Method for biosynthesising diosmetin and/or hesperetin in a microorganism

Abstract

The present invention relates to a recombinant microorganism which is modified to be capable of producing diosmetin and/or hesperetin and to the use thereof for producing diosmetin and/or hesperetin.

Claims

1. A recombinant microorganism comprising a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT) which methylates eriodictyol and/or luteolin in position 4, wherein the O-methyltransferase (OMT) is selected from an enzyme comprising a sequence selected from the group consisting of SEQ ID NOs: 87, 89, 91 and 93 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 87, 89, 91 and 93 and having O-methyltransferase activity, and wherein the microorganism is a yeast.

2. The microorganism as claimed in claim 1, wherein said microorganism comprises a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT) which methylates eriodictyol and/or luteolin in position 4, said O-methyltransferase (OMT) being selected from an enzyme comprising a sequence selected from the group consisting of SEQ ID NOs: 87, 89, 91 and 93 or comprising a sequence having at least 95% identity with a sequence selected from the group consisting of SEQ ID NOs: 87, 89, 91 and 93 and having O-methyltransferase activity.

3. The microorganism as claimed in claim 1, wherein the microorganism also comprises a heterologous or endogenous nucleic acid sequence coding for an S-adenosylmethionine synthetase (SAMT).

4. The microorganism as claimed in claim 1, wherein the microorganism also comprises an endogenous or heterologous nucleic acid sequence coding for a flavone synthase (FNS).

5. The microorganism as claimed in claim 4, wherein the flavone synthase (FNS) is selected from enzymes comprising a sequence selected from the group consisting of SEQ ID NOs: 37, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 37, 33, 35, 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133 and having flavone synthase activity.

6. The microorganism as claimed in claim 1, wherein the microorganism also comprises: a heterologous nucleic acid sequence coding for a tyrosine ammonia lyase (TAL); a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL); a heterologous nucleic acid sequence coding for a chalcone synthase (CHS); and a heterologous nucleic acid sequence coding for a chalcone isomerase (CHI).

7. The microorganism as claimed in claim 6, wherein the microorganism comprises: a heterologous nucleic acid sequence coding for a tyrosine ammonia lyase (TAL) comprising a sequence selected from the group consisting of SEQ ID NOs: 41 and 39 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 41 and 39 and having tyrosine ammonia lyase activity; a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL) comprising a sequence selected from the group consisting of SEQ ID NOs: 97, 99, 43, 45, 47 and 49 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 97, 99, 43, 45, 47 and 49 and having 4-coumarate-CoA ligase activity; a heterologous nucleic acid sequence coding for a chalcone synthase (CHS) comprising a sequence selected from the group consisting of chosen from SEQ ID NOs: 53, 51, 55 and 57 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 53, 51, 55 and 57 and having chalcone synthase activity; and a heterologous nucleic acid sequence coding for a chalcone isomerase (CHI) comprising a sequence selected from the group consisting of SEQ ID NOs: 61 and 59 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of 61 and 59 and having chalcone isomerase activity.

8. The microorganism as claimed in claim 7, wherein the microorganism comprises: a heterologous nucleic acid sequence coding for a tyrosine ammonia lyase (TAL) comprising SEQ ID NO: 41 or a TAL comprising a sequence having at least 90% identity with SEQ ID NO: 41 and having tyrosine ammonia lyase activity; and a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL) comprising SEQ ID NO: 45 or a 4CL comprising a sequence having at least 90% identity with SEQ ID NO: 45 and having 4-coumarate-CoA ligase activity; a heterologous nucleic acid sequence coding for a chalcone synthase (CHS) comprising SEQ ID NO: 53 or a CHS comprising a sequence having at least 90% identity with SEQ ID NO: 53 and having chalcone synthase activity; and a heterologous nucleic acid sequence coding for a chalcone isomerase (CHI) comprising SEQ ID NO: 61 or a CHI comprising a sequence having at least 90% identity with SEQ ID NO: 61 and having chalcone isomerase activity.

9. The microorganism as claimed in claim 1, wherein the microorganism also comprises a heterologous nucleic acid sequence coding for a flavonoid 3-monooxygenase (F3H) comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 or comprising a sequence having at least 90% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 and having flavonoid 3-monooxygenase activity.

10. The microorganism as claimed in claim 9, wherein the microorganism comprises a heterologous nucleic acid sequence coding for a flavonoid 3-monooxygenase (F3H) comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 or comprising a sequence having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 and having flavonoid 3-monooxygenase activity.

11. The microorganism as claimed in claim 1, wherein the microorganism also comprises a heterologous or endogenous nucleic acid sequence coding for a cytochrome P450 reductase (CPR).

12. The microorganism as claimed in claim 11, wherein the microorganism comprises a heterologous nucleic acid sequence coding for a cytochrome P450 reductase (CPR) comprising a sequence selected from the group consisting of SEQ ID NOs: 25, 23, 27, 29 and 31 or a CPR comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 25, 23, 27, 29 and 31 and having cytochrome P450 reductase activity.

13. The microorganism as claimed in claim 1, wherein the microorganism also comprises a heterologous nucleic acid sequence coding for a 4-methoxybenzoate O-demethylase which converts tyrosine into L-DOPA and also p-coumaric acid into caffeic acid, comprising a sequence selected from the group consisting of SEQ ID NOs: 73 and 75 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 73 and 75 and having L-tyrosine hydroxylase activity; or a heterologous nucleic acid sequence coding for a p-coumarate 3-hydroxylase which converts p coumaric acid into caffeic acid, comprising SEQ ID NO: 71 or a sequence having at least 90% identity with SEQ ID NO: 71 and having p-coumarate 3-hydroxylase activity.

14. The microorganism as claimed in claim 1, wherein the microorganism also comprises: a heterologous nucleic acid sequence coding for a phenylalanine ammonia lyase (PAL), comprising a sequence selected from the group consisting of SEQ ID NOs: 63, 65 and 77 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 63, 65 and 77 and having phenylalanine ammonia lyase activity; and a heterologous nucleic acid sequence coding for a cinnamate 4-hydroxylase (C4H) comprising a sequence selected from the group consisting of SEQ ID NOs: 67, 69 and 79 or comprising a sequence having at least 90% identity with a sequence selected from the group consisting of SEQ ID NOs: 67, 69 and 79 and having cinnamate 4-hydroxylase activity.

15. The microorganism as claimed in claim 1, wherein the microorganism comprises: a heterologous nucleic acid sequence coding for a phenylalanine ammonia lyase (PAL) comprising SEQ ID NO: 65 or a PAL comprising a sequence having at least 90% identity with SEQ ID NO: 65 and having phenylalanine ammonia lyase activity; a heterologous nucleic acid sequence coding for a cinnamate 4-hydroxylase (C4H), comprising SEQ ID NO: 79 or a C4H comprising a sequence having at least 90% identity with SEQ ID NO: 79 and having cinnamate 4-hydroxylase activity; a heterologous nucleic acid sequence coding for a tyrosine ammonia lyase (TAL) comprising SEQ ID NO: 41 or a TAL comprising a sequence having at least 90% identity with SEQ ID NO: 41 and having tyrosine ammonia lyase activity; a heterologous nucleic acid sequence coding for a 4-coumaroyl-CoA ligase (4CL) comprising SEQ ID NO: 45 or 97 or a 4CL comprising a sequence having at least 90% identity with SEQ ID NO: 45 or 97 and having 4-coumarate-CoA ligase activity; a heterologous nucleic acid sequence coding for a chalcone synthase (CHS) comprising SEQ ID NO: 53 or a CHS comprising a sequence having at least 90% identity with SEQ ID NO: 53 and having chalcone synthase activity; a heterologous nucleic acid sequence coding for a chalcone isomerase (CHI) comprising SEQ ID NO: 61 or a CHI comprising a sequence having at least 90% identity with SEQ ID NO: 61 and having chalcone isomerase activity; a heterologous nucleic acid sequence coding for a flavonoid 3-monooxygenase (F3H) comprising SEQ ID NO: 7 or a F3H comprising a sequence having at least 90% identity with SEQ ID NO: 7 and having flavonoid 3-monooxygenase activity; a heterologous nucleic acid sequence coding for a flavone synthase (FNS) comprising SEQ ID NO: 37 or a FNS comprising a sequence having at least 90% identity with SEQ ID NO: 37 and having flavone synthase activity; and a heterologous nucleic acid sequence coding for a cytochrome P450 reductase (CPR) comprising SEQ ID NO: 25 or a CPR comprising a sequence having at least 90% identity with SEQ ID NO: 25 and having cytochrome P450 reductase activity; and a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT) comprising SEQ ID NO: 91 or 93 or an OMT comprising a sequence having at least 90% identity with SEQ ID NO: 91 or 93 and having O-methyltransferase activity.

16. The microorganism as claimed in claim 1, wherein the microorganism is a yeast of the genus Saccharomyces.

17. A method for producing diosmetin and/or hesperetin, comprising the cultivation of a microorganism as claimed in claim 1 and optionally the harvesting of the diosmetin and/or hesperetin.

18. The method as claimed in claim 17, wherein no naringenin, apigenin, eriodictyol and/or luteolin is supplied to the culture medium.

19. The microorganism as claimed in claim 2, wherein the microorganism also comprises a heterologous nucleic acid sequence coding for a flavonoid 3-monooxygenase (F3H) comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 or comprising a sequence having at least 95% sequence identity with a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 95 and having flavonoid 3-monooxygenase activity.

Description

DESCRIPTION OF THE FIGURES

(1) FIG. 1: Description of the metabolic pathways for producing hesperetin and diosmetin.

(2) FIG. 2: Production of eriodictyol from naringenin by the strain FL_405 (F3H4+CPR2). Control strain: CF235. Observation of disappearance of the naringenin peak and appearance of an eriodictyol peak in the strain FL-405.

(3) FIG. 3: Production of luteolin from apigenin by the strain FL_405 (F3H4+CPR2). Control strain: CF235. Observation of disappearance of the apigenin peak and appearance of a luteolin peak in the strain FL-405.

(4) FIG. 4: Production of apigenin from naringenin by the strain SC744 (FNSII1+CPR2). Control strain: CF234. Observation of disappearance of the naringenin peak and appearance of an apigenin peak in the strain.

(5) FIG. 5: Production of luteolin from eriodictyol SC744 (FNSII1+CPR2). Control strain: CF234. Observation of disappearance of the eriodictyol peak and appearance of a luteolin peak in the strain.

(6) FIG. 6: Production of eriodictyol and luteolin by the strain SC1500. Control strain: CF237. Observation of the eriodictyol and luteolin peaks.

(7) FIG. 7: Production of hesperetin from eriodictyol by the strains SC 1612 (MET+SAM) and SC 1614 (MET+SAM). Control strain: CF235. Observation of disappearance of the eriodictyol peak and appearance of a hesperetin peak in the strains.

(8) FIG. 8: Production of diosmetin from luteolin by the strains SC 1612 (MET+SAM) and SC 1614 (MET+SAM). Control strain: CF235. Observation of disappearance of the luteolin peak and appearance of a diosmetin peak in the strains.

(9) FIG. 9: Production of diosmetin from hesperetin by the strain SC744 (FNSII+CPR). Control strain: CF234. Observation of disappearance of the hesperetin peak and appearance of a diosmetin peak in the strain.

(10) FIG. 10: Production of hesperetin from eriodictyol by E. coli EC26 (MET+SAM). Control strain: E. coli MH1. Observation of disappearance of the eriodictyol peak and appearance of a hesperetin peak in the strain.

(11) FIG. 11: Production of diosmetin from luteolin by E. coli EC26 (MET+SAM). Control strain: E. coli MH1. Observation of disappearance of the luteolin peak and appearance of a diosmetin peak in the strain.

(12) FIG. 12: Production of diosmetin from hesperetin by E. coli EC30 (FNSII). Control strain: E. coli MH1. Observation of disappearance of the hesperetin peak and appearance of a diosmetin peak in the strain.

(13) FIG. 13: Production of hesperetin and diosmetin by the strain SC1508. Control strain: CF237. Observation of the hesperetin and diosmetin peaks.

(14) FIG. 14: Production of hesperetin from eriodictyol by E. coli EC41 (MET+SAM). Control strain: E. coli MH1. Observation of disappearance of the eriodictyol peak and appearance of a hesperetin peak in the strain.

(15) FIG. 15: Production of hesperetin from eriodictyol by E. coli EC43 (MET+SAM). Control strain: E. coli MH1. Observation of disappearance of the eriodictyol peak and appearance of a hesperetin peak in the strain.

(16) FIG. 16: Production of diosmetin from luteolin by E. coli EC43 (MET+SAM). Control strain: E. coli MH1. Observation of disappearance of the luteolin peak and appearance of a diosmetin peak in the strain.

(17) FIG. 17: Production of hesperetin and diosmetin by the strain SC2408. Control strain: CF237. Observation of the hesperetin and diosmetin peaks.

(18) FIG. 18: Production of hesperetin and diosmetin by the strain SC2409. Control strain: CF237. Observation of the hesperetin and diosmetin peaks.

(19) FIG. 19: Production of hesperetin and homoeriodictyol by the strains SC2147, SC2151, SC1612 and SC1614. Control strain: CF235.

(20) FIG. 20: Production of diosmetin and chrysoeriol by the strains SC2147, SC2151, SC1612 and SC1614. Control strain: CF235.

(21) FIG. 21: Production of hesperetin and diosmetin by the strains SC2408, SC2409 and SC1508. Control strain: CF237.

(22) FIG. 22: Production of eriodictyol and luteolin by the strains SC2424, SC2425, SC2426, SC2427, SC2428 and SC1500. Control strain: CF237.

(23) FIG. 23: Production of diosmetin from naringenin by the strains SC2429 to SC2434, SC2436 to SC2444, SC2446 to SC2454, SC2456 to SC2464 and SC2466.

(24) TABLE-US-00001 TABLE 1 SEQUENCE DESCRIPTION SEQ ID NO. Description 39 Amino acid sequence of tyrosine ammonia lyase from Flavobacterium johnsoniae 40 Nucleic acid sequence coding for tyrosine ammonia lyase from Flavobacterium johnsoniae 41 Amino acid sequence of tyrosine ammonia lyase from Rhodotorula glutinis 42 Nucleic acid sequence coding for tyrosine ammonia lyase from Rhodotorula glutinis 43 Amino acid sequence of 4-coumarate-CoA ligase from Arabidopsis thaliana 44 Nucleic acid sequence coding for 4-coumarate-CoA ligase from Arabidopsis thaliana 45 Amino acid sequence of 4-coumarate-CoA ligase from Petroselinum crispum 46 Nucleic acid sequence coding for 4-coumarate-CoA ligase from Petroselinum crispum 47 Amino acid sequence of 4-coumarate-CoA ligase from Petroselinum crispum 48 Nucleic acid sequence coding for 4-coumarate-CoA ligase from Petroselinum crispum 49 Amino acid sequence of 4-coumarate-CoA ligase from Streptomyces clavuligerus 50 Nucleic acid sequence coding for 4-coumarate-CoA ligase from Streptomyces clavuligerus 51 Amino acid sequence of chaicone synthase from Hordeum vulgare 52 Nucleic acid sequence coding for chaicone synthase from Hordeum vulgare 53 Amino acid sequence of chaicone synthase from Citrus sinensis 54 Nucleic acid sequence coding for chaicone synthase from Citrus sinensis 55 Amino acid sequence of chaicone synthase from Citrus sinensis 56 Nucleic acid sequence coding for chaicone synthase from Citrus sinensis 57 Amino acid sequence of chaicone synthase from Streptomyces clavuligerus 58 Nucleic acid sequence coding for chaicone synthase from Streptomyces clavuligerus 59 Amino acid sequence of chaicone isomerase from Streptomyces clavuligerus 60 Nucleic acid sequence coding for chaicone isomerase from Streptomyces clavuligerus 61 Amino acid sequence of chaicone isomerase from Arabidopsis thaliana 62 Nucleic acid sequence coding for chaicone isomerase from Arabidopsis thaliana 33 Amino acid sequence of flavone synthase from Lonicera japonica 34 Nucleic acid sequence coding for flavone synthase from Lonicera japonica 35 Amino acid sequence of flavone synthase from Lonicera macranthoides 36 Nucleic acid sequence coding for flavone synthase from Lonicera macranthoides 37 Amino acid sequence of flavone synthase from Petroselinum crispum 38 Nucleic acid sequence coding for flavone synthase from Petroselinum crispum 1 Amino acid sequence of flavonoid 3-monooxygenase from Perilla frutescens var. crispa 2 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Perilla frutescens var. crispa 3 Amino acid sequence of flavonoid 3-monooxygenase from Phanerochaete chrysosporium 4 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Phanerochaete chrysosporium 5 Amino acid sequence of flavonoid 3-monooxygenase from Petunia hybrida 6 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Petunia hybrida 7 Amino acid sequence of flavonoid 3-monooxygenase from Callistephus chinensis 8 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Callistephus chinensis 9 Amino acid sequence of flavonoid 3-monooxygenase from Callistephus chinensis 10 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Callistephus chinensis 11 Amino acid sequence of flavonoid 3-monooxygenase from Gerbera hybrida 12 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Gerbera hybrida 13 Amino acid sequence of flavonoid 3-monooxygenase from Osteospermum hybrid cultivar 14 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Osteospermum hybrid cultivar 15 Amino acid sequence of flavonoid 3-monooxygenase from Citrus Clementina 16 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Citrus Clementina 17 Amino acid sequence of flavonoid 3-monooxygenase from Citrus sinensis 18 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Citrus sinensis 19 Amino acid sequence of flavonoid 3-monooxygenase from Pilosella officinarum 20 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Pilosella officinarum 21 Amino acid sequence of flavonoid 3-monooxygenase from Streptomyces avermitilis 22 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Streptomyces avermitilis 23 Amino acid sequence of cytochrome P450 reductase from Catharanthus roseus 24 Nucleic acid sequence coding for cytochrome P450 reductase from Catharanthus roseus 25 Amino acid sequence of cytochrome P450 reductase from Saccharomyces cerevisiae 26 Nucleic acid sequence coding for cytochrome P450 reductase from Saccharomyces cerevisiae 27 Amino acid sequence of chimeric cytochrome P450 reductase 28 Nucleic acid sequence coding for chimeric cytochrome P450 reductase 29 Amino acid sequence of cytochrome P450 reductase from Arabidopsis thaliana 30 Nucleic acid sequence coding for cytochrome P450 reductase from Arabidopsis thaliana 31 Amino acid sequence of cytochrome P450 reductase from Arabidopsis thaliana 32 Nucleic acid sequence coding for cytochrome P450 reductase from Arabidopsis thaliana 63 Amino acid sequence of phenylalanine ammonia lyase from Citrus sinensis 64 Nucleic acid sequence coding for phenylalanine ammonia lyase from Citrus sinensis 65 Amino acid sequence of phenylalanine ammonia lyase from Citrus sinensis 66 Nucleic acid sequence coding for phenylalanine ammonia lyase from Citrus sinensis 67 Amino acid sequence of cinnamate 4-hydroxylase from Citrus sinensis 68 Nucleic acid sequence coding for cinnamate 4-hydroxylase from Citrus sinensis 69 Amino acid sequence of cinnamate 4-hydroxylase from Citrus sinensis 70 Nucleic acid sequence coding for cinnamate 4-hydroxylase from Citrus sinensis 71 Amino acid sequence of coumarate 3-hydroxylase from Saccharothrix espanaensis 72 Nucleic acid sequence coding for coumarate 3-hydroxylase from Saccharothrix espanaensis 73 Amino acid sequence of 4-methoxybenzoate O-demethylase from Beta vulgaris 74 Nucleic acid sequence coding for 4-methoxybenzoate O-demethylase from Beta vulgaris 75 Amino acid sequence of 4-methoxybenzoate O-demethylase from Rhodopseudomonas palustris 76 Nucleic acid sequence coding for 4-methoxybenzoate O-demethylase from Rhodopseudomonas palustris 77 Amino acid sequence of phenylalanine ammonia lyase from Arabidopsis thaliana 78 Nucleic acid sequence coding for phenylalanine ammonia lyase from Arabidopsis thaliana 79 Amino acid sequence of cinnamate 4-hydroxylase from Arabidopsis thaliana 80 Nucleic acid sequence coding for cinnamate 4-hydroxylase from Arabidopsis thaliana 81 Amino acid sequence of S-adenosylmethionine synthetase from Saccharomyces cerevisiae 82 Nucleic acid sequence coding for S-adenosylmethionine synthetase from Saccharomyces cerevisiae 83 Amino acid sequence of 4-hydroxyphenylacetate 3-monooxygenase oxygenase from Escherichia coli 84 Nucleic acid sequence coding for 4-hydroxyphenylacetate 3-monooxygenase oxygenase from Escherichia coli 85 Amino acid sequence of 4-hydroxyphenylacetate 3-monooxygenase reductase from Escherichia coli 86 Nucleic acid sequence coding for 4-hydroxyphenylacetate 3-monooxygenase reductase from Escherichia coli 87 Amino acid sequence of O-methyltransferase from Arabidopsis thaliana 88 Nucleic acid sequence coding for O-methyltransferase from Arabidopsis thaliana 89 Amino acid sequence of O-methyltransferase from Homo sapiens 90 Nucleic acid sequence coding for O-methyltransferase from Homo sapiens 91 Amino acid sequence of O-methyltransferase from Citrus Clementina 92 Nucleic acid sequence coding for O-methyltransferase from Citrus Clementina 93 Amino acid sequence of O-methyltransferase from Citrus sinensis 94 Nucleic acid sequence coding for O-methyltransferase from Citrus sinensis 95 Amino acid sequence of flavonoid 3-monooxygenase from Arabidopsis thaliana 96 Nucleic acid sequence coding for flavonoid 3-monooxygenase from Arabidopsis thaliana 97 Amino acid sequence of a 4-coumarate-CoA ligase from Arabidopsis thaliana 98 Nucleic acid sequence coding for a 4-coumarate-CoA ligase from Arabidopsis thaliana 99 Amino acid sequence of a 4-coumarate-CoA ligase from Citrus Clementina 100 Nucleic acid sequence coding for 4-coumarate-CoA ligase from Citrus Clementina 101 Amino acid sequence of flavone synthase from Angelica archangelica 102 Nucleic acid sequence coding for flavone synthase from Angelica archangelica 103 Amino acid sequence of flavone synthase from Cynara cardunculus var. scolymus 104 Nucleic acid sequence coding for flavone synthase from Cynara cardunculus var. scolymus 105 Amino acid sequence of flavone synthase from Perilla frutescens var. crispa 106 Nucleic acid sequence coding for flavone synthase from Perilla frutescens var. crispa 107 Amino acid sequence of flavone synthase from Dahlia pinnata 108 Nucleic acid sequence coding for flavone synthase from Dahlia pinnata 109 Amino acid sequence of flavone synthase from Callistephus chinensis 110 Nucleic acid sequence coding for flavone synthase from Callistephus chinensis 111 Amino acid sequence of flavone synthase from Apium graveolens 112 Nucleic acid sequence coding for flavone synthase from Apium graveolens 113 Amino acid sequence of flavone synthase from Medicago truncatula 114 Nucleic acid sequence coding for flavone synthase from Medicago truncatula 115 Amino acid sequence of flavone synthase from Cuminum cyminum 116 Nucleic acid sequence coding for flavone synthase from Cuminum cyminum 117 Amino acid sequence of flavone synthase from Aethusa cynapium 118 Nucleic acid sequence coding for flavone synthase from Aethusa cynapium 119 Amino acid sequence of flavone synthase from Conium maculatum 120 Nucleic acid sequence coding for flavone synthase from Conium maculatum 121 Amino acid sequence of flavone synthase from Camellia sinensis 122 Nucleic acid sequence coding for flavone synthase from Camellia sinensis 123 Amino acid sequence of flavone synthase from Saussurea medusa 124 Nucleic acid sequence coding for flavone synthase from Saussurea medusa 125 Amino acid sequence of flavone synthase from Plectranthus barbatus 126 Nucleic acid sequence coding for flavone synthase from Plectranthus barbatus 127 Amino acid sequence of flavone synthase from Scutellaria baicalensis 128 Nucleic acid sequence coding for flavone synthase from Scutellaria baicalensis 129 Amino acid sequence of flavone synthase from Dorcoceras hygrometricum 130 Nucleic acid sequence coding for flavone synthase from Dorcoceras hygrometricum 131 Amino acid sequence of flavone synthase from Antirrhinum majus 132 Nucleic acid sequence coding for flavone synthase from Antirrhinum majus 133 Amino acid sequence of flavone synthase from Erythranthe lewisii 134 Nucleic acid sequence coding for flavone synthase from Erythranthe lewisii

EXAMPLES

(25) Materials and Methods

(26) Strains

(27) The yeasts used in the examples were obtained from Saccharomyces cerevisiae FY1679-28A (Tettelin et al., 1995 https://doi.org/10.1016/S1067-2389(06)80008-7). This yeast is quadruply auxotrophic for uracil, tryptophan, histidine and leucine.

(28) The bacterial strains used in the examples were obtained from Escherichia coli MH1.

(29) Standards

(30) The standards were acquired from the supplier Extrasynthse, France (naringenin, apigenin, eriodictyol, luteolin, hesperetin and diosmetin).

(31) Gene Cloning

(32) The genes optimized to express in the yeast were synthesized by Eurofins Genomics, Ebersberg, Germany or Biomatik, Cambridge, Canada or Twist Biosciences, San Francisco, USA or DC Biosciences, Dundee, UK. By PCR, the gene cpr2 (SEQ ID NO: 26) from S. cerevisiae was amplified from the genomic DNA.

(33) The genes obtained by synthesis or by PCR comprise at the 5 and 3 ends a BbsI (GAAGAC) or BsaI (GGTCTC) restriction site.

(34) All the genes, promoters and terminators were restriction-cloned in the vector pSBK for expression in the yeast or in the vector pSB1K3 for expression in E. coli. The promoters and terminators (Wargner et al., 2015 DOI: 10.1016/j.fgb.2015.12.001) were recovered by PCR from the genomic DNA of the yeast S. cerevisiae or of E. coli.

(35) The vector pSBK comprises a URA or LEU or TRP or HIS selection marker for the yeast and the vector pSB1K3 comprises a kanamycin-resistance marker.

(36) Culture Conditions

(37) The strains were cultivated in 1 ml of minimum nitrogen base medium (Dutscher, Brumath, Fr) supplemented with glucose at 20 g/l for the yeasts and in 1 ml of M9 supplemented with glucose at 4 g.Math.l.sup.1 for E. coli in 24-well plates (Starlab, Orsay, Fr) at 30 C. for 72 hours with continuous stirring at 200 rpm. In certain cases, naringenin or apigenin was added at a concentration of 100 mg.Math.l.sup.1 to determine the activity of the F3Hs, naringenin or eriodictyol was added at a concentration of 100 mg.Math.l.sup.1 to determine the activity of the FNSIIs, eriodictyol or luteolin was added at a concentration of 100 mg.Math.l.sup.1 to determine the activity of the METs. Each strain was inoculated at an OD of 0.2 using a 24-hour preculture cultivated under the same conditions.

(38) Analytical Method:

(39) Preparation of the samples: The 1 mL cultures are frozen at 80 C. and then lyophilized for 12 hours at 0.10 mbar. The samples are then taken up in 1 mL of dimethyl sulfoxide (DMSO), stirred for 30 seconds at 1000 rpm and then centrifuged for 5 minutes at 3000 rpm at room temperature. After centrifugation, a known volume of supernatant is added to a known volume of a mixture of internal standards dissolved in methanol.

(40) The final concentrations of the internal standards are:

(41) TABLE-US-00002 Diosmin C13 0.5 mg/L Diosmetin C13 0.015 mg/L

(42) Analysis by UHPLC-TQ: The samples were analyzed using a Vanquish-H UHPLC machine (Thermo) coupled to a Quantis triple-quadrupole MS (Thermo). The column is a Waters Acquity UPLC@ USST3 column (8 m 2.1100 mm) combined with an HSST3 1.8 m 2.15 mm precolumn.

(43) The mobile phase A is a 0.1% solution of formic acid in LC/MS-grade water and the 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 temperature of the sample changer is 10 C.

(44) Two chromatographic conditions were used for detecting the flavonoids of interest:

(45) TABLE-US-00003 TABLE 2 Chromatographic conditions method 1 Flow rate Mobile Mobile Time (min) (ml/min) phase A (%) phase B (%) 0 0.5 73 27 8 0.5 73 27

(46) TABLE-US-00004 TABLE 3 Chromatographic conditions method 2 Flow rate Mobile Mobile Time (min) (ml/min) phase A (%) phase B (%) 0 0.5 83 17 3.75 0.5 83 17 4 0.5 73 27 8.5 0.5 73 27 11.0 0.5 50 50 13.0 0.5 0 100 13.5 0.5 83 17 15.0 0.5 83 17

(47) The ions monitored and the fragmentation conditions for the molecules of interest are:

(48) TABLE-US-00005 TABLE 4 For method 1 Reference Retention Precursor Daughter Collision Lens RF internal Molecules time (min) Polarity ion ion energy (V) standard Naringenin 3.3 Negative 271.0 119.0 27 169 Diosmetin 150.9 18 169 C13 Apigenin 3.5 Negative 269.0 117.1 35 201 Diosmetin 150.9 24 201 C13 Eriodictyol 1.9 Negative 287.1 135.1 26 147 Diosmetin 150.9 14 147 C13 Luteolin 2.1 Negative 285.0 133.0 34 213 Diosmetin C13

(49) TABLE-US-00006 TABLE 5 For method 2 Reference Retention Precursor Daughter Collision Lens RF internal Molecules time (min) Polarity ion ion energy (V) standard Naringenin 7.8 Negative 271.0 119.0 27 169 Diosmetin 150.9 18 169 C13 Apigenin 8.2 Negative 269.0 117.1 35 201 Diosmetin 150.9 24 201 C13 Eriodictyol 6.2 Negative 287.1 135.1 26 147 Diosmetin 150.9 14 147 C13 Luteolin 6.6 Negative 285.0 133.0 34 213 Diosmetin 150.9 25 213 C13 Hesperetin 8.7 Negative 301.0 164.0 24 169 Diosmetin 150.9 17 169 C13 Diosmetin 9.1 Negative 299.0 256.0 30 192 Diosmetin 284.1 21 192 C13
F3H

(50) Constructs for each of the F3Hs were made in a vector bearing the URA selection marker (Table 6). Constructs including each SAM2 and only one of the various CPRs were created in a vector bearing the LEU selection marker (Table 7). Two vectors including only the URA or LEU selection marker were also created as controls. The marker genes make it possible to detect and to select the cells that have incorporated the gene of interest.

(51) TABLE-US-00007 TABLE 6 List of the various F3H constructs tested Names Assembled genes Markers FL 23 F3H from Perilla frutescens var. crispa URA (SEQ ID NO: 2) FL 24 F3H from Phanerochaete chrysosporium URA (SEQ ID NO: 4) FL 25 F3H from Petunia hybrida (SEQ ID NO: 6) URA FL 26 F3H from Callistephus chinensis (SEQ ID NO: 8) URA FL 27 F3H from Callistephus chinensis (SEQ ID NO: 10) URA FL 28 F3H from Gerbera hybrida (SEQ ID NO: 12) URA FL 29 F3H from Osteospermum hybrid cultivar URA (SEQ ID NO: 14) FL 30 F3H from Citrus Clementina (SEQ ID NO: 16) URA FL 31 F3H from Citrus sinensis (SEQ ID NO: 18) URA FL 32 F3H from Pilosella officinarum (SEQ ID NO: 20) URA FL 1031 F3H from Arabidopsis thaliana (SEQ ID NO: 96) URA TT URA URA

(52) TABLE-US-00008 TABLE 7 List of constructs made with the various CPRs Names Assembled genes Markers FL 121 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 274 Chimeric CPR (SEQ ID NO: 28), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 275 ATR from Arabidopsis thaliana (SEQ ID NO: 30), LEU (ATR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 401 CPR from Saccharomyces cerevisiae (SEQ ID NO: 26), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 463 ATR from Arabidopsis thaliana (SEQ ID NO: 32), LEU (ATR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) TT LEU LEU

(53) Several strains were created with, respectively, all the F3Hs listed in Table 6 so that they could each be tested with the constructs of Table 7.

(54) These various assemblies make it possible to check the enzymatic activity of the F3Hs and also make it possible to determine the most efficient F3H-CPR pairs.

(55) For example, the strain FL 405 contains the constructs FL 26 and FL 401.

(56) The control strain (without the genes) containing the constructs TT URA and TT LEU is called CF235.

(57) FNSII

(58) For each of the following FNSIIs, constructs in a TRP vector were prepared (Table 8). The same vectors with the LEU selection marker each containing SAM2 and a different CPR were used to test the FNSIIs (Table 9).

(59) TABLE-US-00009 TABLE 8 Constructs including the various FNSIIs tested Names Assembled genes Markers FL 620 TAL from Rhodotorula glutinis (SEQ ID NO: 42), TRP (TAL + 4CL + CHS + 4CL from Petroselinum crispum (SEQ ID NO: 46), CHI + FNSII) CHS from Citrus sinensis (SEQ ID NO: 54), CHI from Arabidopsis thaliana (SEQ ID NO: 62) FNSII from Lonicera japonica (SEQ ID NO: 34) FL 621 TAL from Rhodotorula glutinis (SEQ ID NO: 42), TRP (TAL + 4CL + CHS + 4CL from Petroselinum crispum (SEQ ID NO: 46), CHI + FNSII) CHS from Citrus sinensis (SEQ ID NO: 54), CHI from Arabidopsis thaliana (SEQ ID NO: 62) FNSII from Lonicera macranthoides (SEQ ID NO: 36) FL 112 TAL from Flavobacetrium jonhsoniae (SEQ ID NO: 40), TRP (TAL + 4CL + CHS + 4CL from Petroselinum crispum (SEQ ID NO: 46), CHI + FNSII) CHS from Citrus sinensis (SEQ ID NO: 54), CHI from Arabidopsis thaliana (SEQ ID NO: 62) FNSII from Petroselinum crispum (SEQ ID NO: 38) TT TRP TRP

(60) TABLE-US-00010 TABLE 9 List of constructs made with the various CPRs Names Assembled genes Markers FL 121 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 274 Chimeric CPR (SEQ ID NO: 28), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 275 ATR from Arabidopsis thaliana (SEQ ID NO: 30), LEU (ATR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 401 CPR from Saccharomyces cerevisiae (SEQ ID NO: 26), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 463 ATR from Arabidopsis thaliana (SEQ ID NO: 32), LEU (ATR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) TT LEU LEU

(61) Several strains were created with, respectively, each of the constructs of the FNSIIs listed in Table 8 and each of the constructs of the CPRs of Table 9.

(62) These various assemblies make it possible to check the enzymatic activity of the FNSIIs and also make it possible to determine the most efficient FNSIIs.

(63) For example, the strain SC 744 contains the constructs FL 620 and FL 401.

(64) The control strain (without the genes) containing the constructs TT TRP and TT LEU is called CF234.

(65) Similar constructs were made to test the FNSIIs of SEQ ID NOs: 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.

(66) Yeast Up to Eriodictyol/Luteolin

(67) Strains including the pathway up to eriodictyol and luteolin were also tested: the strain SC1500 comprises the constructs FL 26, FL 602, FL 808 and FL 822; and the strain SC2424 comprising the constructs FL 1031+FL 602+FL 822+TT HIS; the strain SC2425 comprising the constructs FL 26+FL 602+FL 822+TT HIS; the strain SC2426 comprising the constructs FL 31+FL 602+FL 822+TT HIS; the strain SC2427 comprises the constructs FL 1031, FL 602, FL 808 and FL 822; and the strain SC2428 comprising the constructs FL 31+FL 602+FL 808+FL 822.

(68) TABLE-US-00011 TABLE 10 Lists of constructs used for the strains including the pathway up to eriodictyol and luteolin Names Assembled genes Markers FL 26 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H) FL 1031 F3H from Arabidopsis thaliana (SEQ ID NO: 96) URA (F3H) FL 31 (F3H) F3H from Citrus sinensis (SEQ ID NO: 18) URA FL 602 TAL from Rhodotorula glutinis (SEQ ID NO: 42), TRP (TAL + 4CL + CHS + 4CL from Petroselinum crispum (SEQ ID NO: 46), CHI + FNS) CHS from Citrus sinensis (SEQ ID NO: 54), CHI from Arabidopsis thaliana (SEQ ID NO: 62) FNSII from Petroselinum crispum (SEQ ID NO: 38) FL 808 PAL from Arabidopsis thaliana (SEQ ID NO: 78), HIS (PAL + C4H) C4H from Arabidopsis thaliana (SEQ ID NO: 80), FL 822 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + CAF) CAF from Rhodopseudomonas palustris (SEQ ID NO: 76) TT HIS HIS

(69) The control strain (without the genes) containing the constructs TT URA, TT TRP, TT HIS and TT LEU is called CF237.

(70) MET:

(71) In order to test each of the METs, constructs were made and are presented in Table 11. The marker genes make it possible to detect and to select the cells that have incorporated the gene of interest.

(72) TABLE-US-00012 TABLE 11 List of constructs made to test the various METs Names Assembled genes Markers FL 121 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 266 F3H from Callistephus chinensis (SEQ ID NO: 8), URA (F3H + MET) MET from Arabidopsis thaliana (SEQ ID NO: 88) FL 268 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H + MET) MET from Homo sapiens (SEQ ID NO: 90) FL 469 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 475 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H + MET) MET from Citrus sinensis (SEQ ID NO: 94)

(73) Four strains SC 1612, SC 1614, SC 2147 and SC 2151 were created, with FL 121 and FL 266 for SC 1612, FL 121 and FL 268 for SC 1614, FL 475 and FL 121 for SC 2147 and FL 469 and FL 121 for SC 2151 for the conversion of eriodictyol into hesperetin in order to determine which MET is the most efficient.

(74) The control strain (without the genes) containing the constructs TT LEU and TT URA is called CF235.

(75) F3H, MET, FNS, CPR: Production of Diosmetin from Naringenin

(76) TABLE-US-00013 TABLE 12 List of constructs used to test the enzymes in Saccharomyces cerevisiae (SC) Names Assembled genes Markers FL 121 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) TT LEU LEU FL_26 F3H from Callistephus chinensis (SEQ ID NO: 8), URA (F3H) FL 1031 F3H from Arabidopsis thaliana (SEQ ID NO: 96), URA (F3H) FL 1111 FNSII from Petroselinum crispum (SEQ ID NO: 33) TRP (FNS + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 1112 FNSII from Angelica archangelica (SEQ ID NO: 102) TRP (FNS + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 1113 FNSII from Cynara cardunculus var. scolymus (SEQ ID NO: 104) TRP (FNS + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 1114 FNSII from Perilla frutescens var. crispa (SEQ ID NO: 106) TRP (FNS + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 1115 FNSII from Dahlia pinnata (SEQ ID NO: 108) TRP (FNS + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 1116 FNSII from Petroselinum crispum (SEQ ID NO: 33) TRP (FNS + MET) MET from Citrus sinensis (SEQ ID NO: 94) FL 1118 FNSII from Cynara cardunculus var. scolymus (SEQ ID NO: 104) TRP (FNS + MET) MET from Citrus sinensis (SEQ ID NO: 94) FL 1119 FNSII from Perilla frutescens var. crispa (SEQ ID NO: 106) TRP (FNS + MET) MET from Citrus sinensis (SEQ ID NO: 94) FL 1120 FNSII from Dahlia pinnata (SEQ ID NO: 108) TRP (FNS + MET) MET from Citrus sinensis (SEQ ID NO: 94)

(77) The following strains were constructed: SC2429: FL 1111+FL 1031+FL 121 SC2443: FL 1115+FL 1031+TT LEU SC2430: FL 1112+FL 1031+FL 121 SC2434: FL 1116+FL 1031+FL 121 SC2431: FL 1113+FL 1031+FL 121 SC2436: FL 1118+FL 1031+FL 121 SC2432: FL 1114+FL 1031+FL 121 SC2437: FL 1119+FL 1031+FL 121 SC2433: FL 1115+FL 1031+FL 121 SC2438: FL 1120+FL 1031+FL 121 SC2439: FL 1111+FL 1031+TT LEU SC2444: FL 1116+FL 1031+TT LEU SC2440: FL 1112+FL 1031+TT LEU SC2446: FL 1118+FL 1031+TT LEU SC2441: FL 1113+FL 1031+TT LEU SC2447: FL 1119+FL 1031+TT LEU SC2449: FL 1111+FL 26+FL 121 SC2463: FL 1115+FL 26+TT LEU SC2450: FL 1112+FL 26+FL 121 SC2454: FL 1116+FL 26+FL 121 SC2451: FL 1113+FL 26+FL 121 SC2456: FL 1118+FL 26+FL 121 SC2452: FL 1114+FL 26+FL 121 SC2457: FL 1119+FL 26+FL 121 SC2453: FL 1115+FL 26+FL 121 SC2458: FL 1120+FL 26+FL 121 SC2459: FL 1111+FL 26+TT LEU SC2464: FL 1116+FL 26+TT LEU SC2460: FL 1112+FL 26+TT LEU SC2466: FL 1118+FL 26+TT LEU SC2461: FL 1113+FL 26+TT LEU SC2467: FL 1119+FL 26+TT LEU SC2462: FL 1114+FL 26+TT LEU SC2468: FL 1120+FL 26+TT LEU

(78) The control strain (without the genes) containing the constructs TT URA, TT TRP, TT HIS and TT LEU is called CF237.

(79) E. Coli Up to Hesperetin/Diosmetin

(80) TABLE-US-00014 TABLE 13 List of constructs used to test the enzymes in E. coli Names Assembled genes EC26 SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) (SAM + MET) MET from Homo sapiens (SEQ ID NO: 90) EC41 SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) (SAM + MET) MET from Citrus Clementina (SEQ ID NO: 92) EC43 SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) (SAM + MET) MET from Citrus sinensis (SEQ ID NO: 94) EC30 (FNSII) FNSII from Petroselinum crispum (SEQ ID NO: 38)
Yeast Up to Hesperetin/Diosmetin

(81) Three strains including the pathway up to hesperetin/diosmetin were also tested. The strain SC 1508 comprises the constructs FL 121+FL 268+FL 602+FL 808 of Table 14. The strain SC 2408 comprises the constructs FL 121+FL 469+FL 602+FL 808 of Table 14. The strain SC 2409 comprises the constructs FL 121+FL 475+FL 602+FL 808 of Table 14.

(82) TABLE-US-00015 TABLE 14 List of constructs used in the examples Names Assembled genes Markers FL 121 CPR from Catharanthus roseus (SEQ ID NO: 24), LEU (CPR + SAM) SAM from Saccharomyces cerevisiae (SEQ ID NO: 82) FL 268 F3H from Callistephus chinensis (SEQ ID NO: 8), URA (F3H + MET) MET from Homo sapiens (SEQ ID NO: 90) FL 469 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H + MET) MET from Citrus Clementina (SEQ ID NO: 92) FL 475 F3H from Callistephus chinensis (SEQ ID NO: 8) URA (F3H + MET) MET from Citrus sinensis (SEQ ID NO: 94) FL 602 TAL from Rhodotorula glutinis (SEQ ID NO: 42), TRP (TAL + 4CL + CHS + 4CL from Petroselinum crispum (SEQ ID NO: 46), CHI + FNSII) CHS from Citrus sinensis (SEQ ID NO: 54), CHI from Arabidopsis thaliana(SEQ ID NO: 62) FNSII from Petroselinum crispum (SEQ ID NO: 38) FL 808 PAL from Arabidopsis thaliana (SEQ ID NO: 78), HIS (PAL + C4H) C4H from Arabidopsis thaliana (SEQ ID NO: 80), TT LEU LEU TT URA URA TT TRP TRP TT HIS HIS

(83) The control strain (without the genes) containing the constructs TT LEU, TT URA, TT TRP and TT HIS is called CF237.

(84) Results

(85) F3H

(86) Tables 15 and 16 below show the production of eriodictyol (Table 15) and of luteolin (Table 16) obtained by cultivating the strains comprising the F3Hs listed in Table 6 and the constructs of Table 7, in the presence of naringenin and apigenin, respectively.

(87) TABLE-US-00016 TABLE 15 Concentration of eriodictyol (in mg .Math. l.sup.1) WITHOUT CPR CPR CPR ATR ATR F3H CPR (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No) (TT LEU) 24; FL121) 26; FL401) 28; FL274) 30; FL275) 32; FL463) 2 (FL23) 35.5 2.9 42.1 4.3 49.9 4.2 43.6 4.2 38.8 4.1 43.3 5.1 4 (FL24) 1 0.8 6.4 0.5 4.0 0.5 4.7 0.3 5.1 0.4 5.3 0.4 6 (FL25) 115.2 3.2 76.8 4.2 42.3 2.6 70.2 8.6 71.1 8.7 71.3 7.4 8 (FL26) 108.3 4.0 71.1 7.1 89.2 9.5 87.4 5.0 75.8 5.2 90.0 6.1 10 (FL27) 28.8 1.2 57.7 2.6 69.3 10.6 79.1 4.2 52.3 0.5 69.7 2.3 12 (FL28) 108.0 2.0 7.0 1.4 9.1 5.9 4.6 0.3 7.4 2.8 9.2 0.6 14 (FL29) 119.9 1.1 39.9 4.7 56.1 16.3 64.8 4.1 36.8 4.4 46.1 5.5 16 (FL30) <QL 76.3 2.6 70.9 6.2 70.4 4.4 58.5 10.9 76.9 1.7 18 (FL31) 107.3 8.0 82.3 17.2 102.2 7.1 98.8 5.9 96.6 4.7 101.3 4.0 20 (FL32) 33.7 4.0 68.9 2.7 81.5 3.4 63.6 3.7 69.5 0.9 69.7 1.1 96 (FL1031) 4.8 0.3 60.5 3.4 34.4 2.8 25.8 5.8 59.0 1.7 40.0 9.5 QL: below the quantification limit

(88) The various strains are indeed capable of producing eriodictyol from naringenin, in different concentrations according to the F3Hs and the CPR used (see FIG. 2).

(89) TABLE-US-00017 TABLE 16 Concentration of luteolin (in mg .Math. l.sup.1) WITHOUT CPR CPR CPR ATR ATR F3H CPR (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No) (TT LEU) 24; FL121) 26; FL401) 28; FL274) 30; FL275) 32; FL463) 2 (FL23) 3.5 0.1 11.7 0.7 9.1 2.1 11.01 0.4 10.8 1.7 10.2 1.4 4 (FL24) <QL <QL <QL <QL <QL <QL 6 (FL25) 10.2 0.9 12.8 0.7 7.8 1.4 11.9 0.8 10.1 1.2 12.9 1.4 8 (FL26) 9.5 0.4 13.2 1.1 8.2 0.7 10.9 0.7 12.2 0.4 12.1 0.7 10 (FL27) <QL 2.5 0.3 <QL 0.5 0 2.7 0.1 2.77 0.4 12 (FL28) 12.1 0.4 13.3 1.2 14.7 1.8 14.1 1.7 12.5 3.8 15.3 0.9 14 (FL29) 1.5 0.1 0.6 0.04 1.1 0.2 0.8 0.03 0.7 0.06 1.0 0.08 16 (FL30) 0.5 0.02 1.3 0.1 2.5 1.5 1.6 0.1 1.5 0.5 2.0 0.1 18 (FL31) 12.2 0.7 13.2 0.8 13.7 1.2 12.7 0.4 14.0 1.8 12.7 0.6 20 (FL32) 1.2 0.2 9.9 1.4 2.8 0.4 4.3 0.1 11.0 0.9 9.3 1.8 96 (FL1031) 0.4 0.1 10.9 0.1 3.0 0.6 3.0 0.9 11.4 0.4 9.5 1.6 QL: below the quantification limit

(90) The various strains are indeed capable of producing luteolin from apigenin, in different concentrations according to the F3Hs and the CPR used (see FIG. 3).

(91) FNS

(92) Tables 17 and 18 below show the production of apigenin (Table 17) and of luteolin (Table 18) obtained by cultivating the strains comprising the FNSIIs listed in Table 8 and the constructs of Table 9, in the presence of naringenin and eriodictyol, respectively.

(93) TABLE-US-00018 TABLE 17 Concentration of apigenin (in mg .Math. l.sup.1) WITHOUT CPR CPR CPR ATR ATR FNSII CPR (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No) (TT LEU) 24; FL121) 26; FL401) 28; FL274) 30; FL275) 32; FL463) 34 (FL620) 11.6 0.3 34.7 1.0 47.6 5.7 37.7 1.6 50.5 1.5 51.3 3.4 36 (FL621) 3.5 0.1 35.6 0.2 14.9 1.3 16.4 1.4 29.8 3.9 33.2 1.5 38 (FL112) 2.9 0.1 40.7 1.2 41.4 1.5 34.2 1.7 38.0 0.9 43.5 0.0

(94) TABLE-US-00019 TABLE 18 Concentration of luteolin (in mg .Math. l.sup.1) WITHOUT CPR CPR CPR ATR ATR FNSII CPR (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No (SEQ ID No) (TT LEU) 24; FL121) 26; FL401) 28; FL274) 30; FL275) 32; FL463) 34 (FL620) 2.5 0.2 1.4 0.3 7.8 1.4 4.5 0.9 4.5 2.9 8.5 0.8 36 (FL621) 0.2 0.2 1.5 0.1 1.3 0.2 0.9 0.1 1.3 0.3 1.2 0.2 38 (FL112) 0.2 0.0 4.5 1.9 2.3 0.5 2.6 1.2 1.4 0.0 1.6 0.0

(95) The various strains are indeed capable of producing apigenin and luteolin from naringenin and eriodictyol, in different concentrations according to the FNS used (FIGS. 4 and 5). Similar results were obtained with the FNSIIs of SEQ ID NOs: 101, 103, 105, 107, 109, 111, 113, 115, 117, 119, 121, 123, 125, 127, 129, 131 and 133.

(96) F3H, MET, FNS, CPR: Production of Diosmetin from Naringenin

(97) The results for the production of diosmetin from naringenin by the strains SC2429 to SC2434, SC2436 to SC2444, SC2446 to SC2454, SC2456 to SC2464 and SC2466 to SC2468 are presented in FIG. 23.

(98) All the strains are capable of producing diosmetin from naringenin. The production of diosmetin is largely increased by adding a CPR.

(99) Strain Up to Eriodictyol/Luteolin

(100) The strains SC2424, SC2425, SC2426, SC2427, SC1500 and SC2428 contain all the enzymes of the pathway and are capable of producing luteolin and eriodictyol from glucose.

(101) The results for the strain SC1500 correspond to FIG. 6, in which the eriodictyol and luteolin peaks are observed. Similar results are obtained for the strains SC2424, SC2425, SC2426, SC2427 and SC2428. The production of eriodictyol and of luteolin for each of the strains SC2424, SC2425, SC2426, SC2427, SC1500 and SC2428 is presented in FIG. 22.

(102) It should be noted that the addition of the enzymes PAL and C4H to the biosynthetic pathway makes it possible to obtain markedly higher eriodictyol and luteolin concentrations. These concentrations may be up to six times higher than the concentrations obtained with the strains containing the same enzymes with the exception of PAL and C4H (cf. FIG. 22, for example by comparing the strain SC2425 without PAL/C4H and the strain SC1500 with PAL/C4H or the strain SC2426 without PAL/C4H and the strain SC2428 with PAL/C4H).

(103) MET

(104) The results for the production of hesperetin and diosmetin from eriodictyol and luteolin by the strains SC1612, SC1614, SC2147 and SC2151 are presented, respectively, in FIGS. 7, 8, 19 and 20.

(105) The yeast strains SC1612, SC1614, SC2147 and SC2151 are indeed capable of producing hesperetin and/or diosmetin.

(106) Starting with eriodictyol, the strains SC2147, SC2151 and SC1612 are capable of specifically producing hesperetin, i.e. of specifically methylating the hydroxyl in position 4 of eriodictyol (FIG. 19). The strain SC1614 produces, for its part, a mixture of hesperetin and of homoeriodictyol.

(107) In a noteworthy manner, the strain SC2151 is moreover capable of producing about 40 mg/L of hesperetin (FIG. 19). The strains SC2147, SC1612 and SC1614, for their part, are capable of producing diosmetin from luteolin (FIG. 20).

(108) FNSII

(109) The results for the production of diosmetin from hesperetin by the strain SC744 are presented in FIG. 9.

(110) The yeast strain SC744 is indeed capable of producing diosmetin from hesperetin.

(111) E. Coli

(112) The results for the production of hesperetin from eriodictyol by the strains EC26, EC41 and EC43 are presented in FIGS. 10, 14 and 15 and the production of diosmetin from luteolin by the strains EC26 and EC43 are presented in FIGS. 11 and 16.

(113) The E. coli strains EC26, EC41 and EC43 are indeed capable of producing hesperetin and/or diosmetin.

(114) The results for the production of diosmetin from hesperetin by the strain EC30 are presented in FIG. 12.

(115) The E. coli strain EC30 is indeed capable of producing diosmetin from hesperetin.

(116) Strain Up to Hesperetin/Diosmetin

(117) The results for the production of hesperetin and diosmetin from glucose by the yeast strains SC1508, SC2408 and SC2409 are presented in FIGS. 13, 17 and 18.

(118) The yeast strains SC1508, SC2408 and SC2409 containing all the enzymes of the pathway are capable of producing hesperetin and/or diosmetin from glucose (FIG. 21). In a noteworthy manner, the strain SC 2408 produces about 25 mg/L of hesperetin and about 5 mg/L of diosmetin.