RECOMBINANT YEAST CAPABLE OF PRODUCING CAFFEIC ACID AND/OR FERULIC ACID

Abstract

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 of 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 microorganisms, preferably yeasts, according to the invention. Finally, the invention also relates to the use of microorganisms, preferably yeasts, according to the invention to produce caffeic acid and/or ferulic acid.

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 of hydrolyzing, the caffeoyl-shikimate bond to produce caffeic acid from caffeoyl-shikimate.

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 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 the caffeoyl-shikimate esterase (CSE) of Medicago truncatula or Arabidopsis thaliana and exhibiting 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 (ChIE).

6. Recombinant yeast according to claim 5, characterized in that the heterologous gene coding for chlorogenic acid esterase (ChIE) is the ChIE gene of Bifidobacterium animalis subsp. Lactis, Ustilago maydis, Lactobacillus johnsonii or Salinibacter ruber, preferably Lactobacillus johnsonii.

7. Recombinant yeast according to claim 6, characterized in that the ChIE is chosen from SEQ ID No. 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 the chlorogenic acid esterase (ChIE) of Bifidobacterium animalis subsp. Lactis, Ustilago maydis, Lactobacillus johnsonii or Salinibacter ruber and exhibiting caffeoyl-shikimate esterase activity.

8. Recombinant yeast according to claim 1, characterized in that said recombinant yeast further comprises: A heterologous gene coding for an enzyme capable of catalyzing the formation of the bond between coumaric acid and coenzyme A.

9. Recombinant yeast according to claim 8, 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).

10. Recombinant yeast according to claim 9, characterized in that the heterologous gene coding for the 4CL is the 4CL gene of Populus tomentosa, Arabidopsis thaliana, or Streptomyces coelicolor, preferably Populus tomentosa.

11. Recombinant yeast according to claim 9, characterized in that 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 an unmutated 4CL.

12. Recombinant yeast according to claim 1, 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 Ammonia Lyase (TAL), and/or a heterologous gene coding for a Phenylalanine Ammonia-Lyase (PAL) and a heterologous gene coding for a Cinnamate 4-Hydroxylase (C4H); and A heterologous gene coding for a Cytochrome P450 reductase (CPR1).

13. Recombinant yeast according to claim 1, 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 invalidation of: a gene coding for a Phenylpyruvate decarboxylase (ARO10).

14. Recombinant yeast according to claim 1, capable of producing ferulic acid from the caffeic acid obtained, characterized in that it further comprises: A heterologous gene coding for a caffeoyl-O-methyl Transferase (COMT).

15. Recombinant yeast according to claim 14, characterized in that it further comprises the invalidation of the gene coding for a ferulic acid decarboxylase 1 (FDC1).

16. Recombinant yeast according to claim 1, 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, and still more preferably from the species Saccharomyces cerevisiae.

17. Method for producing caffeic acid and/or ferulic acid, comprising a step of: a. Cultivating recombinant yeasts capable of producing caffeic acid as defined in claim 1 in a culture medium, or of a′. Cultivating recombinant yeasts capable of producing ferulic acid from the caffeic acid obtained in a culture medium; step a or a′ preferably being followed by a step of: b. Recovering the caffeic acid and/or the ferulic acid obtained in step a. or a′.

18. Production method according to claim 17, characterized in that the caffeic acid and/or the ferulic acid are produced from glucose, p-coumaric acid, p-coumaroyl-shikimate and/or caffeoyl-shikimate, added in the culture medium before or in step a.

19. Use of the recombinant yeast according to claim 1 to produce caffeic acid.

20. Use of the recombinant yeast capable of producing ferulic acid from the caffeic acid obtained according to claim 14 to produce caffeic acid and/or ferulic acid.

Description

DESCRIPTION OF THE FIGURES

[0275] FIG. 1 illustrates the hydrolysis reaction of caffeoyl-shikimate to caffeic acid and shikimate using caffeoyl-shikimate esterase (CSE) according to the invention.

[0276] FIG. 2 illustrates the metabolic pathways for the production of caffeic acid and ferulic acid in yeast according to one embodiment of the invention.

[0277] FIG. 3 illustrates the production of caffeic acid in a recombinant yeast according to one embodiment of the invention from p-coumaric or glucose via a CSE (UHPLC-TQ method), at 24 h, 48 h and 72 h.

[0278] FIG. 4 illustrates the production of the various intermediates produced by the recombinant yeast according to one embodiment of the invention, analyzed by a qualitative method (UHPLC-HRMS method (high resolution mass spectrometry)).

[0279] FIG. 5 illustrates the analysis of the compounds present in the recombinant yeast culture supernatant 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.

[0280] FIG. 6 is a chromatograph characterizing the production of ferulic acid in recombinant yeast according to one 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.

[0281] FIG. 7 is a chromatogram characterizing the hydrolysis of caffeoyl-shikimate into caffeic acid and shikimate using 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.

[0282] FIG. 8 is a chromatogram characterizing the hydrolysis of caffeoyl-shikimate into caffeic acid and shikimate owing to ChIE (from Lactobacillus johnsonii, LaChIE) in recombinant yeast according to one embodiment of the invention. The peak with a retention time of 3.19 min corresponds to caffeic acid.

[0283] FIG. 9 is a chromatogram characterizing the presence of caffeoyl-shikimate in the control samples. The peak with a retention time of 3.88 min corresponds to caffeoyl-shikimate.

EXAMPLES

Example 1: Materials and Methods

[0284] The standards for p-coumaric acid, caffeic acid and ferulic acid were obtained from the supplier Sigma-Aldrich.

[0285] Gene Cloning:

[0286] 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 G141S mutation.

[0287] The genes obtained by synthesis or PCR comprise, at the 5′ and 3′ end, a Bbs I (GAAGAC) or Bsa I (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.

[0288] The pSBK vector includes a yeast selection marker: URA3, LEU2 or TRP1.

TABLE-US-00001 TABLE 1 The various genes used to produce a recombinant yeast according to the invention. Accession Gene number, bank Origin TAL (Tyrosine Ammonia Lyase) KF765779.1, Rhodotorula glutinis GenBank, SEQ ID NO. 14 4CL (4 Coumarate-CoA Ligase) AY043495, Populus tomentosa Genbank, SEQ ID NO. 10 HCT (Hydroxycinnamoyl-Transferase) At5g48930, Arabidopsis thaliana GenBank, SEQ ID NO. 7 C3H (Coumarate 3 Hydroxylase) At2g40890, Arabidopsis thaliana GenBank, SEQ ID NO. 5 MtCSE (Caffeoyl-Shikimate Esterase) XM_003609990.3, Medicago truncatula Genbank, SEQ ID NO. 9 AtCSE At1g52760, Arabidopsis thaliana GenBank, SEQ ID NO. 3 COMT (Caffeoyl-O-Methyl Transferase) At5g54160, Arabidopsis thaliana GenBank, SEQ ID NO. 2 CPR1 (Cytochrome P450 reductase) X69791.1, Catharanthus roseus GenBank, SEQ ID NO. 6 SAM2 (S-Adenosylmethyltransferase) YDR502C, SGD Saccharomyces cerevisiae Database, SEQ ID NO. 11 Mutation of 4CL: P. tomentosa 4CL mutants Y236A and Y236F were constructed by PCR.

[0289] Deletion of Genes:

[0290] The ARO10 (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 regions upstream and downstream of the gene.

[0291] Strains:

[0292] The yeast model used in this study is the FY1679-28A strain of Saccharomyces cerevisiae (Tettelin et al., 1995—table 1 page 85), auxotrophic for uracil, tryptophan and leucine. The constructions were produced in the strain of Escherichia coli MH1 before their transfer to the yeast.

TABLE-US-00002 TABLE 2 List of strains used Name Constructions Markers L16B5 ARO4.sup.K229L-ARO7.sup.G141S -TAL URA3 4CL.sup.Y236A-HCT-C3H TRP1 MtCSE-CPR1 LEU2 L16D5 ARO4.sup.K229L- ARO7.sup.G141S -TAL URA3 4CL.sup.Y236F-HCT-C3H TRP1 MtCSE-CPR1 LEU2 L16A3 ARO4.sup.K229L -ARO7.sup.G141S -TAL URA3 4CL.sup.Y236A-HCT-C3H TRP1 AtCSE-CPR1 LEU2 L16C2 ARO4.sup.K229L -ARO7.sup.G141S-TAL URA3 4CL.sup.Y236F-HCT-C3H TRP1 AtCSE-CPR1 LEU2 L93-2D3 fdc1Δ::(ARO4.sup.K229L - HPH ARO7.sup.G141S -TAL)~HPH (hygromycin) 4CL.sup.Y236A-HCT-C3H TRP1 MtCSE-COMT- CPR1-SAM2 URA3 All strains listed in this table have been invalidated for the ARO10 gene.

[0293] Cultivation Conditions:

[0294] The yeast strains were cultured for 72 h 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 (Complete 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.Math.l-1.

[0295] Analytical method: UHPLC-TQ method:

[0296] Sample preparation: Samples of 100 μL are collected for each experiment. 50 μL is transferred to a new plate, to which 50 μL of the internal standard solution is added. Each sample is then homogenized by suction-discharge, then centrifuged for 5 min at 3000 rpm at room temperature. The final concentration of the internal standard (Protocatechuic Acid) is 0.5 mg/L.

[0297] 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 μm 2.1×100 mm) associated with an HSST3 1.8 μm 2.1×5 mm pre-column.

[0298] 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 column temperature is 50° C. and the autosampler temperature is 10° C.

TABLE-US-00003 TABLE 3 Chromatographic conditions for the detection of molecules of interest: Mobile phase A Mobile phase B Time (min) Flow (mL/min) (%) (%) 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

[0299] The parameters of the electrospray source are: [0300] positive mode spray voltage at 4000 V [0301] curtain gas: at 50 Arb [0302] auxiliary gas at 15 Arb [0303] temperature of the transfer tube at 300° C. [0304] vaporizer temperature at 300° C.

TABLE-US-00004 TABLE 4 Ions monitored and fragmentation conditions for the molecules of interest: Retention Precursor Daughter Collision RF lens Molecules time (min) Polarity ion ion energy (V) P-Coumaric 2.21 Negative 162.9 119.054 14.55 87 Acid 93 31.15 87 Trans-ferulic 2.67 Negative 192.95 149.06 11.33 93 Acid 178.018 12.46 93 Caffeic 2.69 Negative 178.9 135 15.31 91 Acid 107.071 21.34 91

[0305] UHPLC-HRMS method (High Resolution Mass Spectrometry):

[0306] Sample preparation: Samples of 100 μL are collected for each experiment. 50 μL is transferred to a new plate, to which 50 μL of acetonitrile is added. Each sample is then homogenized by suction-discharge, then centrifuged for 5 min at 3000 rpm at room temperature.

[0307] Analysis by UHPLC-HRMS: The samples were analyzed by a UHPLC Vanquish-H (Thermo) coupled to a UHPLC-HRMS. The column is a Waters Acquity UPLC® USST3 column (8 μm 2.1×100 mm) associated with an HSST3 1.8 μm 2.1×5 mm pre-column.

[0308] 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 column temperature is 50° C. and the autosampler temperature is 10° C.

TABLE-US-00005 TABLE 5 Chromatographic conditions for the detection of molecules of interest: Mobile phase A Mobile phase B Time (min) Flow (mL/min) (%) (%) 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

[0309] The parameters of the electrospray source are: [0310] positive mode spray voltage at 3.10 kV [0311] curtain gas at 50 Arb [0312] auxiliary gas at 20 Arb [0313] capillary temperature at 350° C. [0314] auxiliary gas heating temperature at 500° C. [0315] S-lens RF at 55 V [0316] collision energy (NCE in ramp): 20, 40, 60

TABLE-US-00006 TABLE 6 Tracked ions and fragmentation conditions for molecules of interest: Retention Molecules time (min) Polarity Precursor ion Fragments 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

Example 2: Production of Caffeic Acid from Glucose or p-Coumaric Acid

[0317] 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 the mutated 4CLs (either Y236A or Y236F) and the CSEs used (A. thaliana or M. truncatula).

[0318] FIG. 3 describes the results of the production of caffeic acid from p-coumaric or glucose by passing through a 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).

[0319] The results presented in FIG. 3 show that the combination of these different enzymes allows the production of caffeic acid.

[0320] The best condition is obtained with the 4CL Y236A mutation and the CSE of M. truncatula (L16B5).

Example 3: Production of the Various Intermediaries

[0321] The results presented in FIG. 4 show the characterization of the production of the various 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).

[0322] These results show the accumulation of each of the intermediates, i.e. p-coumaric acid, p-coumaroyl-shikimate, caffeoyl-shikimate and caffeic acid (see FIG. 4).

[0323] The accumulation of the various intermediates demonstrates the possibility of producing caffeic acid owing to the yeast according to the invention, as indicated by the presence of p-coumaroyl-shikimate and caffeoyl-shikimate.

Example 4: Production of Ferulic Acid from p-Coumaric Acid

[0324] The yeast according to the invention capable of producing ferulic acid possesses the methytransferase of Arabidopsis thaliana (COMT) and is invalidated for the FDC1 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.

[0325] The chromatogram shows 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.

[0326] 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).

Example 5: Production of Ferulic Acid from Glucose

[0327] The yeast according to the invention capable of producing ferulic acid possesses the methytransferase of Arabidopsis thaliana (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.

[0328] The chromatogram shows 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.

[0329] 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).

Example 6: Test of the Hydrolysis of Caffeoyl-Shikimate into Caffeic Acid and Shikimate Using CSE

[0330] 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).

[0331] 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.

[0332] A production of caffeic acid from caffeoyl-shikimate in the presence of MtCSE is observed.

Example 7: Test of the Hydrolysis of Caffeoyl-Shikimate into Caffeic Acid and Shikimate Using ChLE

[0333] 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 ChIE-containing strain of Lactobacillus johnsonii.

[0334] The results are shown 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, it is possible to verify the presence of caffeoyl-shikimate, for which a peak is observed at 3.88 min, and the absence of caffeic acid.

[0335] A production of caffeic acid from caffeoyl-shikimate is observed in the presence of LaChIE.