Method for the biosynthesis of diosmin and/or hesperidin in a microorganism

Patent metadata

Field	Value
Publication number	US-2022042061-A1
Application number	US-202017429459-A
Country	US
Kind code	A1
Filing date	Feb 11, 2020
Priority date	Feb 11, 2019
Publication date	Feb 10, 2022
Grant date	—

Abstract

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

First claim

1 - 32 . (canceled)

33 . A recombinant microorganism comprising:
a heterologous nucleic acid sequence coding for a flavanone 7-O-beta-D-glucosyltransferase (UGT) which is capable of adding a glucose in position 7 of hesperetin and/or diosmetin; and a heterologous nucleic acid sequence coding for a 6″-O-rhamnosyltransferase (RhaT) which is capable of transferring a rhamnose into position 6 of the glucose of hesperetin-7-O-glucoside and/or diosmetin-7-O-glucoside; and a heterologous nucleic acid sequence coding for a UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase (RHM) which is capable of producing UDP-rhamnose.

34 . The microorganism as claimed in claim 33 , in which the flavanone 7-O-beta-D-glucosyltransferase is selected from the group consisting of SEQ ID NOs: 113, 115, 91, 93, 95, 97, 99 and 101 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having flavanone 7-O-beta-D-glucosyltransferase activity.

35 . The microorganism as claimed in claim 33 , in which the 6″-O-rhamnosyltransferase is selected from the group consisting of SEQ ID NOs: 103 and 105 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having 6″-O-rhamnosyltransferase activity.

36 . The microorganism as claimed in claim 33 , in which the UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase is selected from the group consisting of SEQ ID NOs: 107, 109 and 111 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase activity.

37 . The microorganism as claimed in claim 33 , in which
the flavanone 7-O-beta-D-glucosyltransferase is selected from the group consisting of SEQ ID NOs: 113 and 95 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having flavanone 7-O-beta-D-glucosyltransferase activity; and the 6″-O-rhamnosyltransferase is selected from the group consisting of SEQ ID NO: 103 and polypeptides comprising a sequence having at least 60% sequence identity with this sequence and having 6″-O-rhamnosyltransferase activity; and the UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase is selected from the group consisting of SEQ ID NO: 107 and polypeptides comprising a sequence having at least 60% sequence identity with this sequence and having UDP-glucose 4,6-dehydratase/UDP-4-keto-6-deoxy-D-glucose 3,5-epimerase/UDP-4-keto-L-rhamnose-reductase activity.

38 . The microorganism as claimed in claim 33 , the microorganism also comprising:
a heterologous nucleic acid sequence coding for a tyrosine ammonia lyase (TAL); a heterologous nucleic acid sequence coding for a 4-coumarin-CoA ligase (4CL); a heterologous nucleic acid sequence coding for a naringenin-chalcone synthase (CHS); and a heterologous nucleic acid sequence coding for a chalcone isomerase (CHI).

39 . The microorganism as claimed in claim 38 , characterized in that it 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 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences 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: 123, 125, 43, 45, 47 and 49 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences 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 SEQ ID NOs: 53, 51, 55 and 57 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences 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 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having chalcone isomerase activity.

40 . The microorganism as claimed in claim 33 , characterized in that it also comprises a heterologous nucleic acid sequence coding for a flavonoid 3′-monooxygenase (F3′H).

41 . The microorganism as claimed in claim 40 , characterized in that the microorganism comprises a heterologous nucleic acid sequence coding for a flavonoid 3′-monooxygenase (F3′H) comprising a sequence selected from the group consisting of SEQ ID NOs: 7, 1, 3, 5, 9, 11, 13, 15, 17, 19, 21 and 121 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having flavonoid 3′-monooxygenase activity.

42 . The microorganism as claimed in claim 33 , characterized in that it also comprises a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT).

43 . The microorganism as claimed in claim 42 , characterized in that the microorganism comprises a heterologous nucleic acid sequence coding for an O-methyltransferase (OMT) comprising a sequence selected from the group consisting of SEQ ID NOs: 119, 117, 87 and 89 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having O-methyltransferase activity.

44 . The microorganism as claimed in claim 33 , also comprising:
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 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences 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 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having cinnamate 4-hydroxylase activity.

45 . The microorganism as claimed in claim 33 , also comprising a heterologous or endogenous nucleic acid sequence coding for a flavone synthase (FNS).

46 . The microorganism as claimed in claim 45 , characterized in that it comprises a heterologous nucleic acid sequence coding for a flavone synthase (FNS) comprising a sequence selected from the group consisting of SEQ ID NOs: 33, 35, 37, 127, 129, 131, 133, 135, 137, 139, 141, 143, 145, 147, 149, 151, 153, 155, 157 and 159 and polypeptides comprising a sequence having at least 60% sequence identity with one of these sequences and having flavone synthase activity.

47 . The microorganism as claimed in claim 33 , also comprising:
a heterologous nucleic acid sequence coding for a cytochrome P450 reductase (CPR); and/or a heterologous or endogenous nucleic acid sequence coding for an S-adenosylmethionine synthetase (SAMT).

48 . The microorganism as claimed in claim 47 , characterized in that 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 and polypeptides compris

Assignees

• Servier Lab

Inventors

• Pauthenier Cyrille
• Le Jeune André
• Scornec Hélène
• Roussel Célia
• Joubert Laetitia

Classifications

• C12N9/0006

  acting on CH-OH groups as donors (1.1) · CPC title

• C12Y204/01

  Hexosyltransferases (2.4.1) · CPC title

• C12N9/1085

  transferring alkyl or aryl groups other than methyl groups (2.5) · CPC title

• C12Y205/01006

  Methionine adenosyltransferase (2.5.1.6), i.e. adenosylmethionine synthetase · CPC title

• C12N9/0071

  acting on paired donors with incorporation of molecular oxygen (1.14) · CPC title