0150] According to a fifth aspect, the invention relates to a method for producing caffeic acid and / or ferulic acid, comprising a step of: a. Cultivation of recombinant microorganisms, preferably recombinant yeasts, capable of producing ferulic acid from the caffeic acid obtained as defined according to the invention in a culture medium.
[0151] Preferably, said method for producing caffeic acid and / or ferulic acid further comprises a step of: b. Recovery of caffeic acid and / or ferulic acid obtained in step a.
[0152] Preferably according to the invention, caffeic acid and / or ferulic acid are produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caffeoyl-shikimate, added to the culture medium before or at step a.
[0153] Preferably, caffeic acid and / or ferulic acid are produced from glucose.
[0154] According to a sixth aspect, the invention relates to the use of the recombinant microorganism, preferably recombinant yeast, capable of producing ferulic acid from the caffeic acid obtained according to the invention for producing caffeic acid and / or ferulic acid.
[0155] According to a seventh aspect, the invention relates to the use of the recombinant microorganism, preferably recombinant yeast, producing caffeic acid according to the invention for producing caffeic acid.
[0156] According to an eighth aspect, the invention relates to the use of the recombinant microorganism, preferably recombinant yeast, producing ferulic acid according to the invention for producing ferulic acid.
[0157] Preferably, ferulic acid is produced from caffeic acid produced by said recombinant microorganism, preferably said recombinant yeast, according to the invention.
[0158] According to a ninth aspect, 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 recombinant host microorganism, preferably to the recombinant host yeast, coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikhnate bond to produce caffeic acid from caffeoyl-shikimate.
[0159] Preferably, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a caffeoyl-shikimate esterase (CSE).
[0160] Preferably according to the invention, the heterologous gene coding for a CSE is a gene originating from a prokaryotic or eukaryotic organism.
[0161] According to one embodiment, the heterologous gene encoding a CSE is the Medicago truncatula CSE (MtCSE) gene (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 Medicago truncatula CSE (MtCSE) and exhibiting caffeoyl-shikimate esterase activity.
[0162] According to one embodiment, the heterologous gene encoding a CSE is the Arabidopsis thaliana CSE gene (AtCSE) (Atlg52760, 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 Arabidopsis thaliana CSE (AtCSE) and exhibiting caffeoyl-shikimate esterase activity.
[0163] According to one embodiment, the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a chlorogenic acid esterase (ChlE).
[0164] According to one embodiment, 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 of Bifidobacterium animalis subsp. Lactis (BiChlE) and having chlorogenic acid esterase activity.
[0165] According to one embodiment, the heterologous gene for a ChlE is the gene for the Ustilago maydis ChlE (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 Ustilago maydis ChlE (UmChlE) and exhibiting chlorogenic acid esterase activity.
[0166] According to one embodiment, the heterologous gene for a ChlE is the Lactobacillus johnsonii ChlE (LaChlE) gene (SPPI01000004.1:37780-38526, GenBank, SEQ ID NO. 8) or a gene encoding a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of Lactobacillus johnsonii ChlE (LaChlE) and exhibiting chlorogenic acid esterase activity.
[0167] According to one embodiment, the heterologous gene for a ChlE is the Salinibacter ruber ChlE (SrChlE) gene (CP030369.1:2322200-2323400, GenBank, SEQ ID NO. 13) or a gene encoding a sequence having 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.
[0168] Preferably according to the invention, 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.
[0169] Preferably, the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
[0170] Preferably according to the invention, the heterologous gene coding for a 4CL is a gene originating from a prokaryotic or eukaryotic organism.
[0171] Preferably according to the invention, the heterologous gene coding for a 4CL is mutated in order to reduce the affinity of said mutated 4CL for caffeic acid and to increase its specificity for p-coumaric acid compared to the parent gene coding for a non-mutated 4CL.
[0172] According to one embodiment, the 4CL is the 4CL of Populus tomentosa (AY043495, Genbank, SEQ ID NO. 10).
[0173] According to one embodiment, 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 - at position 329 is an Alanine (G329A)
[0174] According to one embodiment, the 4CL is the 4CL of Arabidopsis thaliana (Atlg51680, GenBank, SEQ ID NO. 1).
[0175] According to one embodiment, 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 - at position 353 is an Alanine (G353A).
[0176] According to one embodiment, the 4CL is the 4CL of Streptomyces coelicolor (CAB95894.1, Genbank, SEQID NO. 12).
[0177] Preferably according to the invention, 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 encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene coding for a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene coding for a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene coding for a Cinnamate 4-Hydroxylase (C4H).
[0178] Preferably according to the invention, 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 encoding a Cytochrome P450 reductase (CPR1).
[0179] According to one embodiment of the invention, 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 encoding a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene encoding a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene encoding a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene encoding a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene encoding a Cinnamate 4-Hydroxylase (C4H); and - A heterologous gene encoding a Cytochrome P450 reductase (CPR1).
[0180] Preferably according to the invention, the heterologous gene coding for an HCT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At5g48930, GenBank, SEQ ID NO. 7).
[0181] Preferably according to the invention, the heterologous gene coding for a C3H is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis thaliana (At2g40890, GenBank, SEQ ID NO. 5).
[0182] Preferably according to the invention, the heterologous gene coding for a CPR1 is a gene originating from a prokaryotic or eukaryotic organism, preferably Ca-tharanthus roseus (X69791.1, GenBank, SEQ ID NO. 6).
[0183] Preferably according to the invention, the heterologous gene coding for a TAL is a gene originating from a prokaryotic or eukaryotic organism, preferably Rho-dotorula glutinis (KF765779.1, GenBank, SEQ ID NO. 14).
[0184] Preferably according to the invention, 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 gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition compared to the parent gene.
[0185] Preferably according to the invention, 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: - a gene coding for Phenylpyruvate decarboxylase (ARO10).
[0186] According to one embodiment of the invention, 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 gene encoding a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or - A gene encoding a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition relative to the parent gene; and / or invalidation of: - a gene coding for a Phenylpyruvate decarboxylase (ARO10).
[0187] Preferably, the ARO4 gene is mutated so that the amino acid at position 229 is a Leucine (K229L).
[0188] Preferably, the ARO7 gene is mutated so that the amino acid at position 141 is a Serine (G141S).
[0189] According to one embodiment, said at least one expression vector in a recombinant host microorganism, preferably a recombinant host yeast, allows the production of ferulic acid from the caffeic acid obtained, and further comprises a coding sequence for: - A heterologous gene encoding caffeoyl-O-methyl Transferase (COMT).
[0190] Preferably, 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: - A gene encoding S-Adenosylmethyltransferase (SAM2).
[0191] Preferably, said yeast is a species of the As-comycota phylum, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygo-saccharomyces.
[0192] Preferably, said yeast is of the species Saccharomyces Cerevisiae.
[0193] Preferably, the SAM2 gene is the Saccharomyces cerevisiae SAM2 gene and is a copy of the naturally occurring one (YDR502C, SGD Database, SEQ ID NO. H).
[0194] Preferably according to the invention, the heterologous gene coding for COMT is a gene originating from a prokaryotic or eukaryotic organism, preferably Arabidopsis Thaliana (At5g54160, UniProtKB).
[0195] Preferably, 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 sequence allowing the invalidation of the gene coding for a ferulic acid decarboxylase 1 (FDC1).
[0196] The invention may be better understood by reading the detailed description which follows, of non-limiting examples of implementation of the invention, and by examining the appended figures, in which: DESCRIPTION OF THE FIGURES
[0197] [fig. 1] illustrates the hydrolysis reaction of caffeoyl-shikimate into caffeic acid and shikimate using caffeoyl-shikimate esterase (CSE) according to the invention.
[0198] [fig-2] illustrates the metabolic pathways for the production of caffeic acid and ferulic in yeast according to one embodiment of the invention.
[0199] [fig.3] illustrates the production of caffeic acid in a recombinant yeast according to a embodiment of the invention from p-coumaric acid or glucose via CSE (UHPLC-TQ method), at 24h, 48h and 72h.
[0200] [fig-4] illustrates the production of the different intermediates produced by yeast re combining according to an embodiment of the invention, analyzed by a qualitative method (UHPLC-HRMS method (High Resolution Mass Spectrometry).
[0201] [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.
[0202] [fig.6] is a chromatograph characterizing the production of ferulic acid in the recombinant yeast according to one embodiment of the invention from glucose. The peak at 2.01 corresponds to caffeic acid and peak at 3.15 corresponds to ferulic acid.
[0203] [fig.7] is a chromatogram characterizing the hydrolysis of caffeoyl-shikimate in caffeic acid and shikimate by CSE (from Medicago truncatula, MtCSE) in recombinant yeast according to one embodiment of the invention. The peak with a retention time of 3.23 min corresponds to caffeic acid and the second at 3.78 min to caffeoyl-shikimate.
[0204] [fig.8] is a chromatogram characterizing the hydrolysis of caffeoyl-shikimate in caffeic acid and shikimate by ChlE (from Lactobacillus johnsonii, LaChlE) in recombinant yeast according to one embodiment of the invention. The peak with a retention time of 3.19 min corresponds to caffeic acid.
[0205] [fig.9] is a chromatogram characterizing the presence of caffeoyl-shikimate in control samples. The peak with a retention time of 3.88 min corresponds to caffeoyl-shikimate. EXAMPLES
[0206] Example 1: Materials and methods:
[0207] Standards for p-coumaric acid, caffeic acid and ferulic acid were obtained from the supplier Sigma-Aldrich. Gene cloning:
[0208] The ARO4 (NP_009808, Genbank) and ARO7 (NP_015385, Genbank) 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. Microbial Cell Factories (2015)). 14:73, see pages 11 to 16 and additional files). For ARO4, this corresponds to the K229L mutation, and for ARO7 to the G141 S mutation.
[0209] The genes obtained by synthesis or PCR include at the 5' and 3' end, a Bbsl (GAAGAC) or Bsal (GGTCTC) restriction site, compatible with the cloning system used. All genes, promoters and terminators were cloned by restriction in the pSBK vector. The promoters and terminators (Wargner et al., 2015) were amplified by PCR from the genomic DNA of the yeast S. cerevisiae.
[0210] The pSBK vector comprises a yeast selection marker: URA3, LEU2 or TRP1.
[0211] Table 1: The different genes used to produce a recombinant yeast according to the invention. [Tables 1] Gene Accession number, bank Provenance TAL (Tyrosine Amonia Lyase) KF765779.1, GenBank, SEQ ID NO. 14 Rhodotorula glutinis 4CL (4 Coumarate-CoA Ligase) AY043495, Genbank, SEQ ID NO. 10 Populus tomentosa HCT (Hydroxycinnamoyl-Transferase) At5g48930, GenBank, SEQ ID NO. 7 Arabidopsis thaliana C3H (Coumarate 3 Hydroxylase) At2g40890, GenBank, SEQ ID NO. 5 Arabidopsis thaliana MtCSE (caffeoyl-Shikimate Esterase) XM_00360999 0.3, Genbank, SEQ ID NO. 9 Medicago truncatula AtCSE Atlg52760, GenBank, SEQ ID NO. 3 Arabidopsis thaliana COMT (caffeoyl-O-Methyl Transferase) At5g54160, GenBank, SEQ ID NO. 2 Arabidopsis thaliana CPR1 (Cytochrome P450 reductase) X69791.1, GenBank, SEQ ID NO. 6 Catharanthus roseus SAM2 (S-Adenosylmethyltransferase) YDR502C, SGD Database, SEQ ID NO. 11 Saccharomyces cerevisiae
[0213] Mutation of 4CL:
[0214] The Y236A and Y236F mutants of P. tomentosa 4CL were constructed by PCR.
[0215] Deletion of genes:
[0216] The ARO10 (YDR380W) and FDC1 (YDR539W) genes were invalidated by deletion, that is to say 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.
[0217] Strains:
[0218] The yeast model used in this study is Sac-charomyces cerevisiae strain FY1679-28A (Tettelin et al., 1995 - Table 1 page 85), auxotrophic for uracil, tryptophan, and leucine. The constructs were made in Escherichia coli strain MH1 before transfer to yeast.
[0219] Table 2: List of strains used
[0220] [Tables2] Name Constructions Markers L16B5 ARO4 K229L -ARO7Gi^ -TAL 4CL ^a -HCT-C3H MtCSE-CPRl URA3 TRP1 LEU2 L16D5 ARO4^l -ARO7C^S-TAL 4CL y236f -HCT-C3H MtCSE-CPRl URA3 TRP1 LEU2 L16A3 ARO4^l -ARO7 GWS -TAL 4CL y236a -HCT-C3H AtCSE-CPRl URA3 TRP1 LEU2 L16C2 ARO4^l .aro7g'^ -TAL 4CL y236f -HCT-C3H AtCSE-CPRl URA3 TRP1 LEU2 L93-2D3 fdclA::(ARO4 K229L -ARO7GWS-TAL)~HPH 4CL ^236a -HCT-C3H MtCSE-COMT-CPRl-SAM2 7 / P / / (hygromycin e) TRP1 URA3
[0221] All strains listed in this table have been invalidated for the ARO10 gene.
[0222] Cultivation conditions:
[0223] 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, Fr) supplemented or not with CSM (Complemented Supplement Mixture; Formedium, UK). Glucose was added at 20 g / L. or p-coumaric acid was added to the medium at a concentration of 100 mg.1-1.
[0224] Analytical method: UHPLC-TQ' method.
[0225] 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 are 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.
[0226] Analysis by UHPLC-TQ: The samples were analyzed by a Vanquish-H UHPLC (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 1.8 pm 2.1 X 5 mm pre-column.
[0227] 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.
[0228] Table 3: chromatographic conditions for the detection of molecules of interest:
[0229] [Tables3] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.5 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
[0230] The parameters of the electrospray source are: - positive mode spray voltage at 4000V - curtain gas: at 50 Arb - auxiliary gas at 15 Arb - transfer tube temperature at 300°C - vaporizer temperature at 300°C
[0231] Table 4: The ions monitored and the fragmentation conditions for the molecules of interest: [Tables 4] Molecules Retention time (min) Polarity Precursor ion Daughter ion Collision energy RF lens (V) P-Coumaric Acid 2.21 Negative 162.9 119.054 14.55 87 93 31.15 87 Trans-Ferulic Acid 2.67 Negative 192.95 149.06 11.33 93 178.018 12.46 93 Caffeic Acid 2.69 Negative 178.9 135 15.31 91 107.071 21.34 91
[0233] UHPLC-HRMS (High Resolution Mass Spectrometry) method:
[0234] Sample preparation: 100 pL samples are collected for each experiment. 50 pL are transferred to a new plate, to which 50 pL of acetonitrile is added. Each sample is then homogenized by suction-discharge, then centrifuged for 5 min at 3000 rpm at room temperature.
[0235] Analysis by UHPLC-HRMS: The samples were analyzed by a Vanquish-H UHPLC (Thermo) coupled with a UHPLC-HRMS. The column is a Waters Acquity UPLC@ USST3 column (8 pm 2.1 X 100 mm) associated with a HSST3 1.8 pm 2.1X5 mm pre-column.
[0236] 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 pure LC / MS grade acetonitrile. The column temperature is 50°C and the sample changer temperature is 10°C.
[0237] Table 5: Chromatographic conditions for the detection of molecules of interest: [Tables 5] Time (min) Flow rate (ml / min) Mobile phase A (%) Mobile phase B (%) 0 0.5 100 0 0.5 0.5 100 0 1 0.5 88 12 4.7 0.5 84 16 5.5 0.5 55 45 6.5 0.5 55 45 9 0.5 0 100 10 0.5 0 100 10.5 0.5 100 0 12 0.5 100 0
[0239] The parameters of the electrospray source are: - positive mode spray voltage at 3.10 kV - curtain gas at 50 Arb - auxiliary gas at 20 Arb - capillary temperature at 350°C - auxiliary gas heating temperature at 500°C - S-lens RF at 55 V - collision energy (NCE ramp): 20, 40, 60
[0240] Table 6: Tracked ions and fragmentation conditions for molecules of interest
[0241] [Tableauxô] Molecules Retention Time Polarity Ion Fragments (min) precursor P-Coumaric Acid 4.18 Negative 163.04007 119.0502 93.0346 Coumaroyl Shikimate 5.09 Negative 319.08233 163.04007 119.05024 93.03459 Caffeic Acid 3.05 Negative 179.03498 135.0452 Caffeoyl Shikimate 3.70 Negative 335.07724 135.0452 179.0351
[0242] Example 2: Production of caffeic acid from glucose or p-coumaric acid.
[0243] The production of caffeic acid from glucose or p-coumaric acid was tested in 4 strains, the differences of which relate to the choice of mutated 4CLs (either Y236A or Y236F) and the CSEs used (A. thaliana or M. truncatula).
[0244] [Fig. 3] describes the results of the production of caffeic acid from p-coumaric acid or glucose by CSE (UHPLC-TQ method) using the yeast according to the invention. In all the strains tested the ARO4K229L-ARO7G141S-TAL-HCT-C3H-CPR1 genes were added and the ARO10 gene was invalidated by deletion. These strains therefore diverge only by the 4CL and CSE used (L16A3: 4CLY236A-AtCSE; L16B5: 4CLY236A-MtCSE; L16C2: 4CLY236F-AtCSE; L16D5: 4CLY236F-MtCSE).
[0245] The results presented in [fig.3] show that the association of these different enzymes allows the production of caffeic acid.
[0246] The best condition is obtained with the 4CL Y236A mutation and the CSE of M. truncatula (L16B5).
[0247] Example 3: Production of the different intermediaries.
[0248] The results presented in [fig.4] show the characterization of the production of the different intermediates produced by the caffeic acid-producing yeast according to the invention from glucose or p-coumaric acid, by a qualitative method (UHPLC-HRMS method).
[0249] These results make it possible to show the accumulation of each of the intermediates, i.e. p-coumaric acid, p-coumaroyl-shikimate, caffeoyl-shikimate and caffeic acid (see [fig.4]).
[0250] The accumulation of the different 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 caffeoyl-shikimate.
[0251] Example 4: Production of ferulic acid from p-coumaric acid
[0252] The yeast according to the invention capable of producing ferulic acid possesses the Arabidobsis thaliana methyltransferase (COMT) and is invalidated for the FDCL gene. This strain was incubated for 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.
[0253] The chromatogram shows a production of caffeic acid and ferulic acid ( [fig.5]). 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.
[0254] These tests show that caffeic acid, produced from p-coumaric acid, can be efficiently converted into ferulic acid, when a methyl-transferase is added to the producing strain ([fig.5]).
[0255] Example 5: Production of ferulic acid from glucose:
[0256] The yeast according to the invention capable of producing ferulic acid possesses the Arabidobsis thaliana methyltransferase (COMT) and is invalidated for the FDCL 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.
[0257] The chromatogram shows a production of caffeic acid and ferulic acid ( [fig.6]). The first peak with a retention time of 2.01 min corresponds to caffeic acid and the second at 3.15 min to ferulic acid.
[0258] These tests show that caffeic acid produced from p-coumaric acid can be efficiently converted into ferulic acid, when a methyl-transferase is added to the producing strain ([fig.6]).
[0259] Example 6: Test of the hydrolysis of caffeoyl-shikimate into caffeic acid and shikimate using CSE:
[0260] The caffeoyl-shikimate sample was prepared from the culture supernatant of a producer strain. The release of caffeic acid from caffeoyl-shikimate was tested using a CSE-containing strain from Medicago truncatula (MtCSE).
[0261] The results are shown in [fig.7], the first peak with a retention time of 3.23 min corresponding to caffeic acid and the second at 3.78 min to caffeoyl-shikimate.
[0262] Production of caffeic acid from caffeoyl-shikimate in the presence of MtCSE is observed.
[0263] Example 7: Test of the hydrolysis of caffeoyl-shikimate into caffeic acid and shikimate using ChLE:
[0264] The caffeoyl-shikimate sample was prepared from the culture supernatant of a producing strain. The release of caffeic acid from caffeoyl-shikimate was tested using a strain containing Lactobacillus johnsonii ChlE.
[0265] The results are presented in [fig.8]. A peak is observed at a retention time of 3.19 min corresponding to caffeic acid, all the caffeoyl-shikimate having been consumed. In [fig.9] representing a control sample, the presence of caffeoyl-shikimate can be verified for which a peak is observed at 3.88 min and the absence of caffeic acid.
[0266] Production of caffeic acid from caffeoyl-shikimate is observed in the presence of LaChlE.
Claims
Claims
1. Recombinant yeast capable of producing caffeic acid, characterized in that it comprises: - A heterologous gene coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate; and - A heterologous gene coding for an enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A.
2. Recombinant yeast according to claim 1, characterized in that the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a caffeoyl-shikimate esterase (CSE).
3. Recombinant yeast according to claim 2, characterized in that the heterologous gene coding for caffeoyl-shikimate esterase (CSE) is the CSE gene of Medicago truncatula or Arabidopsis thaliana, preferably of Medicago truncatula.
4. Recombinant yeast according to claim 3, characterized in that the CSE is chosen from SEQ ID NOs. 9 and 3 or a gene coding for a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of caffeoyl-shikimate esterase (CSE) of Medicago truncatula or Arabidopsis thaliana and having caffeoyl-shikimate esterase activity.
5. Recombinant yeast according to claim 1, characterized in that the enzyme capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate is a chlorogenic acid esterase (ChlE).
6. Recombinant yeast according to claim 5, characterized in that the heterologous gene coding for chlorogenic acid esterase (ChlE) is the ChlE gene of Bifidobacterium animalis subsp. Lactis of Ustilago maydis, of Lactobacillus johnsonii or of Salinibacter ruber, preferably of Lactobacillus johnsonii.
7. Recombinant yeast according to claim 6, characterized in that the ChlE is chosen from SEQ ID NOs. 4 and 8 or a gene coding for a sequence having at least 55, 60, 70, 80, 85, 90 or 95% identity with the amino acid sequence of chlorogenic acid esterase (ChlE) of Bifidobacterium animalis subsp. Lactis, of Ustilago maydis, of Lactobacillus johnsonii or Salinibacter ruber and exhibiting caffeoyl-shikimate esterase activity.
8. Recombinant yeast according to any one of claims 1 to 7, characterized in that the enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A is a 4-coumarate-CoA ligase (4CL).
9. Recombinant yeast according to claim 8, characterized in that the heterologous gene coding for 4CL is the 4CL gene of Populus tomentosa, Arabidopsis thaliana, or Streptomyces coelicolor, preferably Populus tomentosa.
10. Recombinant yeast according to claims 8 or 9, characterized in that the heterologous gene coding for 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 compared to the parent gene coding for a non-mutated 4CL.
11. Recombinant yeast according to any one of claims 1 to 10, characterized in that said recombinant yeast further comprises: - A heterologous gene coding for a Hydroxycinnamoyl-Transferase (HCT); and - A heterologous gene coding for a Coumarate 3 Hydroxylase (C3H); and - A heterologous gene coding for a Tyrosine Amonia Lyase (TAL), and / or a heterologous gene coding for a Phenylalanine Amonia-Lyase (PAL) and a heterologous gene coding for a Cinnamate 4-Hydroxylase (C4H); and - A heterologous gene coding for a Cytochrome P450 reductase (CPR1).
12. Recombinant yeast according to any one of claims 1 to 11, characterized in that said recombinant yeast further comprises: - A gene coding for a 3-deoxy-7-phosphoheptulonate synthase (ARO4) mutated so that the product is resistant to feedback inhibition compared to the parent gene; and / or - A gene coding for a chorismate mutase (ARO7) mutated so that the product is resistant to feedback inhibition compared to the parent gene; and / or the invalidation of: - a gene coding for a Phenylpyruvate decarboxylase (ARO10).
13. Recombinant yeast capable of producing ferulic acid from of caffeic acid, comprising a heterologous gene coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate; or recombinant yeast capable of producing ferulic acid from the caffeic acid obtained according to any one of claims 1 to 12; characterized in that it further comprises: - A heterologous gene coding for a caffeoyl-O-methyl Transferase (COMT).
14. Recombinant yeast according to claim 13, characterized in that it further comprises the invalidation of the gene coding for a ferulic acid decarboxylase 1 (FDC1)
15. Recombinant yeast according to any one of claims 1 to 14, characterized in that said yeast is a species of the Ascomycota phylum, preferably chosen from the genera Schizosaccharomycetes, Saccharomyces, Kluyveromyces, Komagataella, Scheffersomyces, Torulaspora and / or Zygosaccharomyces, even more preferably from the species Saccharomyces cerevisiae.
16. A method for producing caffeic acid and / or ferulic acid, comprising a step of: a. Culturing a recombinant yeast capable of producing caffeic acid, said yeast comprising a heterologous gene coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate, or culturing the recombinant yeasts capable of producing caffeic acid as defined in any one of claims 1 to 15 in a culture medium, or a'. Culturing the recombinant yeasts capable of producing ferulic acid from the obtained caffeic acid as defined in any one of claims 13 to 15 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'.
17. Production method according to claim 16, characterized in that caffeic acid and / or ferulic acid are produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and / or caffeoyl-shikimate, added to the culture medium before or at step a.
18. Use of a recombinant yeast capable of producing caffeic acid, said yeast comprising a heterologous gene coding for an enzyme of the hydrolase family capable of breaking, preferably hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate, or of the recombinant yeast according to any one of claims 1 to 15 to produce caffeic acid.
19. Use of recombinant yeast capable of producing ferulic acid from caffeic acid obtained according to any one of claims 13 to 15 for producing caffeic acid and / or ferulic acid.