Process for preparation of flavanone derivatives

Abstract

The invention generally provides a method for preparing a class of flavanone derivatives and application of the flavanone derivatives as a sweetness enhancer. In particular, the present invention provides a method of preparing a compound of Formula (I) as defined herein by reacting a precursor compound of Formula (Ia) as defined herein with an acyltransferase. In some embodiments, the compound of formula (Ia) is hesperidin, taxifolin, dihydrotamaristin, 3 '-O-methyl taxifolin, pinocembrin, 5-deoxyhesperidin, 5-deoxytaxifolin, 5-deoxydihydrotamaristin, 5-deoxy-3'-O-methyl taxifolin, or 5-deoxypinocembrin. In some embodiments, the compound shown in the formula (I) is hesperidin-3-O-acetate, taxifolin-3-O-acetate, dihydrotaxifolin-3-O-acetate, 3 '-O-methyl taxifolin-3-O-acetate, pinocembrin-3-O-acetate, 5-deoxyhesperidin-3-O-acetate and 5-deoxytaxifolin-3-O-acetate, and the structural formula of the compound shown in the formula (I) is shown in the description. The compound is prepared from 5-deoxytamarix flavin-3-O-acetate, 5-deoxydihydrotamarix flavin-3-O-acetate, 5-deoxy-3 '-O-methyl taxifolin-3-O-acetate or 5-deoxybrepinin-3-O-acetate. The invention also relates to acyltransferases useful in this method and certain compositions comprising such flavanone derivatives, such as compositions comprising such flavanone derivatives and one or more other sweeteners.

Description

Technical Field

[0001] The present disclosure generally provides a method for preparing a class of flavanone derivatives and their use as sweetness enhancers. In certain aspects, the present disclosure provides certain compositions comprising such flavanone derivatives, for example, compositions comprising such flavanone derivatives and one or more other sweeteners. Background Art

[0002] The taste system provides sensory information about the chemical composition of the external world. Taste transduction is one of the more complex forms of chemically triggered sensation in animals. Taste signals are found throughout the animal kingdom, from simple metazoans to the most complex vertebrates. Mammals are believed to possess five basic forms of taste: sweet, bitter, sour, salty, and umami.

[0003] Sweetness is the taste most commonly experienced when consuming foods rich in sugar. Mammals generally perceive sweetness as a pleasant sensation, unless consumed in excess. Caloric sweeteners, such as sucrose and fructose, are typical examples of sweet substances. Despite the availability of a variety of non-caloric and low-calorie alternatives, these caloric sweeteners remain the primary means of eliciting sweetness in edible products.

[0004] Metabolic disorders and related conditions, such as obesity, diabetes, and cardiovascular disease, are major public health concerns worldwide. Their prevalence is increasing at an alarming rate in nearly every developed country. Caloric sweeteners are a key factor driving this trend, as they are included in a variety of packaged food and beverage products, making them more palatable to consumers. In many cases, no- or low-calorie alternatives can be used to replace sucrose or fructose in food and beverages. Even so, the sweetness imparted by these compounds differs from that of caloric sweeteners, and many consumers fail to perceive them as suitable substitutes. Moreover, such compounds can be difficult to incorporate into certain products. In some cases, they can be used as partial replacements for caloric sweeteners, but their presence alone can lead to unpleasant off-flavors perceived by many consumers, including astringency, bitterness, metallicity, and licorice. Consequently, low-calorie sweeteners face certain challenges in their adoption.

[0005] Sweetness enhancement offers an alternative approach to overcoming some of the adoption challenges faced by low-calorie sweeteners. Such compounds can be used in combination with sucrose or fructose to enhance their sweetness, thereby allowing the use of lower amounts of these caloric sweeteners in various food or beverage products. However, in addition to enhancing the perceived sweetness of the primary sweetener, these compounds still alter the perceived taste of the sweetener. Consequently, many consumers find consuming such sweetness-enhanced products less enjoyable than unenhanced alternatives with higher calories.

[0006] WO2021043842 discloses natural flavanone derivatives that are particularly suitable for enhancing the sweetness of natural sugars. Summary of the Invention

[0007] The present invention claims a method for preparing a compound of formula (I):

[0008]

[0009] in:

[0010] R 1 A hydrogen atom, -OH or -OR 1A ;

[0011] R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0012] R 2 A hydrogen atom, -OH or -OR 2A ;

[0013] R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0014] R 3 A hydrogen atom, -OH or -OR 3A ;

[0015] R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0016] R 4 A hydrogen atom, -OH or -OR 4A ;

[0017] R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0018] R 5 -OC(O)-(C 1-24 alkyl);

[0019] R 6 and R 7 Independently from C 1-6 Alkyl, -OH, C1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group;

[0020] m is 0, 1, or 2; and

[0021] n is 0, 1, 2, or 3;

[0022] The method comprises reacting a precursor compound of formula (Ia) with an acyltransferase to form a compound of formula (I):

[0023]

[0024] in:

[0025] R 1 A hydrogen atom, -OH or -OR 1A ;

[0026] R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0027] R 2 A hydrogen atom, -OH or -OR 2A ;

[0028] R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0029] R 3 A hydrogen atom, -OH or -OR 3A ;

[0030] R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0031] R 4 A hydrogen atom, -OH or -OR 4A ;

[0032] R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0033] R 6 and R 7 Independently from C1-6 Alkyl, -OH, C 1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group;

[0034] m is 0, 1, or 2; and

[0035] n is 0, 1, 2 or 3.

[0036] A preferred embodiment of the process of the present invention is that wherein the process is carried out in the presence of acyl-CoA.

[0037] A preferred embodiment of the method of the present invention is wherein the acyltransferase is an acetyltransferase.

[0038] A preferred embodiment of the method of the present invention is wherein the acetyltransferase comprises the amino acid sequence HXXXD (SEQ ID NO: 29) and / or the amino acid sequence [DN]FGxG (SEQ ID NO: 30). Another preferred embodiment of the method of the present invention is wherein the acetyltransferase comprises the amino acid sequence [ST]S[WL] (SEQ ID NO: 94).

[0039] A preferred embodiment of the method of the present invention is wherein the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0040] A preferred embodiment of the method of the present invention is that the compound of formula (Ia) is aromandrin, taxifolin, dihydrotamarixetin, 3'-O-methyltaxifolin, pinobanksin, 5-deoxyaromadendrin, 5-deoxytaxifolin, 5-deoxydihydrotamarixetin, 5-deoxy-3'-O-methyltaxifolin or 5-deoxypinobanksin.

[0041] A preferred embodiment of the method of the present invention is that the compound of formula (I) is agaretin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltamarixanthin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxyagaretin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, or 5-deoxy-3'-O-methyltamarixanthin-3-O-acetate or 5-deoxybrevicornin-3-O-acetate.

[0042] A preferred embodiment of the method of the present invention is wherein the method is performed in vivo.

[0043] The present invention also claims a recombinant polypeptide having acyltransferase activity, comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0044] The present invention also claims a recombinant cell comprising the compound of formula (I).

[0045] In one embodiment, the recombinant cell further comprises an acyltransferase, preferably an acetyltransferase. More preferably, the recombinant cell further comprises a recombinant acyltransferase, even more preferably, a recombinant acetyltransferase.

[0046] In another embodiment, the recombinant cell further comprises a recombinant nucleic acid sequence encoding an acyltransferase, preferably an acetyltransferase. More preferably, the recombinant cell further comprises a recombinant nucleic acid sequence encoding an acetyltransferase having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or a nucleotide sequence comprising any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or a reverse complement thereof.

[0047] In yet another embodiment, the recombinant cell further comprises the following enzyme:

[0048] (a) flavanone 3-hydroxylase (F3H),

[0049] (b) chalcone isomerase (CHI),

[0050] (c) chalcone synthase (CHS),

[0051] (d) 4-Coumarate CoA ligase (4CL),

[0052] (e) cytochrome P450 reductase (CPR),

[0053] (f) tyrosine ammonia lyase (TAL),

[0054] (g) chalcone isomerase-like (CHIL),

[0055] (h) cinnamate-4-hydroxylase (C4H),

[0056] (I) phenylalanine ammonia lyase (PAL),

[0057] (j) flavonoid 3'-hydroxylase (F3'H),

[0058] (k) 3'-O-methyltransferase (3'-MT),

[0059] (1) 4'-O-methyltransferase (4'-MT),

[0060] (m) 3-O-methyltransferase (3-MT),

[0061] (n) 4-O-methyltransferase (4-MT),

[0062] (o) 3-OH-specific P450 monooxygenases,

[0063] (p) glycosidase, and / or

[0064] (q) Polyketide reductase (PKR).

[0065] The cell can be a prokaryotic cell, an archaebacterial cell, or a eukaryotic cell.

[0066] Prokaryotic cells may be, but are not limited to, bacterial cells.

[0067] The eukaryotic cell may be, but is not limited to, a fungus (such as yeast or filamentous fungi), an algae, a plant cell, or a cell line.

[0068] Preferably, the cell is a bacterium, an archaeon, a fungus such as a yeast, an algae cell or a plant cell.

[0069] The present invention also claims a growth medium comprising the recombinant cells of the present invention and a compound of formula (I).

[0070] The present invention also claims a method for preparing a compound of formula (I), which method comprises growing the recombinant cell of the present invention under growth conditions suitable for producing the compound of formula (I).

[0071] The present invention also claims a compound of formula (I) obtained or obtainable by a process as claimed in any one of the preceding claims.

[0072] The present invention also claims the use of a compound of formula (I) obtained or obtainable by the process of any one of the preceding claims for (a) enhancing the sweetness of an ingestible composition, (b) reducing the bitterness of an ingestible composition, or (c) reducing the sourness of an ingestible composition.

[0073] The present invention also claims a method for a) enhancing the sweetness of an ingestible composition of a product, (b) reducing the bitterness of an ingestible composition of a product and / or (c) reducing the sourness of an ingestible composition of a product, which method comprises introducing into the product a compound of formula (I) obtained or obtainable by the method of any of the preceding claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0074] Figure 1 .Schematic diagram of the biosynthesis of flavanones and flavanone derivatives.

[0075] Figure 2 Examples of negative and positive ion ESI-MS / MS spectra of citronellin-3-O-acetate. Part A shows the negative ion ESI-MS / MS spectrum of citronellin-3-O-acetate. Part B shows the positive ion ESI-MS / MS spectrum of citronellin-3-O-acetate. DETAILED DESCRIPTION

[0076] The following detailed description describes various aspects and embodiments provided herein. This description should be read from the perspective of one of ordinary skill in the relevant art. Therefore, information known to such persons of ordinary skill is not necessarily included.

[0077] Abbreviations used

[0078] bp——base pair

[0079] kb - kilobase

[0080] DNA - deoxyribonucleic acid

[0081] cDNA – complementary DNA

[0082] DTT - dithiothreitol

[0083] GC – Gas Chromatography

[0084] HPLC – High Performance Liquid Chromatography

[0085] IPTG——Isopropyl-D-thiogalactopyranoside

[0086] LB - Lysogeny Broth

[0087] MS – Mass Spectrometer / Mass Spectrometry

[0088] PCR — polymerase chain reaction

[0089] RNA – Ribonucleic Acid

[0090] mRNA – messenger ribonucleic acid

[0091] miRNA – microRNA

[0092] siRNA – small interfering RNA

[0093] rRNA – ribosomal RNA

[0094] tRNA – transfer RNA

[0095] SMM - Supplementary Minimal Medium

[0096] BLAST - Basic Local Alignment Search Tool

[0097] ESI – Electrospray Ionization

[0098] OD – Optical density

[0099] SDS-PAGE - sodium dodecyl sulfate polyacrylamide gel electrophoresis

[0100] definition

[0101] As used herein, "solvate" refers to a compound formed by the interaction of one or more solvent molecules with one or more compounds described herein. In some embodiments, the solvate is an ingestibly acceptable solvate, such as a hydrate.

[0102] As used herein, "C" wherein "a" and "b" are integers a to C b ” or “C a-b " refers to the number of carbon atoms in the specified group. That is, the group can contain from "a" to "b" (including the end values) carbon atoms. Thus, for example, "C1 to C4 alkyl" or "C 1-4 "Alkyl" refers to all alkyl groups having 1 to 4 carbons, i.e., CH3-, CH3CH2-, CH3CH2CH2-, (CH3)2CH-, CH3CH2CH2CH2-, CH3CH2CH(CH3)-, and (CH3)3C-.

[0103] As used herein, "halogen" or "halo" refers to any of the radiostable atoms in column 7 of the periodic table, such as fluorine, chlorine, bromine, or iodine. In some embodiments, "halogen" or "halo" refers to fluorine or chlorine.

[0104] As used herein, "alkyl" refers to a fully saturated straight or branched hydrocarbon chain (i.e., containing no double or triple bonds). In some embodiments, an alkyl group has 1 to 20 carbon atoms (when appearing herein, a numerical range such as "1 to 20" refers to each integer in the given range; for example, "1 to 20 carbon atoms" means that the alkyl group can consist of 1 carbon atom, 2 carbon atoms, 3 carbon atoms, etc., up to and including 20 carbon atoms, although this definition also covers occurrences of the term "alkyl" in which no numerical range is specified). An alkyl group can also be a medium-sized alkyl group having 1 to 9 carbon atoms. An alkyl group can also be a lower alkyl group having 1 to 4 carbon atoms. An alkyl group can be referred to as a "C 1-4 Alkyl" or similar designations. For example only, "C 1-4 "Alkyl" means that there are 1 to 4 carbon atoms in the alkyl chain, i.e., the alkyl chain is selected from the group consisting of methyl, ethyl, propyl, isopropyl, n-butyl, isobutyl, sec-butyl and tert-butyl. Typical alkyl groups include, but are in no way limited to, methyl, ethyl, propyl, isopropyl, butyl, isobutyl, tert-butyl, pentyl, hexyl, and the like. Unless otherwise indicated, the term "alkyl" refers to a group that is not further substituted.

[0105] As used herein, "substituted alkyl" refers to an alkyl group substituted with one or more substituents independently selected from the group consisting of C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogen, cyano, hydroxy, C1-C6 alkoxy, aryloxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyloxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-oxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl-oxy (optionally substituted with halogen , C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6 alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6-alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6) C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogenated (C1-C6) alkyl (e.g. -CF3), halogenated (C1-C6) alkoxy (e.g. -OCF3), C1-C6 alkylthio, arylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclic thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-thio (optionally substituted with halogen, C1-C6 alkyl,C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl-thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6 alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkyl thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), amino, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxyl, O-carboxyl, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, and oxo (═O). ,

[0106] As used herein, "alkoxy" refers to a radical of the formula -OR, wherein R is an alkyl radical as defined above, e.g., "C 1-9 "Alkoxy" includes, but is not limited to, methoxy, ethoxy, n-propoxy, 1-methylethoxy (isopropoxy), n-butoxy, isobutoxy, sec-butoxy and tert-butoxy.

[0107] As used herein, "alkylthio" refers to a radical of the formula -SR, wherein R is an alkyl radical as defined above, e.g., "C 1-9 "alkylthio" and the like, including but not limited to methylthio, ethylthio, n-propylthio, 1-methylethylthio (isopropylthio), n-butylthio, isobutylthio, sec-butylthio, tert-butylthio and the like.

[0108] As used herein, "alkenyl" refers to a straight or branched hydrocarbon chain containing one or more double bonds. In some embodiments, an alkenyl group has 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkenyl" where no numerical range is specified. An alkenyl group may also be a medium-sized alkenyl group having 2 to 9 carbon atoms. An alkenyl group may also be a lower alkenyl group having 2 to 4 carbon atoms. An alkenyl group may be referred to as a "C 2-4 "C-alkenyl" or similar names. For example only, "C 2-4"Alkenyl" means that there are 2 to 4 carbon atoms in the alkenyl chain, i.e., the alkenyl chain is selected from the group consisting of ethenyl, propen-1-yl, propen-2-yl, propen-3-yl, buten-1-yl, buten-2-yl, buten-3-yl, buten-4-yl, 1-methyl-propen-1-yl, 2-methyl-propen-1-yl, 1-ethyl-ethen-1-yl, 2-methyl-propen-3-yl, buta-1,3-dienyl, buta-1,2-dienyl, and buta-1,2-dien-4-yl. Typical alkenyl groups include, but are in no way limited to, ethenyl, propenyl, butenyl, pentenyl, hexenyl, and the like. Unless otherwise indicated, the term "alkenyl" refers to a group that is not further substituted.

[0109] As used herein, "alkynyl" refers to a straight or branched hydrocarbon chain containing one or more triple bonds. In some embodiments, an alkynyl group has 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term "alkynyl" where no numerical range is specified. An alkynyl group may also be a medium-sized alkynyl group having 2 to 9 carbon atoms. An alkynyl group may also be a lower alkynyl group having 2 to 4 carbon atoms. An alkynyl group may be referred to as a "C 2-4 Alkynyl" or similar designations. For example only, "C 2-4 "Alkynyl" means that there are 2 to 4 carbon atoms in the alkynyl chain, i.e., the alkynyl chain is selected from the group consisting of ethynyl, propyn-1-yl, propyn-2-yl, butyn-1-yl, butyn-3-yl, butyn-4-yl, and 2-butynyl. Typical alkynyl groups include, but are in no way limited to, ethynyl, propynyl, butynyl, pentynyl, hexynyl, and the like. Unless otherwise indicated, the term "alkynyl" refers to a group that is not further substituted.

[0110] As used herein, "heteroalkyl" refers to a straight or branched hydrocarbon chain containing one or more heteroatoms, i.e., elements other than carbon, in the backbone of the chain, including, but not limited to, nitrogen, oxygen, and sulfur. In some embodiments, a heteroalkyl group has from 1 to 20 carbon atoms, although this definition also encompasses occurrences of the term "heteroalkyl" where no numerical range is specified. A heteroalkyl group may also be a medium-sized heteroalkyl group having from 1 to 9 carbon atoms. A heteroalkyl group may also be a lower heteroalkyl group having from 1 to 4 carbon atoms. A heteroalkyl group may be referred to as a "C 1-4 Heteroalkyl" or similar designations. A heteroalkyl group may contain one or more heteroatoms. For example only, "C 1-4 "Heteroalkyl" means that there are one to four carbon atoms in the heteroalkyl chain and one or more additional heteroatoms in the backbone of the chain. Unless otherwise indicated, the term "heteroalkyl" refers to a group that is not further substituted.

[0111] As used herein, "alkylene" refers to a fully saturated, branched or straight-chain diradical chemical group containing only carbon and hydrogen, which is connected to the rest of the molecule (i.e., an alkanediyl group) by two points of attachment. In some embodiments, an alkylene group has from 1 to 20 carbon atoms, although this definition also encompasses occurrences of the term alkylene where no numerical range is specified. An alkylene group may also be a medium-sized alkylene group having from 1 to 9 carbon atoms. An alkylene group may also be a low-sized alkylene group having from 1 to 4 carbon atoms. An alkylene group may be referred to as a "C 1-4 Alkylene" or similar designations. For example only, "C 1-4 “Alkylene” means that there are 1 to 4 carbon atoms in the alkylene chain, i.e., the alkylene chain is selected from the group consisting of methylene, ethylene, ethane-1,1-diyl, propylene, propylene-1,1-diyl, propylene-2,2-diyl, 1-methyl-ethylene, butylene, butane-1,1-diyl, butane-2,2-diyl, 2-methyl-propylene-1,1-diyl, 1-methyl-propylene, 2-methyl-propylene, 1,1-dimethyl-ethylene, 1,2-dimethyl-ethylene and 1-ethyl-ethylene. Unless otherwise indicated, the term “alkylene” refers to groups that are not further substituted.

[0112] As used herein, "alkenylene" refers to a straight or branched diradical chemical group containing only carbon and hydrogen and containing at least one carbon-carbon double bond connected to the rest of the molecule by two points of attachment. In some embodiments, alkenylene has 2 to 20 carbon atoms, although this definition also encompasses occurrences of the term alkenylene where no numerical range is specified. Alkenylene can also be a medium-sized alkenylene having 2 to 9 carbon atoms. Alkenylene can also be a lower alkenylene having 2 to 4 carbon atoms. Alkenylene groups can be referred to as "C 2-4 Alkenylene" or similar names. For example only, "C 2-4"Alkenylene" means that there are 2 to 4 carbon atoms in the alkenylene chain, i.e., the alkenylene chain is selected from the group consisting of vinylene, ethylene-1,1-diyl, propenylene, propen-1,1-diyl, prop-2-ene-1,1-diylidene, 1-methylvinylene, but-1-enylidene, but-2-enylidene, buta-1,3-dienyl, butene-1,1-diyl, buta-1,3-diene-1,1-diyl, but-2-ene-1,1-diyl, buta-3-ene-1, ... 1-diyl, 1-methyl-1-propen-2-ene-1,1-diyl, 2-methyl-1-propen-2-ene-1,1-diyl, 1-ethyl-vinylene, 1,2-dimethyl-vinylene, 1-methyl-propenylene, 2-methyl-propenylene, 3-methyl-propenylene, 2-methyl-propenylene-1,1-diyl and 2,2-dimethyl-vinylene-1,1-diyl. Unless otherwise indicated, the term "alkenylene" refers to a group that is not further substituted.

[0113] As used herein, "aromatic" refers to a ring or ring system having a conjugated π electron system and includes carbocyclic aromatic groups (e.g., phenyl) and heterocyclic aromatic groups (e.g., pyridine). The term includes monocyclic or fused-ring polycyclic (i.e., rings that share adjacent pairs of atoms) groups, provided that the entire ring system is aromatic.

[0114] As used herein, "aryl" refers to an aromatic ring or ring system (i.e., two or more fused rings that share two adjacent carbon atoms) that contains only carbon in the ring backbone. When aryl is a ring system, each ring in the system is aromatic. In some embodiments, aryl has 6 to 18 carbon atoms, although this definition also encompasses occurrences of the term "aryl" where no numerical range is specified. In some embodiments, aryl has 6 to 10 carbon atoms. Aryl may be referred to as "C 6-10 Aryl", "C6-C 10 Examples of aryl groups include, but are not limited to, phenyl, naphthyl, azulenyl, and anthracenyl. In some embodiments, the term "aryl" refers to phenyl. Unless otherwise indicated, the term "aryl" refers to a group that is not further substituted.

[0115] As used herein, "aryloxy" and "arylthio" refer to moieties of the formula RO- and RS-, respectively, wherein R is an aryl group as defined above, e.g., "C 6-10 Aryloxy" or "C 6-10 "arylthio" and the like, including but not limited to phenoxy and phenylthio.

[0116] As used herein, "aralkyl" or "arylalkyl" refers to an aryl group attached as a substituent through an alkylene group, for example, "C 7-14"Aralkyl" and the like, including but not limited to benzyl, 2-phenylethyl, 3-phenylpropyl, etc. In some embodiments, the alkylene group is a lower alkylene group (i.e., C 1-4 alkylene).

[0117] As used herein, "heteroaryl" refers to an aromatic ring or ring system (i.e., two or more fused rings sharing two adjacent atoms) containing one or more heteroatoms, i.e., elements other than carbon (including but not limited to nitrogen, oxygen and sulfur) in the ring backbone. When heteroaryl is a ring system, each ring in the system is aromatic. In some embodiments, heteroaryl has 5 to 18 ring members (i.e., the number of atoms making up the ring backbone, including carbon atoms and heteroatoms), although this definition also covers the occurrence of the term "heteroaryl" in which no numerical range is specified. In some embodiments, heteroaryl has 5 to 10 ring members or 5 to 7 ring members. Heteroaryl may be referred to as "5 to 7-membered heteroaryl", "5 to 10-membered heteroaryl" or similar names. Examples of heteroaryl rings include, but are not limited to, furanyl, thienyl, phthalazinyl, pyrrolyl, oxazolyl, thiazolyl, imidazolyl, pyrazolyl, isoxazolyl, isothiazolyl, triazolyl, thiadiazolyl, pyridyl, pyridazinyl, pyrimidinyl, pyrazinyl, triazinyl, quinolyl, isoquinolyl, benzimidazolyl, benzoxazolyl, benzothiazolyl, indolyl, isoindolyl, and benzothienyl. Unless otherwise indicated, the term "heteroaryl" refers to a group that is not further substituted.

[0118] As used herein, "heteroaralkyl" or "heteroarylalkyl" refers to a heteroaryl group attached as a substituent via an alkylene group. Examples include, but are not limited to, 2-thienylmethyl, 3-thienylmethyl, furanylmethyl, thienylethyl, pyrrolylalkyl, pyridylalkyl, isoxazolylalkyl, and imidazolylalkyl. In some cases, the alkylene group is a lower alkylene group (i.e., C 1-4 alkylene).

[0119] As used herein, "carbocyclyl" refers to a non-aromatic ring or ring system that contains only carbon atoms in the ring system backbone. When a carbocyclyl is a ring system, two or more rings can be joined together in a fused, bridged, or spiro manner. A carbocyclyl can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. Thus, carbocyclyl includes cycloalkyl, cycloalkenyl, and cycloalkynyl groups. In some embodiments, a carbocyclyl has 3 to 20 carbon atoms, although this definition also encompasses occurrences of the term "carbocyclyl" where no numerical range is specified. A carbocyclyl can also be a medium-sized carbocyclyl having 3 to 10 carbon atoms. A carbocyclyl can also be a carbocyclyl having 3 to 6 carbon atoms. A carbocyclic group can be referred to as a "C 3-6Examples of carbocyclyl rings include, but are not limited to, cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cyclohexenyl, 2,3-dihydroindene, bicyclo[2.2.2]octyl, adamantyl, and spiro[4.4]nonyl. Unless otherwise indicated, the term "carbocyclyl" refers to a group that is not further substituted.

[0120] As used herein, "(carbocyclyl)alkyl" refers to a carbocyclyl group attached as a substituent through an alkylene group, for example, "C 4-10 (Carbocyclyl)alkyl" and the like, including but not limited to cyclopropylmethyl, cyclobutylmethyl, cyclopropylethyl, cyclopropylbutyl, cyclobutylethyl, cyclopropylisopropyl, cyclopentylmethyl, cyclopentylethyl, cyclohexylmethyl, cyclohexylethyl, cycloheptylmethyl and the like. In some cases, alkylene is lower alkylene.

[0121] As used herein, "cycloalkyl" refers to a fully saturated carbocyclyl ring or ring system according to any of the embodiments described above for carbocyclyl. Examples include cyclopropyl, cyclobutyl, cyclopentyl, and cyclohexyl.

[0122] As used herein, "cycloalkenyl" refers to a carbocyclyl ring or ring system having at least one double bond, wherein no ring in the ring system is aromatic, and according to any of the embodiments described above for carbocyclyl. An example is cyclohexenyl.

[0123] As used herein, "heterocyclyl" refers to a non-aromatic ring or ring system containing at least one heteroatom in the ring backbone. The heterocyclyl group can be connected together in a fused, bridged or spirally connected manner. The heterocyclyl group can have any degree of saturation, provided that at least one ring in the ring system is not aromatic. The heteroatom can be present in the non-aromatic or aromatic ring of the ring system. In some embodiments, the heterocyclyl group has 3 to 20 ring members (i.e., the number of atoms that make up the ring backbone, including carbon atoms and heteroatoms), although this definition also covers the occurrence of the term "heterocyclyl" in which no numerical range is specified. The heterocyclyl group can also be a medium-sized heterocyclyl group with 3 to 10 ring members. The heterocyclyl group can also be a heterocyclyl group with 3 to 6 ring members. The heterocyclyl group can be referred to as a "3 to 6-membered heterocyclyl" or similar names. In preferred six-membered monocyclic heterocyclyls, the heteroatoms are selected from one to up to three of O, N or S, and in preferred five-membered monocyclic heterocyclyls, the heteroatoms are selected from one or two heteroatoms selected from O, N or S. Examples of heterocyclyl rings include, but are not limited to, azepinyl, acridinyl, carbazolyl, cinnolinyl, dioxolanyl, imidazolinyl, imidazolidinyl, morpholinyl, oxiranyl, oxepanyl, thiepanyl, piperidinyl, piperazinyl, dioxopiperazinyl, pyrrolidonyl, pyrrolidionyl, 4-piperidonyl, pyrazolinyl, pyrazolidinyl, 1,3-dioxinyl, 1,3-dioxinyl, 1,4-dioxinyl, 1,4-dioxinyl, 1,3-oxathianyl, 1,3-dioxan ... yl), 1,4-oxathiinyl, 1,4-oxathiinyl, 2H-1,2-oxazinyl, trioxanyl, hexahydro-1,3,5-triazinyl, 1,3-dioxolyl, 1,3-dioxolane, 1,3-dithiolyl, 1,3-dithiolanyl, isoxazoline 1,4-thiazinyl, thiamorpholinyl, dihydrobenzofuranyl, benzimidazolidinyl, and tetrahydroquinoline.

[0124] As used herein, "(heterocyclyl)alkyl" refers to a heterocyclyl group attached via an alkylene group as a substituent. Examples include, but are not limited to, imidazolinylmethyl and indolinylethyl.

[0125] "Acyl" refers to -C(=O)R, where R is hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein. Non-limiting examples include formyl, acetyl, propionyl, benzoyl, and acryloyl.

[0126] "O-carboxyl" refers to a "-OC(=O)R" group, wherein R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0127] "C-carboxyl" refers to a "-C(=O)OR" group, where R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein. Non-limiting examples include carboxyl (ie, -C(=O)OH).

[0128] A "cyano" group refers to a "-CN" group.

[0129] A "cyanate" group refers to a "-OCN" group.

[0130] An "isocyanate" group refers to a "-NCO" group.

[0131] A "thiocyanate" group refers to a "-SCN" group.

[0132] An "isothiocyanate" group refers to a "-NCS" group.

[0133] "Sulfinyl" refers to a "-S(=O)R" group, where R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0134] "Sulfonyl" refers to a "-SO2R" group where R is selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0135] "S-sulfonamido" refers to "-SO2NR A R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0136] "N-sulfonamido" refers to "-N(R A )SO2R B ”, where R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0137] "O-carbamoyl" refers to "-OC(=O)NR A R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0138] "N-carbamoyl" refers to "-N(R A )C(=O)OR B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0139] "O-thiocarbamoyl" refers to "-OC(=S)NR A R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0140] "N-thiocarbamoyl" refers to "-N(R A )C(=S)OR B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0141] "C-amido" refers to "-C(=O)NR A R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0142] "N-amido" refers to "-N(R A )C(=O)R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein.

[0143] "Amino" refers to "-NR A R B " group, wherein R A and R B are each independently selected from hydrogen, C 1-6 Alkyl, C 2-6 Alkenyl, C 2-6 Alkynyl, C 3-7 Carbocyclic group, C 6-10 Aryl, 5- to 10-membered heteroaryl, and 3- to 10-membered heterocyclyl are as defined herein. Non-limiting examples include free amino (ie, -NH2).

[0144] "Aminoalkyl" refers to an amino group linked through an alkylene group.

[0145] "Alkoxyalkyl" refers to an alkoxy group linked through an alkylene group, for example, "C 2-8 Alkoxyalkyl" etc.

[0146] As used herein, a "glucosyl moiety" is a monovalent moiety in which one of the hydroxyl groups of a glucose is replaced by a bond, functional group, or moiety to another atom. Unless otherwise indicated, the glucose can have any suitable stereochemistry. Thus, the term includes moieties having a D stereochemistry as well as moieties having an L stereochemistry. Additionally, the term includes moieties having an α stereochemistry as well as moieties having a β stereochemistry. The carbon atoms of the glucosyl moiety follow conventional numbering, as shown below. The figure shows β-D glucose, but applies in a similar manner to glucosyl moieties having an α and / or L stereochemistry:

[0147]

[0148] As used herein, a "glucuronyl moiety" is a monovalent moiety in which one of the hydroxyl groups of the glucuronic acid is replaced by a bond, functional group, or moiety to another atom. Unless otherwise indicated, the glucuronic acid can have any suitable stereochemistry. Thus, the term includes moieties having a D stereochemistry as well as moieties having an L stereochemistry. Additionally, the term includes moieties having an α stereochemistry as well as moieties having a β stereochemistry. The carbon atoms of the glucuronyl moiety follow conventional numbering, as shown below. The figure shows β-D glucuronic acid, but applies in a similar manner to glucuronyl moieties having an α and / or L stereochemistry:

[0149]

[0150] The term "C 1-6 An "alkyl glucuronyl ester moiety" is a glucuronyl moiety (as defined in this paragraph) in which the carboxylic acid group of the glucuronic acid has a C 1-6 In any of the following embodiments, C1-6 The alkyl moiety can have any suitable value, such as methyl, ethyl, isopropyl, propyl, butyl, pentyl, etc. In some embodiments, C 1-6 In some other embodiments, the alkyl moiety is methyl. 1-6 The alkyl portion is ethyl.

[0151] As used herein, a substituted group is derived from an unsubstituted parent group wherein one or more hydrogen atoms have been exchanged for another atom or group. Unless otherwise specified, when a group is referred to as being "substituted by...", it means that the group is substituted with one or more substituents independently selected from the group consisting of C1-C6 alkyl, C1-C6 alkenyl, C1-C6 alkynyl, C1-C6 heteroalkyl, C3-C7 carbocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclic-C1-C6-alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), halogen, cyano, hydroxy, C1-C6 alkoxy, C1-C6 alkoxy(C1-C6)alkyl (i.e., ether), aryloxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclyl (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 haloalkoxy), 3- to 10-membered heterocyclyloxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryloxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7-carbocyclyl-C1-C6 alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6-alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy,C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl(C1-C6)alkoxy (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), sulfhydryl (mercapto), halo(C1-C6)alkyl (e.g. -CF3), halo(C1-C6)alkoxy (e.g. -OCF3), C1-C6 alkylsulfhydryl C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C3-C7 carbocyclic thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 5- to 10-membered heteroaryl-thio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), C1-C6 alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), 3- to 10-membered heterocyclyl-C1-C6 alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), aryl(C1-C6)alkylthio (optionally substituted with halogen, C1-C6 alkyl, C1-C6 alkoxy, C1-C6 haloalkyl and C1-C6 haloalkoxy), alkyl, C1-C6 haloalkyl, C1-C6 alkylthio, C1-C6 alkyl, C1-C6 haloalkoxy, C1-C6 haloalkyl, and C1-C6 haloalkoxy), amino, amino(C1-C6)alkyl, nitro, O-carbamoyl, N-carbamoyl, O-thiocarbamoyl, N-thiocarbamoyl, C-amide, N-amide, S-sulfonamide, N-sulfonamide, C-carboxy, O-carboxy, acyl, cyanate, isocyanate, thiocyanate, isothiocyanate, sulfinyl, sulfonyl, and oxo (=O). Where a group is described as "optionally substituted with," the group may be substituted with the substituents described above.

[0152] It should be understood that, depending on the context, certain radical naming conventions may include monoradicals or diradicals. For example, when a substituent requires two points of attachment to the rest of the molecule, it is understood that the substituent is a diradical. For example, a substituent that is identified as an alkyl group requiring two points of attachment includes a diradical, such as -CH2-, -CH2CH2-, -CH2CH(CH3)CH2-, etc. Other radical naming conventions clearly indicate that the radical is a diradical, such as "alkylene" or "alkenylene."

[0153] Where a substituent is described as diradical (i.e., having two points of attachment to the rest of the molecule), it is understood that the substituent may be attached in any orientation unless otherwise indicated. Thus, for example, a substituent represented by -AE- or Substituents of include those oriented such that A is attached at the leftmost point of attachment in the molecule as well as those where A is attached at the rightmost point of attachment in the molecule.

[0154] As used herein, a "sweetener," "sweet flavoring," "sweet flavor entity," or "sweet compound" refers to a compound that elicits a detectable sweet taste in a subject, or an ingestibly acceptable salt thereof.

[0155] As used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to "a substituent" encompasses a single substituent as well as two or more substituents, and so forth.

[0156] As used herein, "for example," "such as," "such as," or "including" are intended to introduce examples that further illustrate a more general subject matter. Unless expressly stated otherwise, such examples are provided merely to aid in understanding the embodiments shown in the present disclosure and are not meant to be limiting in any way. These phrases also do not indicate any preference for the disclosed embodiments.

[0157] As used herein, "include," "comprising," "containing," and "comprised of" refer to open groups, meaning that the group may include other members in addition to those explicitly stated. For example, the phrase "comprising A" means that A must be present, but other members may also be present. The terms "including," "having," and "composed of" and their grammatical variations have the same meaning. In contrast, "consist of," "composed of," or "composed of" refer to closed groups. For example, the phrase "consisting of A" means that only A is present.

[0158] As used herein, "optionally" means that the subsequently described event may or may not occur. In some embodiments, the optional event does not occur. In some other embodiments, the optional event does occur one or more times.

[0159] As used herein, "or" is to be given its broadest reasonable interpretation and is not limited to an "either / or" construction. Thus, the phrase "comprising A or B" means that A may be present and B may not be present, or B may be present and A may not be present, or both A and B may be present. Furthermore, for example, if A defines a class that may have multiple members (e.g., A1 and A2), then one or more members of the class may be present at the same time.

[0160] As used herein, the term "flavor-modifying compound" refers to a compound of Formula (I) or Formula (Ia) or a salt thereof, or any embodiment thereof described herein.

[0161] As used herein, certain monovalent or multivalent groups having only a single atom may be referred to by the name of the atom. For example, in some cases, the substituent "-H" may be referred to as "hydrogen" or "hydrogen atom," or the substituent "-F" may be referred to as "fluorine" or "fluorine atom," and the linking group "-O-" may be referred to as "oxygen" or "oxygen atom."

[0162] The point of attachment of a group is usually indicated by a dash (-) or an asterisk (*). For example, groups such as *-CH2-CH3 or -CH2-CH3 both represent ethyl groups.

[0163] Chemical structures are often displayed using a "skeleton" format, so that no carbon atoms are shown explicitly and hydrogen atoms attached to them are omitted entirely. For example, the structure represents butane (i.e., n-butane). In addition, aromatic groups such as benzene are represented by showing a contributing resonance structure. For example, the structure Represents toluene.

[0164] Positions on the flavanone ring may be indicated by numbers, such as "position 2," "position 3," etc. These terms refer to a specific position on the flavanone fused ring structure, even though further substitutions on the fused ring structure may result in a different numbering of the position. Thus, for example, the position of a carbonyl substitution is referred to as occurring at "position 4," regardless of whether, in some embodiments, addition of further substituents would result in it occurring at a position other than position 4 on the fused ring structure. Similarly, the position of a phenyl substituent substituted on the fused ring structure is referred to as occurring at "position 2," regardless of whether, in some embodiments, addition of further substituents would result in it occurring at a position other than position 2 on the fused ring structure.

[0165] The term "dihydroflavonol" or "flavanonol" refers to a flavan-4-one containing an OH group at the 3-position.

[0166] The term "polypeptide" refers to a continuous polymer of amino acid residues, such as an amino acid sequence of at least 15 residues, at least 30 residues, or at least 50 residues. In some embodiments herein, the polypeptide comprises an amino acid sequence that is an enzyme, or a fragment thereof, or a variant or mutant thereof.

[0167] The term "protein" refers to an amino acid sequence of any length in which the amino acids are linked by covalent peptide bonds, and includes oligopeptides, peptides, polypeptides and full-length proteins, whether naturally occurring or synthetic.

[0168] The term "isolated" polypeptide refers to an amino acid sequence that has been removed from its natural environment by any method known in the art, or a combination of methods, including recombinant, biochemical, and synthetic methods.

[0169] The terms "biological function", "function", "biological activity" or "activity" refer to the ability of an acyltransferase to catalyze the formation of a compound of formula (I).

[0170] The terms "nucleic acid sequence," "nucleic acid," "nucleic acid molecule," and "polynucleotide" are used interchangeably to refer to a sequence of nucleotides. A nucleic acid sequence can be single-stranded or double-stranded deoxyribonucleotides or ribonucleotides of any length and includes coding and non-coding sequences of genes, exons, introns, sense and antisense complementary sequences, genomic DNA, cDNA, miRNA, siRNA, mRNA, rRNA, tRNA, recombinant nucleic acid sequences, isolated and purified naturally occurring DNA and / or RNA sequences, synthetic DNA and RNA sequences, fragments, primers, and nucleic acid probes. The skilled artisan understands that the nucleic acid sequence of RNA is identical to the sequence of DNA, except that thymine (T) is replaced by uracil (U). The term "nucleotide sequence" should also be understood to include polynucleotide molecules or oligonucleotide molecules, either in the form of individual fragments or as components of a larger nucleic acid.

[0171] "Isolated nucleic acid" or "isolated nucleic acid sequence" refers to a nucleic acid or nucleic acid sequence that is found in an environment that is different from that in which it occurs in nature, and can include those that are substantially free of contaminating endogenous materials. As used herein, the term "naturally occurring" as applied to nucleic acids refers to a nucleic acid that is found in the cells of an organism in nature and has not been intentionally modified by man in a laboratory.

[0172] A "recombinant nucleic acid sequence" is a nucleic acid sequence produced by combining genetic material from more than one source using laboratory methods (e.g., molecular cloning), thereby creating or modifying a nucleic acid sequence that does not occur in nature and is not otherwise found in a biological organism.

[0173] "Recombinant DNA technology" refers to molecular biology methods for preparing recombinant nucleic acid sequences as described, for example, in Laboratory Manuals, ed., Weigel and Glazebrook, 2002, Cold Spring Harbor Lab Press; and Sambrook et al., 1989 Cold Spring Harbor, NY: Cold Spring Harbor Laboratory Press.

[0174] The term "gene" refers to a DNA sequence that includes a region that is transcribed into an RNA molecule, such as mRNA, in a cell and is operably linked to an appropriate regulatory region (e.g., a promoter). Thus, a gene may include multiple operably linked sequences, such as a promoter, a 5' leader sequence (including, for example, sequences involved in translation initiation), a coding region of cDNA or genomic DNA, introns, exons, and / or a 3' untranslated sequence (including, for example, a transcription termination site).

[0175] "Chimeric gene" refers to any gene that can not usually be found in species in nature, particularly a gene in which one or more parts of the nucleic acid sequence are not associated with each other in nature. For example, a promoter is not associated with part or all of a transcription region or with another regulatory region in nature. The term "chimeric gene" should be understood to include expression constructs in which a promoter or transcriptional regulatory sequence is operably linked to one or more coding sequences or antisense (i.e., the reverse complementary strand of the sense strand) or inverted repeats (sense and antisense, whereby RNA transcripts form double-stranded RNA after transcription). The term "chimeric gene" also includes genes obtained by combining parts of one or more coding sequences to produce new genes.

[0176] "3' URT" or "3' untranslated sequence" (also called "3' untranslated region" or "3' end") refers to a nucleic acid sequence found downstream of the coding sequence of a gene that contains, for example, a transcription termination site and (in most but not all eukaryotic mRNAs) a polyadenylation signal, such as AAUAAA or a variant thereof. Following transcription termination, the mRNA transcript may be cleaved downstream of the polyadenylation signal and a poly(A) tail may be added, which participates in the transport of the mRNA to the site of translation, such as the cytoplasm.

[0177] "Gene expression" encompasses "heterologous expression" and "overexpression" and involves the transcription of a gene and the translation of mRNA into protein. Overexpression refers to the production of a gene product in a transgenic cell or organism, as measured by mRNA, polypeptide, and / or enzyme activity levels, that exceeds the level of production in a non-transformed cell or organism of a similar genetic background.

[0178] As used herein, "expression vector" refers to a nucleic acid molecule that is engineered using molecular biology methods and recombinant DNA technology to deliver foreign or exogenous DNA into a host cell. An expression vector typically includes sequences required for the correct transcription of the nucleotide sequence. The coding region usually encodes a protein of interest, but may also encode RNA, such as antisense RNA, siRNA, etc.

[0179] As used herein, "expression vector" includes any linear or circular recombinant vector, including but not limited to viral vectors, phages and plasmids. The skilled person can select a suitable vector according to the expression system. In one embodiment, the expression vector includes a nucleic acid of the embodiments herein, which is operably linked to at least one "regulatory sequence" that controls transcription, translation, initiation and termination, such as a transcriptional promoter, operator or enhancer, or an mRNA ribosome binding site, and optionally includes at least one selection marker. When a regulatory sequence is functionally related to a nucleic acid of the embodiments herein, the nucleotide sequence is "operably linked".

[0180] "Regulatory sequence" refers to a nucleic acid sequence that determines the expression level of the nucleic acid sequence of the embodiments herein and is capable of regulating the transcription rate of the nucleic acid sequence operably linked to the regulatory sequence. Regulatory sequences include promoters, enhancers, transcription factors, promoter elements, etc.

[0181] "Promoter" refers to a nucleic acid sequence that controls the expression of a coding sequence by providing a binding site for RNA polymerase and other factors required for transcription, including but not limited to transcription factor binding sites, repressor and activator protein binding sites. The term promoter also includes the term "promoter regulatory sequence". The promoter regulatory sequence may include upstream and downstream elements that may affect the stability of transcription, RNA processing or the associated coding nucleic acid sequence. Promoters include sequences of natural origin and synthesis. The coding nucleic acid sequence is usually located downstream of the promoter relative to the transcription direction starting with the transcription start site.

[0182] The term "constitutive promoter" refers to an unregulated promoter that allows for continuous transcription of a nucleic acid sequence to which it is operably linked.

[0183] As used herein, the term "operably connected" refers to the connection of a polynucleotide element in a functional relationship. When a nucleic acid is in a functional relationship with another nucleic acid sequence, the nucleic acid is "operably connected". For example, if a promoter or transcriptional regulatory sequence can affect the transcription of a coding sequence, the promoter or transcriptional regulatory sequence is operably connected to the coding sequence. Operably connected means that the DNA sequence being connected is usually adjacent. The nucleotide sequence associated with the promoter sequence can be of homologous or heterologous origin relative to the plant to be transformed. The sequence can also be fully or partially synthesized. Regardless of the source, the nucleic acid sequence associated with the promoter sequence will be expressed or silenced according to the promoter properties connected after being attached to the polypeptide of this paper's embodiment. The relevant nucleic acid can encode the protein that needs to be expressed or suppressed at all times or alternatively at a specific time in the whole organism or in a specific tissue, cell or cell chamber. This nucleotide sequence encodes the protein that the desired phenotypic traits are given to the host cell or organism that is changed or transformed by it. More particularly, the nucleotide sequence of interest leads to the production of a compound of formula (I) or a mixture comprising a compound of formula (I) and one or more other compounds in a cell or organism. In particular, the nucleotide sequence encodes a polypeptide having acyltransferase activity.

[0184] "Target peptide" refers to an amino acid sequence that targets a protein or polypeptide to an intracellular organelle (i.e., mitochondria or plastids) or the extracellular space (secretion signal peptide). The nucleic acid sequence encoding the target peptide can be fused to the nucleic acid sequence encoding the amino terminus (e.g., N-terminus) of the protein or polypeptide, or can be used to replace the native targeting polypeptide.

[0185] The term "primer" refers to a short nucleic acid sequence that hybridizes to a template nucleic acid sequence and is used to polymerize a nucleic acid sequence complementary to the template.

[0186] As used herein, the term "host cell" or "transformed cell" or "recombinant cell" refers to a cell (or organism) that has been modified to harbor at least one nucleic acid molecule, for example, a recombinant gene encoding a desired protein or nucleic acid sequence, which, upon transcription, can produce an acyltransferase protein for producing a compound of formula (I) or a mixture comprising a compound of formula (I) and one or more other compounds. The host cell may contain the recombinant gene integrated into the host cell's nuclear or organelle genome. Alternatively, the host cell may contain the recombinant gene extrachromosomally.

[0187] The host cell can be a prokaryotic cell, an archaeal cell, or a eukaryotic cell.

[0188] Prokaryotic cells may be, but are not limited to, bacterial cells. Bacterial cells may be Gram-negative bacteria or Gram-positive bacteria. Examples of bacteria include, but are not limited to, bacteria belonging to the following genera: Bacillus (e.g., Bacillus subtilis, Bacillus amyloliquefaciens, Bacillus licheniformis, Bacillus puntis, Bacillus megaterium, Bacillus halodurans, Bacillus pumilus), Acinetobacter (Bacillus spp.), Bacillus spp. ter), Nocardia, Xanthobacter, Escherichia (e.g., E. coli), Streptomyces, Erwinia, Klebsiella, Serratia (e.g., Serratia marcessans), Pseudomonas (e.g., Pseudomonas aeruginosa), inosa), Pseudomonas fluorescens), Salmonella (e.g., S. typhimurium, S. typhi), Anabaena, Caulobacter, Gluconobacter, Rhodobacter, Paracoccus, Brevibacterium, Corynebacterium, Rhizobium (Sinorhizobium), Flavobacterium, Klebsiella, Enterobacter, Lactobacillus, Lactococcus, Methylobacterium, Staphylococcus. Bacteria also include, but are not limited to, photosynthetic bacteria (eg, green non-sulfur bacteria, green sulfur bacteria, purple sulfur bacteria, and purple non-sulfur bacteria).

[0189] The eukaryotic cell may be, but is not limited to, a fungus (such as yeast or filamentous fungi), an algae, a plant cell, or a cell line.

[0190] The eukaryotic cell may be a fungus, such as a filamentous fungus or a yeast. Filamentous fungal strains include, but are not limited to, strains of the genera Acremonium, Aspergillus (e.g., A. niger, A. oryzae, A. oryzae), Aspergillus nidulans), Agaricus, Aureobasidium, Coprinus, Cryptococcus, Corynascus, Chrysosporium, Filibasidium, Fusarium, Humicola, Magnaporthe, Monascus, Mucor, Myceliophthora, Mortierella, Neocallimastix, Neurospora, Paecilomyces, Penicillium and / or fungi. These fungi are, for example, Penicillium chrysogenum, P. Camemberti, Piromyces, Phanerochaete, Pleurotus, Podospora, Pycnoporus, Rhizopus, Schizophyllum, Sordaria, Talaromyces, Rasamsonia (e.g., Rasamsonia emersonii), Thermoascus, Thielavia, Tolypocladium, Trametes, and Trichoderma.

[0191] The yeast cell may be selected from the following genera: Saccharomyces (e.g., S. cerevisiae, S. bayanus, S. pastorianus, S. carlsbergensis), Kluyveromyces, Candida (e.g., C. rugosa, C. revkaufi, C. pulcherrima, C. tropicalis, C. utilis, C. krusei, C. truncatum), C. truncatum (e.g. ... krusei), Pichia (e.g., P. pastoris), Schizosaccharomyces, Issatchenkia (e.g., I. orientalis), Zygosaccharomyces, Hansenula, Kloeckera, Schwanniomyces, and Yarrowia (e.g., Y. lipolytica, formerly classified as Candida lipolytica).

[0192] The cell may be an algae, microalgae, or marine eukaryote. The cell may be a Labyrinthulomycetes cell, preferably belonging to the Thraustochytriales order, more preferably belonging to the Thraustochytriaceae family, more preferably belonging to a member of the genus selected from the group consisting of Aurantiochytrium, Oblongichytrium, Schizochytrium, Thraustochytrium, and Ulkenia, even more preferably belonging to Schizochytrium ATCC #20888.

[0193] Homologous sequences include orthologous or paralogous sequences. Methods for identifying orthologs or paralogs include phylogenetic methods, sequence similarity and hybridization methods known in the art and described herein.

[0194] Paralogs are derived from gene duplication, which produces two or more genes with similar sequences and similar functions. Paralogs are typically clustered together and formed by duplication of genes in related plant species. Paralogs are found in groups of similar genes using paired blast analysis or using programs such as CLUSTAL during phylogenetic analysis of gene families. Among paralogs, a consensus sequence can be identified as a sequence that is characterized by being in a related gene and having a similar function to that of a gene.

[0195] Orthologs or orthologous sequences are sequences similar to each other because they are found in species passed down by a common ancestor. For example, it is known that plant species with a common ancestor contain many enzymes with similar sequences and functions. For example, by using CLUSTAL or BLAST programs to build a phylogenetic tree of the gene family of a species, technicians can identify orthologous sequences and predict the function of orthologs. A method for identifying or confirming similar functions between homologous sequences is by comparing the transcript profiles in the host cell or organism (such as plant) that overexpresses or lacks (in gene knockout / knockdown) related polypeptides. Technicians can understand that genes with similar transcript profiles (having a common transcript greater than 50% regulation, or having a common transcript greater than 70% regulation, or a common transcript greater than 90% regulation) can have similar functions. Homologues, paralogs, orthologs and any other variants of sequences described herein are expected to play a role in a similar manner by making host cells, organisms (such as plants) produce formula (I) compounds.

[0196] The term "selectable marker" refers to any gene that, after expression, can be used to select for one or more cells containing the selectable marker. Examples of selectable markers are described below. Those skilled in the art will appreciate that different antibiotic, fungicide, auxotrophic or herbicide selectable markers may be suitable for use with different target species.

[0197] The term "organism" refers to any non-human multicellular or unicellular organism, such as a plant or a microorganism. In particular, a microorganism refers to a bacterium, an archaea, an algae or a fungus, such as a yeast.

[0198] The term "plant" is used interchangeably to include plant cells, including plant protoplasts, plant tissues, plant cell tissue cultures to produce regenerated plants, or parts of plants, or plant organs such as roots, stems, leaves, flowers, pollen, ovules, embryos, fruits, etc. Any plant can be used to practice the methods of the embodiments herein.

[0199] As used herein, the terms "phenylalanine ammonia lyase" and "PAL" refer to both an encoding nucleic acid and the phenylalanine ammonia lyase enzyme itself. Phenylalanine ammonia lyase (EC 4.3.1.24) catalyzes the conversion of L-phenylalanine to trans-cinnamic acid. GenBank Accession No. AY303128 provides an example of a PAL sequence. The term also includes enzymes of the EC 4.3.1.25 class, which are bifunctional phenylalanine / tyrosine ammonia lyases.

[0200] As used herein, the terms "cinnamate 4-hydroxylase" and "C4H" refer to both an encoding nucleic acid and the cinnamate 4-hydroxylase enzyme itself. Cinnamate 4-hydroxylase (EC 1.14.14.91) catalyzes the conversion of trans-cinnamate to p-coumaric acid. C4H is a P450 monooxygenase that benefits from the regeneration of cytochrome P450 reductase. GenBank Accession No. U71080 provides an example of a C4H sequence.

[0201] As used herein, the terms "cytochrome P450 reductase" and "CPR" refer to both an encoding nucleic acid and the cytochrome P450 reductase enzyme itself. Cytochrome P450 reductase (EC 1.6.2.4) is an enzyme required for electron transfer from NAD(P)H to cytochrome P450 monooxygenases (including C4H and F3'H). GenBank accession numbers X66017 and NM_119167 provide an example of a CPR sequence.

[0202] As used herein, the terms "tyrosine ammonia-lyase" and "TAL" refer to an encoding nucleic acid and the tyrosine ammonia-lyase enzyme itself. Tyrosine ammonia-lyase (EC 4.3.1.23) catalyzes the conversion of L-tyrosine to p-coumaric acid. An example of a TAL sequence is provided by GenBank Accession No. Q3IWB0. The term also includes enzymes of the EC 4.3.1.25 class, which are bifunctional phenylalanine / tyrosine ammonia-lyases.

[0203] As used herein, the terms "4-coumarate-CoA ligase" and "4CL" refer to both an encoding nucleic acid and the enzyme 4-coumarate-CoA ligase itself. 4-Coumarate-CoA ligase (EC 6.2.1.12) catalyzes the conversion of p-coumaric acid to p-coumaroyl-CoA. An example of a 4CL sequence is provided by GenBank Accession No. U18675.

[0204] As used herein, the terms "chalcone synthase" and "CHS" refer to both an encoding nucleic acid and the chalcone synthase enzyme itself. Chalcone synthase (EC 2.3.1.74) catalyzes the condensation of p-coumaroyl-CoA with three malonyl-CoA molecules to produce naringenin chalcone. GenBank accession number AF112086 provides an example of a CHS sequence.

[0205] As used herein, the terms "chalcone isomerase" and "CHI" refer to an encoding nucleic acid and the chalcone isomerase enzyme itself. Chalcone isomerase (EC 5.5.1.6) catalyzes the conversion of naringenin chalcone to naringenin. An example of a CHI sequence is provided by GenBank Accession No. M86358.

[0206] As used herein, the terms "chalcone isomerase-like protein" and "CHIL" refer to an encoding nucleic acid and the chalcone isomerase-like protein itself. Chalcone isomerase-like proteins can enhance the activity of CHS. GenBank accession number NP_850770 provides an example of a CHIL sequence.

[0207] As used herein, the terms "flavanone 3-hydroxylase" and "F3H" refer to an encoding nucleic acid and the enzyme flavanone 3-hydroxylase itself. Flavanone 3-hydroxylase (EC 1.14.11.9) catalyzes the conversion of naringenin to naringenin, eriodictyol to taxifolin, hesperetin to dihydrotamarixanthin, homoeriodictyol to 3'-O-methyldiquixanthin, pincembrin / pinocembrin to brevifolia, liquiritigenin to 5-deoxynaringenin, 5-deoxyeriodictyol to 5-deoxydiquixanthin, 5-deoxyhesperetin to 5-deoxydihydrotamarixanthin, 5-deoxyhomoeriodictyol to 5-deoxy-3'-O-methyl-diquixanthin, or 5-deoxypinus to 5-deoxybrevifolia. An example of an F3H sequence is provided by GenBank accession number U33932.

[0208] As used herein, the terms "flavonoid 3'-hydroxylase" and "F3'H" refer to both an encoding nucleic acid and the enzyme itself. Flavonoid 3'-hydroxylase (EC 1.14.14.82) catalyzes the addition of an OH group to the 3' position of flavanones (e.g., naringenin) or dihydroflavonols (e.g., naringenin). F3'H is a P450 monooxygenase that benefits from the regeneration of cytochrome P450 reductase. An example of an F3'H sequence is provided by GenBank Accession No. AH009204.

[0209] As used herein, the terms "3'-O-methyltransferase" and "3'-MT" refer to both an encoding nucleic acid and the 3'-O-methyltransferase enzyme itself. 3'-O-methyltransferase (EC 2.1.1) catalyzes the transfer of a methyl group to the 3'-OH group of a flavanone (e.g., eriodictyol) or a dihydroflavonol (e.g., taxifolin). An example of a 3'-MT sequence is provided by GenBank Accession No. NP_200227.

[0210] As used herein, the terms "4'-O-methyltransferase" and "4'-MT" refer to both an encoding nucleic acid and the 4'-O-methyltransferase enzyme itself. 4'-O-methyltransferase (EC 2.1.1) catalyzes the transfer of a methyl group to the 4'-OH group of a flavanone (e.g., naringenin or eriodictyol) or a dihydroflavonol (e.g., neritin or taxifolin). An example of a 4'-MT sequence is provided by GenBank Accession No. C6TAY1.

[0211] As used herein, the terms "O-methyltransferase" and "OMT" refer to an encoding nucleic acid and the O-methyltransferase enzyme itself. O-methyltransferases (EC 2.1.1) catalyze the transfer of a methyl group to an OH group of an acceptor molecule, such as tyrosine, (hydroxyphenyl)-2-propenoic acid (e.g., coumaric acid or caffeic acid), a flavanone (e.g., eriodictyol), or a dihydroflavonol (e.g., taxifolin).

[0212] As used herein, the terms "glycosyltransferase" and "GT" refer to an encoding nucleic acid and a glycosyltransferase. Glycosyltransferases catalyze the transfer of a sugar moiety from an activated nucleotide sugar to a nucleophilic glycosyl acceptor molecule, in this case a dihydroflavonol-3-O-acetate, such as aurantin-3-O-acetate, taxifolin-3-O-acetate, dihydrotaciferin-3-O-acetate, 3'-O-methyltaciferin-3-O-acetate, brevifoliain-3-O-acetate, 5-deoxyurantin-3-O-acetate, 5-deoxytaxifolin-3-O-acetate, 5-deoxydihydrotaciferin-3-O-acetate, or 5-deoxy-3'-O-methyltaxifolin-3-O-acetate or 5-deoxybrevifoliain-3-O-acetate.

[0213] As used herein, the term "glycosidase (glycoside hydrolase)" refers to both an encoding nucleic acid and the glycosidase itself. Glycosidases (EC 3.2.1) catalyze the hydrolysis of glycosidic bonds, in this case, glycosylated flavanone precursors (e.g., naringin or hesperidin) to the corresponding aglycones, e.g., naringenin and hesperetin.

[0214] As used herein, the terms "polyketide reductase" and "PKR" refer to an encoding nucleic acid and the polyketide reductase itself. Polyketide reductase (EC 2.3.1.170) couples with CHS and catalyzes the reduction of a specific keto group in a tetraketide intermediate to produce 6'-deoxychalcone. In this context, the term chalcone reductase or CHR is also used in the literature. However, since this term is misleading, its use is not recommended ( (Comprehensive Natural Product Chemistry, 1999, Chapter 1.27.6.1). An example of a PKR sequence is provided by GenBank Accession No. AB263016.

[0215] Other terms are defined in other parts of this specification even if not included in this section.

[0216] Product and precursor compounds

[0217] The present invention relates to a method for preparing a compound of formula (I).

[0218] Compound of formula (I):

[0219]

[0220] in:

[0221] R 1 A hydrogen atom, -OH or -OR 1A ;

[0222] R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0223] R 2 A hydrogen atom, -OH or -OR 2A ;

[0224] R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0225] R 3 A hydrogen atom, -OH or -OR 3A ;

[0226] R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0227] R 4 A hydrogen atom, -OH or -OR 4A ;

[0228] R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0229] R 5 -OC(O)-(C 1-24 alkyl);

[0230] R6 and R 7 Independently from C 1-6 Alkyl, -OH, C 1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group;

[0231] m is 0, 1, or 2; and

[0232] n is 0, 1, 2 or 3.

[0233] The process of the present invention comprises reacting a precursor compound of formula (Ia).

[0234] Compound of formula (Ia):

[0235]

[0236] in:

[0237] R 1 A hydrogen atom, -OH or -OR 1A ;

[0238] R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0239] R 2 A hydrogen atom, -OH or -OR 2A ;

[0240] R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0241] R 3 A hydrogen atom, -OH or -OR 3A ;

[0242] R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times;

[0243] R 4 A hydrogen atom, -OH or -OR 4A ;

[0244] R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6The substituents of the alkoxy group are substituted one or more times;

[0245] R 6 and R 7 Independently from C 1-6 Alkyl, -OH, C 1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group;

[0246] m is 0, 1, or 2; and

[0247] n is 0, 1, 2 or 3.

[0248] R 1 Any suitable value can be taken according to the above parameters. In some embodiments, R 1 is H, -OH or -OCH3. In some further embodiments, R 1 It is -OH.

[0249] R 2 Any suitable value can be taken according to the above parameters. In some embodiments, R 2 is H, -OH or -OCH3. In some further embodiments, R 2 In some embodiments, R 1 and R 2 All are -OH.

[0250] R 3 Any suitable value can be taken according to the above parameters. In some embodiments, R 3 is H, -OH or -OCH3. In some further embodiments, R 3 is H. In some further embodiments, R 3 In some other embodiments, R 3 It is -OCH3.

[0251] R 4 Any suitable value can be taken according to the above parameters. In some embodiments, R 4 is H, -OH or -OCH3. In some further embodiments, R 4 is H. In some further embodiments, R 4 In some other embodiments, R 4 In some embodiments, R 3 and R 4 In some embodiments, R 3 -OH, R 4In some embodiments, R 3 and R 4 In some embodiments, R 3 -OH, R 4 In some embodiments, R 3 -OCH3, R 4 In some embodiments, R 3 and R 4 At least one of them is -OH.

[0252] R 5 Any suitable value can be taken according to the above parameters. In some embodiments, R 5 -OC(O)-(C 1-22 In some embodiments, R 5 -OC(O)-(C 1-18 In some embodiments, R 5 -OC(O)-(C 1-12 In some embodiments, R 5 -OC(O)-(C 1-8 In some embodiments, R 5 -OC(O)-(C 1-6 In some embodiments, R 5 In some embodiments, R 5 In some embodiments, R 5 is -OC(O)-CH(CH3)2. In some embodiments, R 5 In some embodiments, R 5 In some embodiments, R 5 It is -OC(O)-(CH2)4-CH3.

[0253] In some embodiments, R 5 It is -OC(O)-(CH2)5-CH3.

[0254] R 6 and R 7 The above parameters may take any suitable value and may appear any number of times according to the variables m and n. 6 and R 7In some embodiments, m+n is 0, 1, or 2. In some embodiments, m+n is 0 or 1. In some embodiments, m is 0 and n is 0 or 1. In some embodiments, m and n are both 0.

[0255] A preferred embodiment of the present invention is that the compound of formula (Ia) is aurantifoliin, taxifolin, dihydrotamarixanthin, 3'-O-methyltamarixanthin, brevifoliin, 5-deoxyaurantifoliin, 5-deoxytaxifolin, 5-deoxydihydrotamarixanthin, 5-deoxy-3'-O-methyltamarixanthin or 5-deoxybrevifoliin.

[0256] "Aurora cinnamomea", also known as dihydrokaempferol, is a compound known in the art. The preferred IUPAC name is (2R,3R)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one.

[0257] "Taixin" is a compound known in the art. The preferred IUPAC name is (2R,3R)-3,5,7-trihydroxy-2-(3,4-dihydroxyphenyl)-2,3-dihydrochromen-4-one.

[0258] "Dihydrotamaricin" is a compound known in the art. Its IUPAC name is (2R,3R)-3,5,7-trihydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0259] "3'-O-Methyltaxifolin" is a compound known in the art. Its IUPAC name is (2R,3R)-3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0260] "Brevifolia pine" is a compound known in the art. Its IUPAC name is (2R,3R)-3,5,7-trihydroxy-2-phenyl-2,3-dihydrochromen-4-one.

[0261] "5-Deoxyauricularin" is known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrochromen-4-one.

[0262] "5-Deoxytaxifolin" is known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(3,4-dihydroxyphenyl)-2,3-dihydrochromen-4-one.

[0263] "5-Deoxydihydrotamaricin" is known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0264] "5-Deoxy-3'-O-methyltaxifolin" is known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0265] "5-Deoxybreviol" is known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-phenyl-2,3-dihydrochromen-4-one.

[0266] A preferred embodiment of the present invention is that the compound of formula (I) is aurantifoliin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixin-3-O-acetate, 3'-O-methyltamarixin-3-O-acetate, brevifoliin-3-O-acetate, 5-deoxyaurantifoliin-3-O-acetate, 5-deoxytamarixin-3-O-acetate, 5-deoxydihydrotamarixin-3-O-acetate, 5-deoxy-3'-O-methyltamarixin-3-O-acetate or 5-deoxybrevifoliin-3-O-acetate.

[0267] "Aurantium-3-O-acetate" is a compound known in the art. The preferred IUPAC name is [(2R,3R)-5,7-dihydroxy-2-(4-hydroxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0268] "Taixin-3-O-acetate" is a compound known in the art. The preferred IUPAC name is [(2R,3R)-5,7-dihydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0269] "Dihydrotamariscine-3-O-acetate" is a compound known in the art. Its IUPAC name is [(2R,3R)-5,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0270] "3'-O-Methyltaxifolin-3-O-acetate" is a compound known in the art. Its IUPAC name is [(2R,3R)-5,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0271] "Brevipine-3-O-acetate" is a compound known in the art. The IUPAC name is [(2R,3R)-5,7-dihydroxy-2-phenyl-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0272] The preferred IUPAC name of “5-deoxyaurantifolin-3-O-acetate” is [(2R,3R)-7-hydroxy-2-(4-hydroxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0273] The preferred IUPAC name of “5-deoxytaxifolin-3-O-acetate” is [(2R,3R)-7-hydroxy-2-(3,4-dihydroxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0274] The preferred IUPAC name of “5-deoxydihydrotamariscine-3-O-acetate” is [(2R,3R)-7-hydroxy-2-(3-hydroxy-4-methoxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0275] The preferred IUPAC name of “5-deoxy-3′-O-methyltaxifolin-3-O-acetate” is [(2R,3R)-7-hydroxy-2-(4-hydroxy-3-methoxyphenyl)-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0276] The IUPAC name of “5-deoxybreviol-3-O-acetate” is [(2R,3R)-7-hydroxy-2-phenyl-4-oxo-2,3-dihydrochromen-3-yl]acetate.

[0277] By acetylation of the R5 position of the compound of formula (Ia), compounds such as citronellin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltaxifolin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxycitronellin-3-O-acetate, 5-deoxytaxifolin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, 5-deoxy-3'-O-methyltaxifolin-3-O-acetate and 5-deoxybrevicornin-3-O-acetate can be prepared.

[0278] If the compounds disclosed herein possess at least one chiral center not explicitly indicated in the formula, they may exist as individual enantiomers and diastereomers or as mixtures of such isomers. In some embodiments, the sweet taste enhancing compounds have substantial enantiomeric purity.

[0279] The separation of each isomer or the selective synthesis of each isomer is completed by applying various methods well known to those skilled in the art. Unless otherwise stated (for example, in the case of clearly showing the stereochemistry of the chiral center), all such isomers and mixtures thereof are included in the scope of compounds disclosed herein. In addition, compounds disclosed herein can exist in one or more crystalline or amorphous forms. Unless otherwise stated, all these forms are included in the scope of compounds disclosed herein, including any polymorphic form. In addition, some compounds disclosed herein can form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise stated, these solvates are included in the scope of compounds disclosed herein.

[0280] Those skilled in the art will recognize that some of the structures described herein may be resonance forms or tautomers of compounds that can be reasonably represented, even kinetically, by other chemical structures; the skilled artisan recognizes that such structures may represent only a small fraction of such compound samples. Such compounds are considered to be within the scope of the structures shown, even though such resonance forms or tautomers are not shown herein.

[0281] Isotopes may be present in the described compounds. Each chemical element represented in a compound structure may include any isotope of that element. For example, in a compound structure, a hydrogen atom may be explicitly disclosed or understood to be present in the compound. Any position in the compound where a hydrogen atom may be present may include any isotope of hydrogen, including but not limited to hydrogen-1 (protium) and hydrogen-2 (deuterium). Therefore, the compounds mentioned herein encompass all possible isotopic forms unless the context clearly indicates otherwise.

[0282] In some embodiments, the compounds disclosed herein are capable of forming acidic and / or basic salts due to the presence of amino and / or carboxyl groups or groups similar thereto. Physiologically acceptable acid addition salts can be formed with inorganic and organic acids. Inorganic acids from which salts can be derived include, for example, hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid, and the like. Organic acids from which salts can be derived include, for example, acetic acid, propionic acid, glycolic acid, pyruvic acid, oxalic acid, maleic acid, malonic acid, succinic acid, fumaric acid, tartaric acid, citric acid, benzoic acid, cinnamic acid, mandelic acid, methanesulfonic acid, ethanesulfonic acid, p-toluenesulfonic acid, salicylic acid, and the like. Physiologically acceptable salts can be formed with inorganic and organic bases. Inorganic bases from which salts can be derived include, for example, bases containing sodium, potassium, lithium, ammonium, calcium, magnesium, iron, zinc, copper, manganese, aluminum, and the like; particularly preferred are ammonium, potassium, sodium, calcium, and magnesium salts. In some embodiments, treatment of the compounds disclosed herein with an inorganic base causes the compound to lose an unstable hydrogen, thereby obtaining a compound containing an inorganic cation such as Li + 、Na + , K + Mg 2+and Ca 2 + The organic bases from which salts can be derived include, for example, primary, secondary, and tertiary amines, substituted amines, including naturally occurring substituted amines, cyclic amines, basic ion exchange resins, and the like, particularly isopropylamine, trimethylamine, diethylamine, triethylamine, tripropylamine, and ethanolamine. In some embodiments, the salt is an edible salt, which is a salt suitable for inclusion in edible food and / or beverage products.

[0283] When the compounds disclosed herein possess at least one chiral center, they may exist as individual enantiomers and diastereomers or as mixtures of such isomers.In some embodiments related to the second aspect, the sweet taste enhancing compound has substantial enantiomeric purity.

[0284] For example, in some embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2R,3R) enantiomer comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound [i.e., the amount of (2R,3R) + the amount of (2S,3R) + the amount of (2R,3S) + the amount of (2S,3S)]. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2R,3S) enantiomer comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2S, 3R) enantiomer accounts for at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2S, 3S) enantiomer accounts for at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition.

[0285] In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of formula (I or Ia) or a salt thereof, wherein the (2R, 3R) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of formula (I or Ia) or a salt thereof, wherein the (2R, 3S) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of Formula (I or Ia), or a salt thereof, wherein the (2S,3R) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of Formula (I or Ia), or a salt thereof, wherein the (2S,3S) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the compound present in the composition, based on the total weight of the compound in the composition.

[0286] For example, in some embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2R,3R) enantiomer comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of compound present in the composition, based on the total weight of the compound in the composition [i.e., the amount of (2R,3R) + the amount of (2S,3R) + the amount of (2R,3S) + the amount of (2S,3S)]. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2R,3S) enantiomer comprises at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2S, 3R) enantiomer accounts for at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments, in a composition comprising a compound of Formula (I or Ia) or a salt thereof, the (2S, 3S) enantiomer accounts for at least 50% by weight, or at least 60% by weight, or at least 70% by weight, or at least 80% by weight, or at least 90% by weight, or at least 95% by weight, or at least 97% by weight, or at least 99% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition.

[0287] In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of formula (I or Ia) or a salt thereof, wherein the (2R, 3R) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of formula (I or Ia) or a salt thereof, wherein the (2R, 3S) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the amount of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of Formula (I or Ia), or a salt thereof, wherein the (2S,3R) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the compound present in the composition, based on the total weight of the compound in the composition. In some other embodiments of any of the foregoing embodiments, the composition comprises a compound of Formula (I or Ia), or a salt thereof, wherein the (2S,3S) enantiomer comprises no more than 50% by weight, or no more than 40% by weight, or no more than 30% by weight, or no more than 20% by weight, or no more than 10% by weight, or no more than 5% by weight, or no more than 3% by weight, or no more than 1% by weight of the compound present in the composition, based on the total weight of the compound in the composition.

[0288] The separation of each isomer or the selective synthesis of each isomer is completed by applying various methods well known to those skilled in the art. Unless otherwise stated (for example, in the case of clearly showing the stereochemistry of the chiral center), all such isomers and mixtures thereof are included in the scope of compounds disclosed herein. In addition, compounds disclosed herein can exist in one or more crystalline or amorphous forms. Unless otherwise stated, all these forms are included in the scope of compounds disclosed herein, including any polymorphic form. In addition, some compounds disclosed herein can form solvates with water (i.e., hydrates) or common organic solvents. Unless otherwise stated, these solvates are included in the scope of compounds disclosed herein.

[0289] Those skilled in the art will recognize that some of the structures described herein may be resonance forms or tautomers of compounds that can be reasonably represented, even kinetically, by other chemical structures; the skilled artisan recognizes that such structures may represent only a small fraction of such compound samples. Such compounds are considered to be within the scope of the structures shown, even though such resonance forms or tautomers are not shown herein.

[0290] Polypeptides of the present invention

[0291] The present invention relates to a process for preparing compounds of formula (I) by acylation of precursor compounds of formula (Ia).

[0292] It is understood that such reactions can be carried out using known chemical reactions. However, a disadvantage of this approach is that the precursor compound can be acylated at various positions within the structure. Furthermore, due to the chemical reaction conditions, unwanted by-product formation and isomerization may be observed. This is undesirable because these molecules may not function as sweetness enhancers, which is precisely the application of compounds of formula (I).

[0293] Therefore, the inventors sought to identify enzymes that can be used to prepare compounds of formula (I). They identified several acyltransferases that can be used for this purpose, which are numbered herein as SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68. These acyltransferases were produced by recombinant expression techniques and are considered to be polypeptides of the present invention.

[0294] Acyltransferases are enzymes that catalyze the transfer of an acyl group to an acceptor molecular oxygen molecule. Acyltransferases are a large and diverse class of enzymes involved in numerous metabolic pathways within cells.

[0295] The present inventors sought to determine whether acyltransferases could be used in a process for preparing a compound of formula (I) from a compound of formula (Ia). Surprisingly, they identified several enzymes that could be used for this purpose, as shown in the accompanying examples. Furthermore, these enzymes specifically acylate the R5 position of the compound of formula (Ia) and do not acylate other positions in the structure.

[0296] Acyltransferases also have the advantage of high efficiency under aqueous reaction conditions and thus have good practicality in in vivo reactions.

[0297] To the best of the inventors' knowledge, this is the first time that an acyltransferase has been used for a specific reaction at position R5 in formula (Ia). In fact, it has never been reported before that an acyltransferase can accept flavanone alcohols as acceptor molecules.

[0298] The transfer of the acyl group to the oxygen molecule of the acceptor molecule is preferably carried out in the presence of a cofactor, preferably acyl-CoA. These cofactors are non-protein compounds that act as catalysts for the transfer of the acyl group.

[0299] In a preferred embodiment of the present invention, the acyltransferase is an acetyltransferase and the cofactor is acetyl-CoA. More preferably, the acetyltransferase is an O-acetyltransferase.

[0300] In a further preferred embodiment of the present invention, the acetyltransferase comprises the amino acid sequence HXXXD (SEQ ID NO: 29) and / or the amino acid sequence [DN]FGxG (SEQ ID NO: 30) and / or the amino acid sequence [ST]S[WL] (SEQ ID NO: 94).

[0301] The HXXXD, [DN]FGxG, and [ST]S[WL] motifs conform to the Prosite syntax, as defined at https: / / prosite.expasy.org / scanprosite / scanprosite_doc.html , where “X” or “x” represents any amino acid.

[0302] The HXXXD, [DN]FGxG, and [ST]S[WL] motifs are amino acid motifs common to acetyltransferases, and these motifs have been demonstrated in the accompanying Examples to be useful in the methods of the first aspect of the present invention. Thus, these motifs define a group of acetyltransferases that have been demonstrated to be functional in the methods of the present invention, and thus define a subset of these enzymes that can be used in these methods. The histidine (H) in HXXXD and the [ST] and [WL] in the [ST]S[WL] motif are part of the binding pocket of the enzyme.

[0303] Examples of acetyltransferases that can be used in the method of the present invention include polypeptides encoded by any one of the amino acid sequences of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68.

[0304] Therefore, a preferred embodiment of the method of the present invention is wherein the acyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0305] SEQ ID NOs: 1, 4 and 5 encode acyltransferases isolated from Inula viscosa (Dittrichiaviscosa), a highly branched perennial plant widely distributed in the Mediterranean basin. The present inventors have for the first time identified and characterized these polypeptide sequences as enzymes having acyltransferase activity.

[0306] SEQ ID NOs: 2, 3, and 56 encode acyltransferases identified from Erigeron canadensis. Erigeron canadensis is an annual plant with tall, sparsely hairy stems that grows throughout most temperate regions of Asia, Europe, North America, and Australia. The present inventors have for the first time identified and characterized these polypeptide sequences as enzymes with acyltransferase activity.

[0307] SEQ ID NOs: 6 and 7 encode acyltransferases identified from Pulicaria dysenterica, a plant native to Europe and Western Asia. The present inventors have for the first time identified and characterized these polypeptide sequences as enzymes having acyltransferase activity.

[0308] SEQ ID NO: 31 encodes an acyltransferase identified from Cynara cardunculus var. scolymus, commonly known as globe artichoke. The present inventors have for the first time identified and characterized these polypeptide sequences as enzymes having acyltransferase activity.

[0309] SEQ ID NOs: 32, 57, and 58 encode acyltransferases identified from Helianthus annuus (also known as the common sunflower). Sunflower is a large, annual herbaceous plant of the genus Helianthus, cultivated for its edible, oily seeds. The present inventors have for the first time identified and characterized this polypeptide sequence as an enzyme with acyltransferase activity.

[0310] SEQ ID NO:33 encodes an acyltransferase identified from Arctium lappa (also known as great burdock), a Eurasian plant of the Asteraceae family whose roots are grown as a vegetable in gardens. The present inventors have for the first time identified and characterized this polypeptide sequence as an enzyme with acyltransferase activity.

[0311] SEQ ID NOs: 34 and 60 encode an acyltransferase identified from Mikania micrantha. Mikania micrantha, also known as bitter vine, climbing hemp vine, or American rope, belongs to the Asteraceae family. Mikania micrantha is native to subtropical regions of North, Central, and South America, but is also found as a weed in Asia. The present inventors have identified and characterized this polypeptide sequence as an enzyme with acyltransferase activity for the first time.

[0312] SEQ ID NOs: 35 and 36 encode an acyltransferase identified from Smallanthus sonchifolius, also known as yacon, which belongs to the Asteraceae family. Smallanthus sonchifolius is an edible plant traditionally grown in the Andes but also found worldwide. The present inventors have identified and characterized this polypeptide sequence as an enzyme with acyltransferase activity for the first time.

[0313] SEQ ID NO:55 encodes an acyltransferase identified from Artemisia annua (also known as sweet wormwood). Artemisia annua belongs to the Asteraceae family. It is native to temperate Asia but has become naturalized in many countries around the world. Artemisia annua is cultivated agriculturally for the extraction of artemisinin, a drug used to treat malaria.

[0314] SEQ ID NO: 59 encodes an acyltransferase identified from carrot (Daucus carota subsp. Sativus) (also known as wild carrot). Carrot belongs to the Asteraceae family and is found worldwide.

[0315] SEQ ID NO:61 encodes an acyltransferase identified from Tanacetum cinerariifolium (also known as Dalmatian chrysanthemum). Pyrethrum belongs to the Asteraceae family. It is native to the Mediterranean region. However, because it is the natural source of pyrethrum (a type of insecticide), it is cultivated worldwide.

[0316] SEQ ID NO:62 is a variant of SEQ ID NO:1 comprising a P34A substitution.

[0317] SEQ ID NO:63 is a variant of SEQ ID NO:1 comprising a P34H substitution.

[0318] SEQ ID NO:64 is a variant of SEQ ID NO: 1 comprising P34A and F354Y substitutions.

[0319] SEQ ID NO:65 is a variant of SEQ ID NO:1 comprising P34A and F365Y substitutions.

[0320] SEQ ID NO:66 is a variant of SEQ ID NO:1 comprising P34A, F354Y and F365Y substitutions.

[0321] SEQ ID NO:67 is a variant of SEQ ID NO: 1 comprising a F358Y substitution.

[0322] SEQ ID NO:68 is a variant of SEQ ID NO:6 comprising substitutions I302F, L304F, L362F, L403F, and A400N.

[0323] The present inventors also tested the activities of the enzymes encoded by SEQ ID NOs: 1 to 4 on different acyl-CoA compounds. As shown in the accompanying examples, when the acyltransferase has the amino acid sequence shown in SEQ ID NO: 1, the cofactor is acetyl-CoA, propionyl-CoA, or butyryl-CoA, preferably acetyl-CoA.

[0324] When the acyltransferase has the amino acid sequence shown in SEQ ID NO: 2, the cofactor is acetyl-CoA or propionyl-CoA, preferably acetyl-CoA.

[0325] When the acyltransferase has the amino acid sequence shown in SEQ ID NO: 3, the cofactor is acetyl-CoA or propionyl-CoA, preferably acetyl-CoA.

[0326] When the acyltransferase has the amino acid sequence shown in SEQ ID NO: 4, the cofactor is acetyl-CoA, butyryl-CoA, hexanoyl-CoA or octanoyl-CoA, preferably hexanoyl-CoA.

[0327] An alternative aspect of the invention is wherein the process for preparing a compound of formula (I) comprises acylating a precursor compound of formula (Ia) with a hydrolase.

[0328] Carboxylate hydrolases [EC 3.1.1] are a class of hydrolases commonly used as biochemical catalysts that utilize water as a hydroxyl donor in the decomposition of substrates. Furthermore, they are known in the art to be most efficient at catalyzing the synthesis of ester bonds under anhydrous conditions.

[0329] As shown in the accompanying examples, the present inventors have identified hydrolases that can be used in the methods of the present invention, including triacylglycerol lipase [EC 3.1.1.3] and cutinase [EC 3.1.1.74]. This is the first demonstration that a hydrolase can be used to convert a compound of formula (Ia) into a compound of formula (I), wherein the compound of formula (I) is agaretin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltamarixanthin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxyagaretin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, 5-deoxy-3'-O-methyltamarixanthin-3-O-acetate or 5-deoxybrevicornin-3-O-acetate.

[0330] Therefore, another aspect of the present invention provides a method for preparing a compound of formula (I), which comprises reacting a precursor compound of formula (Ia) with a hydrolase to form a compound of formula (I).

[0331] Examples of hydrolases that can be used in this aspect of the invention include:

[0332] IMMLIPX-COV-1, available from Chiralvision, uses the lipase Lipex 100L (Lipex 100L from Novozymes) covalently bound to IB-150A.

[0333] IMMAULI-COV-1, available from Chiralvision, uses a lipase from Bacillus subtilis (Lipase from Aum Enzymes) covalently bound to IB-150A.

[0334] IMML51-COV-1, available from Chiralvision, uses a cutinase from Humicola insolens (NZ51032 from Novozymes) covalently bound to IB-150A.

[0335] IMMRES-COV-1, available from Chiralvision, uses a lipase from Aspergillus oryzae (Resinase HT from Novozymes) covalently bound to IB-150A.

[0336] IMMTLL-COV-1, available from Chiralvision, uses a lipase from Thermomyces lanuginosa (Lipolase from Novozymes) covalently bound to IB-150A.

[0337] IMMCALBY-COV-1, available from Chiralvision, uses universal lipase B from Candida antarctica (CaLB) from c-Lecta, covalently bound to IB-150A.

[0338] IMMCALB-COV-1XL, available from Chiralvision, uses lipase B from Candida antarctica (CaLB) from Novozymes, covalently bound to IB-150A.

[0339] However, a preferred embodiment of the present invention is wherein the lipase is IMMLIPX-COV-1.

[0340] As mentioned above, the present inventors sought to determine whether acyltransferases could be used to acylate compounds of formula (Ia) to form compounds of formula (I). Several enzymes useful for this purpose are disclosed in the accompanying Examples.

[0341] Therefore, another aspect of the present invention provides a recombinant polypeptide having acyltransferase activity, comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0342] Also provided herein is a recombinant nucleic acid molecule comprising a nucleotide sequence encoding a polypeptide having acyltransferase activity and comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0343] In addition to identifying a polypeptide having acyltransferase activity as defined in any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68, the present inventors also identified the native nucleic acid sequence of each of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 61. Furthermore, for all sequences, including SEQ ID NOs: 62 to 68, the present inventors optimized the codons of each nucleic acid sequence to make it suitable for expression in prokaryotic cells (preferably E. coli cells) and eukaryotic cells (preferably Saccharomyces cerevisiae).

[0344] Therefore, SEQ ID NO: 1 is encoded by its native nucleic acid sequence SEQ ID NO: 8. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 9. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 10.

[0345] Therefore, SEQ ID NO: 2 is encoded by its native nucleic acid sequence SEQ ID NO: 11. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 12. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 13.

[0346] Therefore, SEQ ID NO: 3 is encoded by its native nucleic acid sequence SEQ ID NO: 14. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 15. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 16.

[0347] Therefore, SEQ ID NO: 4 is encoded by its native nucleic acid sequence SEQ ID NO: 17. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 18. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 19.

[0348] Therefore, SEQ ID NO: 5 is encoded by its native nucleic acid sequence SEQ ID NO: 20. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 21. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 22.

[0349] Therefore, SEQ ID NO: 6 is encoded by its native nucleic acid sequence SEQ ID NO: 23. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 24. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 25.

[0350] Therefore, SEQ ID NO: 7 is encoded by its native nucleic acid sequence SEQ ID NO: 26. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 27. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 28.

[0351] Therefore, SEQ ID NO: 31 is encoded by its native nucleic acid sequence SEQ ID NO: 37. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 38. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 39.

[0352] Therefore, SEQ ID NO: 32 is encoded by its native nucleic acid sequence SEQ ID NO: 40. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 41. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 42.

[0353] Therefore, SEQ ID NO: 33 is encoded by its native nucleic acid sequence SEQ ID NO: 43. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 44. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 45.

[0354] Thus, SEQ ID NO: 34 is encoded by its native nucleic acid sequence, SEQ ID NO: 46. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 47. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 48.

[0355] Therefore, SEQ ID NO: 35 is encoded by its native nucleic acid sequence SEQ ID NO: 49. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 50. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 51.

[0356] Therefore, SEQ ID NO: 36 is encoded by its native nucleic acid sequence SEQ ID NO: 52. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 53. A nucleic acid sequence optimized for prokaryotic expression is shown in SEQ ID NO: 54.

[0357] Therefore, SEQ ID NO: 55 is encoded by its native nucleic acid sequence SEQ ID NO: 69. A nucleic acid sequence optimized for eukaryotic cell expression is shown in SEQ ID NO: 70.

[0358] Therefore, SEQ ID NO: 56 is encoded by its native nucleic acid sequence SEQ ID NO: 71. A nucleic acid sequence optimized for eukaryotic cell expression is shown in SEQ ID NO: 72.

[0359] Therefore, SEQ ID NO: 57 is encoded by its native nucleic acid sequence SEQ ID NO: 73. A nucleic acid sequence optimized for eukaryotic cell expression is shown in SEQ ID NO: 74.

[0360] Therefore, SEQ ID NO: 58 is encoded by its native nucleic acid sequence SEQ ID NO: 75. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 76.

[0361] Therefore, SEQ ID NO: 59 is encoded by its native nucleic acid sequence SEQ ID NO: 77. A nucleic acid sequence optimized for eukaryotic cell expression is shown in SEQ ID NO: 78.

[0362] Therefore, SEQ ID NO: 60 is encoded by its native nucleic acid sequence SEQ ID NO: 79. A nucleic acid sequence optimized for eukaryotic expression is shown in SEQ ID NO: 80.

[0363] Therefore, SEQ ID NO: 61 is encoded by its native nucleic acid sequence SEQ ID NO: 81. A nucleic acid sequence optimized for eukaryotic cell expression is shown in SEQ ID NO: 82.

[0364] Therefore, SEQ ID NO:62 is encoded by the nucleic acid sequence shown in SEQ ID NO:83 which is optimized for eukaryotic expression or the nucleic acid sequence shown in SEQ ID NO:84 which is optimized for prokaryotic expression.

[0365] Therefore, SEQ ID NO: 63 is encoded by the nucleic acid sequence shown in SEQ ID NO: 85 which is optimized for eukaryotic expression or the nucleic acid sequence shown in SEQ ID NO: 86 which is optimized for prokaryotic expression.

[0366] Thus, SEQ ID NO:64 is encoded by the nucleic acid sequence shown in SEQ ID NO:87 which is optimized for eukaryotic expression.

[0367] Thus, SEQ ID NO:65 is encoded by the nucleic acid sequence shown in SEQ ID NO:88 which is optimized for eukaryotic expression.

[0368] Thus, SEQ ID NO: 66 is encoded by the nucleic acid sequence shown in SEQ ID NO: 89 which is optimized for eukaryotic expression.

[0369] Therefore, SEQ ID NO: 67 is encoded by the nucleic acid sequence shown in SEQ ID NO: 90 which is optimized for eukaryotic expression or the nucleic acid sequence shown in SEQ ID NO: 91 which is optimized for prokaryotic expression.

[0370] Therefore, SEQ ID NO: 68 is encoded by the nucleic acid sequence shown in SEQ ID NO: 92 which is optimized for eukaryotic expression or the nucleic acid sequence shown in SEQ ID NO: 93 which is optimized for prokaryotic expression.

[0371] Thus, further provided is an isolated nucleic acid comprising a nucleotide sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or a nucleotide sequence comprising any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or the reverse complement thereof.

[0372] Further provided is an isolated nucleic acid molecule encoding a recombinant polypeptide provided herein.

[0373] In one embodiment, the present invention provides a vector comprising a nucleic acid molecule described herein. In another embodiment, the vector is an expression vector. In yet another embodiment, the vector is a prokaryotic vector, a viral vector, or a eukaryotic vector.

[0374] The present invention also provides a non-human host organism or host cell comprising (1) the above-mentioned nucleic acid molecule, or (2) an expression vector comprising the nucleic acid molecule. In one embodiment, the non-human organism or host cell is a prokaryotic cell or a eukaryotic cell. In another embodiment, the host cell is a bacterium, an archaeon, a fungus such as a yeast, an algae cell, or a plant cell. In yet another embodiment, the bacterial cell is Escherichia coli, and the yeast cell is Saccharomyces cerevisiae.

[0375] Further provided is the use of a polypeptide described herein for producing a compound of formula (I).

[0376] Also provided are nucleotide sequences obtained by modifying any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 or their reverse complements, encompassing any sequence obtained by modifying any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 or their reverse complements using any method known in the art, for example by introducing any type of mutation, such as deletion, insertion and / or substitution mutations. Nucleic acids comprising a sequence obtained by mutating any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 or their reverse complements are included in one embodiment of the invention, provided that they comprise a sequence having at least the defined sequence identity to any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 or their reverse complements, and provided that they encode a polypeptide having acyltransferase activity as defined in any of the above embodiments. The mutation can be any type of mutation of these nucleic acids, for example, a point mutation, a deletion mutation, an insertion mutation and / or a frameshift mutation of one or more nucleotides in the DNA sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93. In one embodiment, the nucleic acid in the embodiments herein can be truncated, provided that it encodes a polypeptide as described herein.

[0377] Variant nucleic acids can be prepared to adapt their nucleotide sequence to a particular expression system. For example, bacterial and yeast expression systems are known to express polypeptides more efficiently if the amino acids are encoded by specific codons.

[0378] Due to the degeneracy of the genetic code, more than one codon may encode the same amino acid sequence, and multiple nucleic acid sequences may also encode the same protein or polypeptide, all of which DNA sequences are included in an embodiment of this invention. Where appropriate, the nucleic acid sequence encoding the acyltransferase may be optimized to improve its expression in the host cell. For example, the nucleotides in an embodiment of this invention may be synthesized using codons specific to the host cell to improve expression.

[0379] In one embodiment, provided herein is an isolated, recombinant or synthetic nucleic acid sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, which encodes a polypeptide having acyltransferase activity, the polypeptide comprising the amino acid sequence of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or a fragment thereof, which catalyzes the production of a compound of formula (I):

[0380] cDNA, genomic DNA, and RNA sequences are also provided herein.Any nucleic acid sequence encoding an acyltransferase or a variant thereof is also referred to herein as an acyltransferase encoding sequence.

[0381] According to one embodiment, the nucleic acid of any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 is the coding sequence of an acyltransferase gene encoding an acyltransferase obtained as described in the Examples.

[0382] A fragment of a polynucleotide of any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93 refers to a continuous nucleotide, in particular a length of at least 15 bp, at least 30 bp, at least 40 bp, at least 50 bp and / or at least 60 bp of a polynucleotide of the embodiments herein. In particular, the polynucleotide fragment comprises at least 25, more particularly at least 50, more particularly at least 75, more particularly at least 100, more particularly at least 150, more particularly at least 200, more particularly at least 300, more particularly at least 400, more particularly at least 500, more particularly at least 600, more particularly at least 700, more particularly at least 800, more particularly at least 900, more particularly at least 1000 continuous nucleotides of a polynucleotide of the embodiments herein. Without limitation, the polynucleotide fragments herein can be used as PCR primers and / or probes, or for antisense gene silencing or RNAi.

[0383] It will be clear to those skilled in the art that genes, including the polynucleotides described herein, can be cloned by methods known in the art based on available nucleotide sequence information (e.g., information in the attached sequence listing). These methods include, for example, designing DNA primers representing sequences flanking the gene, wherein one primer is generated in the forward direction to initiate synthesis of the forward strand; and another primer is generated in the reverse complementary direction to generate the antisense strand. Such experiments are typically performed using a thermostable DNA polymerase (e.g., an enzyme for the polymerase chain reaction). Alternatively, a DNA sequence representing a gene can be chemically synthesized and then introduced into a DNA vector molecule that can be propagated, for example, by compatible bacteria (e.g., E. coli) or yeast cells.

[0384] In the relevant embodiment provided herein, PCR primers and / or probes for detecting the nucleotide sequence of encoding acyltransferase are provided. Those skilled in the art will understand how to synthesize degenerate or specific PCR primer pairs, based on any one in SEQ ID NO:8 to 28, 37 to 54 and 69 to 93, amplify the nucleotide sequence of encoding acyltransferase or its segmental nucleotide sequence. The test kit for detecting the nucleotide sequence of encoding acyltransferase can comprise primers and / or probes specific for the nucleotide sequence of encoding acyltransferase, and the relevant protocol for using the nucleotide sequence of encoding acyltransferase in the sample using these primers and / or probes. This type of test kit can be used for determining whether plant, organism or cell has been modified, i.e. whether the sequence of encoded acyltransferase has been transformed.

[0385] To test the functionality of a variant DNA sequence according to the embodiments herein, the sequence of interest is tested in an activity assay, examples of which are provided herein.

[0386] Those skilled in the art will appreciate that methods for identifying homologous sequences in other organisms, and methods for determining the sequence identity percentage between the homologous sequences. These newly identified DNA molecules can then be sequenced, and compared with any one's nucleotide sequence with SEQ ID NO:8 to 28, 37 to 54 and 69 to 93.

[0387] The percent identity between two peptides or nucleotide sequences is a function of the number of identical amino acids or nucleotide residues in the two sequences after the alignment is generated. Identical residues are defined as identical residues in the two sequences at a given position in the alignment. The percent identity of the sequence used herein is calculated from the best alignment by dividing the number of identical residues between the two sequences by the total number of residues in the shortest sequence and multiplying by 100. The best alignment is the alignment with the highest probability of percent identity. Rooms can be introduced into one or more positions of the alignment in one or two sequences to obtain the best alignment. These rooms are then considered to be non-identical residues for calculating sequence identity percentages. Alignment for determining amino acid or nucleotide sequence identity percentages can be achieved in a variety of ways using computer programs and, for example, publicly available computer programs on the internet. Preferably, the BLAST program (Tatiana et al., FEMS Microbiol Lett., 1999, 174: 247-250, 1999), available from the National Center for Biotechnology Information (NCBI) at http: / / www.ncbi.nlm.nih.gov / BLAST / bl2seq / wblast2.cgi, set to default parameters can be used to obtain optimal alignment of protein or nucleic acid sequences and calculate percentages of sequence identity.

[0388] Related embodiments provided herein provide a nucleic acid sequence that is complementary to the nucleic acid sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54, and 69 to 93, such as an inhibitory RNA, or a nucleic acid sequence that hybridizes under stringent conditions to at least a portion of the nucleotide sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54, and 69 to 93. An alternative embodiment of the embodiments herein provides a method for altering gene expression in a host cell. For example, the polynucleotides of the embodiments herein can be enhanced, overexpressed, or induced in a host cell or host organism under certain circumstances (e.g., upon exposure to a specific temperature or culture conditions).

[0389] Alteration of expression of the polynucleotides provided herein can also result in ectopic expression, which is a different expression pattern in the altered and control or wild-type organisms. Alteration of expression occurs due to the interaction of a polypeptide of an embodiment herein with an exogenous or endogenous modulator or due to chemical modification of the polypeptide. The term also refers to an altered expression pattern of a polynucleotide of an embodiment herein, i.e., altered to below the level of detection or completely inhibited activity.

[0390] In one embodiment, at least one polypeptide having acyltransferase activity used in any of the embodiments described herein, or a polypeptide encoded by a nucleic acid according to any of the embodiments described herein, comprises a variant amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68 obtained by genetic engineering. In one embodiment, the polypeptide comprises an amino acid sequence encoded by a nucleotide sequence obtained by modifying any one of SEQ ID NOs: 8 to 28, 37 to 54, and 69 to 93, or a reverse complement thereof.

[0391] The polypeptides also include variants and truncated polypeptides, provided they have acyltransferase activity.

[0392] According to another embodiment, the at least one polypeptide having acyltransferase activity for use in any of the embodiments described herein or the at least one polypeptide encoded by the nucleic acid of any of the embodiments described herein comprises an amino acid sequence that is a variant of any of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, obtained by genetic engineering, provided that the variant has acyltransferase activity and has the required percentage identity with any of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68 as described herein.

[0393] According to another embodiment, the at least one polypeptide having acyltransferase activity used in any embodiment described herein or the at least one polypeptide having acyltransferase activity encoded by the nucleic acid of any embodiment described herein is a variant of any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68, which variant may naturally occur in other organisms, provided that it has acyltransferase activity. The polypeptides used herein include polypeptides or peptide fragments comprising the amino acid sequences described herein, as well as truncated or variant polypeptides, provided that they have acyltransferase activity and have at least the specified percentage identity with the corresponding fragment of any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68.

[0394] Examples of variant polypeptides are naturally occurring proteins produced by alternative mRNA splicing events or proteolytic cleavage of polypeptides described herein. Variations attributable to proteolysis include, for example, differences in the N-terminus or C-terminus when expressed in different types of host cells due to proteolytic removal of one or more terminal amino acids in the polypeptides of the embodiments described herein. Polypeptides encoded by nucleic acids obtained by natural or artificial mutations of the nucleic acids of the embodiments described herein (as described below) are also encompassed in the embodiments described herein.

[0395] Polypeptide variants produced by fusing additional peptide sequences at the amino and carboxyl termini can also be used in the methods of the embodiments herein. Specifically, such fusions can enhance the expression of the polypeptide, aid in the purification of the protein, or improve the enzymatic activity of the polypeptide in a desired environment or expression system. For example, such additional peptide sequences can be signal peptides. Another aspect encompasses methods using variant polypeptides, such as polypeptides obtained by fusion with other oligopeptides or polypeptides and / or polypeptides linked to a signal peptide. Polypeptides produced by fusion with another functional protein can also be advantageously used in the methods of the embodiments herein.

[0396] Variants can also differ from the polypeptides of the embodiments herein by the addition of modifying groups covalently or non-covalently attached to the polypeptide backbone. Variants also include polypeptides that differ from the polypeptides provided herein by the introduction of N-linked or O-linked glycosylation sites, and / or the addition of cysteine ​​residues. Those skilled in the art will understand how to modify an amino acid sequence while retaining its biological activity.

[0397] The present invention also relates to "functional equivalents" (also called "analogs" or "functional mutants") of the polypeptides specifically described herein.

[0398] For example, "functional equivalents" refers to polypeptides whose acyltransferase activity is at least 1 to 10%, or at least 20%, or at least 50%, or at least 75%, or at least 90% higher or lower than the corresponding polypeptide as specifically defined herein in a test for determining acyltransferase activity, as well as truncated or variant polypeptides, provided that they have acyltransferase activity and have at least the specified percentage identity with the corresponding fragment of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0399] "Functional equivalents" may also be derived from auxiliary polypeptides as described herein, which facilitate the functional expression of another polypeptide (preferably a polypeptide having enzymatic activity), in particular the correct folding of the expressed polypeptide, such as a polypeptide having acyltransferase activity. Such modified auxiliary polypeptides may still be considered functional as long as they improve the correct expression or folding of the enzymatically active polypeptide relative to the expression of the same enzymatically active polypeptide under otherwise identical conditions but in the absence of such auxiliary polypeptide.

[0400] In addition to the gene sequences shown in the sequences disclosed herein, those skilled in the art will appreciate that DNA sequence polymorphisms may exist within a particular population, which may result in variations in the amino acid sequences of the polypeptides disclosed herein. Such gene polymorphisms may exist in cells from different populations or within the same population due to natural allelic variation. Allelic variants may also include functional equivalents.

[0401] Further embodiments also relate to molecules derived from the specifically disclosed nucleic acid sequence polymorphisms. These natural variations typically result in about 1% to 5% variation in the nucleotide sequence of a gene or the amino acid sequence of a polypeptide disclosed herein. As described above, nucleic acids encoding the polypeptides of the embodiments herein or variants thereof are useful tools for modifying non-human host organisms or cells, as well as for modifying non-human host organisms or cells intended for use in the methods described herein.

[0402] The embodiments provided herein provide amino acid sequences of acyltransferase proteins, including orthologues and paralogues, and methods for identifying and isolating the orthologues and paralogues of the acyltransferases in other organisms. Specifically, orthologues and paralogues of the acyltransferases are identified and are capable of producing compounds of formula (I).

[0403] Acyltransferase polypeptides can be obtained by extraction from any organism expressing the polypeptide using standard protein or enzyme extraction techniques. If the host organism is a unicellular organism or cell and the polypeptide of the embodiments herein is released into the culture medium, the polypeptide can be simply collected from the culture medium, for example by centrifugation, optionally followed by a washing step, and resuspended in a suitable buffer. If the organism or cell accumulates the polypeptide intracellularly, the polypeptide can be obtained by disrupting or lysing the cells and optionally further extracting the polypeptide from the cell lysate.

[0404] According to another embodiment, the at least one polypeptide having an acyltransferase activity can be used in the method of the present invention.

[0405] The functionality or activity of acyltransferase proteins, variants or fragments can be determined by a variety of methods. For example, transient or stable overexpression can be performed in plants, bacteria or yeast cells to detect whether the protein is active, i.e., whether it produces compounds of formula (I). Acyltransferase activity can be assessed by the assays described in the embodiments herein to indicate its functionality. Variants or derivatives of the acyltransferase polypeptides of the embodiments herein retain the ability to produce compounds of formula (I). Amino acid sequence variants of the acyltransferases provided herein may have other desired biological functions, such as altered substrate utilization, reaction kinetics, product distribution or other changes.

[0406] Further provided is at least one vector comprising a nucleic acid molecule described herein.

[0407] Also provided herein are vectors selected from the group consisting of prokaryotic vectors, viral vectors, and eukaryotic vectors.

[0408] This article also provides a vector, which is an expression vector.

[0409] In the embodiments herein, the nucleotide sequence encoding the acyltransferase protein can be inserted into an expression vector and / or be included in a mosaic gene inserted into an expression vector to produce the acyltransferase protein in a host cell or a non-human host organism. Carriers for inserting transgenes into the host cell genome are well known in the art and include plasmids, viruses, cosmids, and artificial chromosomes. Binary vectors or co-integration vectors that insert mosaic genes also can be used to transform host cells. For clarity, multiple copies of the gene can be inserted into the host cell.

[0410] One embodiment provided herein provides a recombinant expression vector comprising a nucleic acid sequence of an acyltransferase gene, or a chimeric gene comprising a nucleic acid sequence of an acyltransferase gene, wherein the chimeric gene is operably linked to a related nucleic acid sequence, such as a promoter sequence. For example, a chimeric gene comprising a nucleic acid sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54, and 69 to 93, or a variant thereof, can be operably linked to a promoter sequence suitable for expression in a plant cell, a bacterial cell, or a fungal cell, wherein the promoter sequence is optionally linked to a 3' non-translated nucleic acid sequence.

[0411] Alternatively, the promoter sequence may already be present in the vector, and the nucleic acid sequence to be transcribed is inserted downstream of the promoter sequence in the vector. The vector may be engineered to have an origin of replication, a multiple cloning site, and a selectable marker.

[0412] In one embodiment, expression vectors comprising the nucleic acids described herein can be used as a means to transform a non-human host organism or host cell suitable for performing the methods of the embodiments herein in vivo.

[0413] The expression vectors provided herein can be used in methods for preparing genetically transformed non-human host organisms and / or host cells, in methods for preparing non-human host organisms and / or host cells carrying nucleic acids of the embodiments herein, and in methods for preparing polypeptides having acyltransferase activity as described herein.

[0414] Recombinant non-human host organisms and host cells, which have been transformed to carry at least one nucleic acid according to the present invention, to heterologously express or overexpress at least one polypeptide according to the present invention, are also very useful tools for practicing the methods of the present invention. Accordingly, such non-human host organisms and host cells are provided herein.

[0415] In one embodiment, a host cell or non-human host organism comprising at least one nucleic acid molecule described herein or comprising at least one vector comprising at least one nucleic acid molecule is provided.

[0416] The nucleic acid according to any of the above embodiments may be used to transform non-human host organisms and cells, and the expressed polypeptide may be any of the polypeptides described above.

[0417] In one embodiment, the non-human host organism or host cell is a prokaryotic cell. In another embodiment, the non-human host organism or host cell is a bacterial cell. In a further embodiment, the non-human host organism or host cell is Escherichia coli.

[0418] In one embodiment, the non-human host organism or host cell is a eukaryotic cell. In another embodiment, the non-human host organism or host cell is a yeast cell. In a further embodiment, the non-human host organism or host cell is Saccharomyces cerevisiae.

[0419] In one embodiment, the non-human host organism or host cell expresses a polypeptide if the organism or cell is transformed to contain a nucleic acid encoding the polypeptide, the nucleic acid is transcribed into mRNA and the polypeptide is present in the host organism or cell.

[0420] Suitable methods for transforming non-human host organisms or host cells have been previously described and are provided herein.

[0421] In order to implement the embodiments herein in vivo, the host organism or host cell is cultured under conditions that are conducive to the production of the compound of formula (I). If the host is a unicellular organism, the conditions that are conducive to the production of the compound of formula (I) may include adding suitable cofactors to the host culture medium. In addition, culture medium may be selected to maximize the synthesis of the compound of formula (I). Examples of optimal culture conditions will be described in more detail in the Examples.

[0422] The non-human host organisms for implementing the method for this paper embodiment in vivo can be any non-human multicellular or unicellular organisms. In one embodiment, the non-human host organisms for implementing the method for this paper embodiment in vivo are plants, prokaryotes or fungi. Any plant, prokaryote or fungi can be used. In another embodiment, the non-human host organisms for implementing the method for this paper embodiment in vivo are microorganisms. Any microorganism can be used, for example, microorganisms can be bacterium or yeast, such as Escherichia coli (E.coli), Corynebacterium glutamicum (Corynebacteriumglutamicum), Pseudomonas putida (Pseudomonas putida), Streptomyces (Streptomyces sp), Saccharomyces cerevisiae (Saccharomyces cerevisiae), Candida krusei (Candida krusei), Issatchenkia orientalis (Issatchenkia orientalis), Pichia pastoris (Pichia pastoris) or Yarrowia lipolytica (Yarrowia lipolytica).

[0423] Isolated higher eukaryotic cells can also be used in place of intact organisms as hosts for performing the methods of the embodiments herein in vivo. Suitable eukaryotic cells can be any non-human cell, such as a plant cell or a fungal cell.

[0424] Further provided herein is a method comprising transforming a host cell or non-human host organism with a nucleic acid encoding a polypeptide having acyltransferase activity, said polypeptide comprising an amino acid sequence having at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

[0425] In one embodiment, the methods provided herein comprise culturing a non-human host organism or host cell transformed to express a polypeptide under conditions permitting production of the polypeptide, wherein the polypeptide comprises an amino acid sequence having at least 75%, 80%, 85%, 90%, 95%, 98%, 99% or 100% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68.

[0426] Recombinant production of peptides

[0427] The present invention further relates to a method for the recombinant production of a polypeptide according to the invention or a functional, biologically active fragment thereof, wherein a microorganism producing the polypeptide is cultured, expression of the polypeptide is optionally induced by administering at least one inducer of gene expression, and the polypeptide is isolated from the culture. If desired, the polypeptide can also be produced on an industrial scale in this manner.

[0428] The microorganism that produces according to the present invention can be cultivated continuously or discontinuously by batch process or fed-batch process or repeated fed-batch process.The general introduction of known culture method can be in the textbook (Bioprozesstechnik 1.Einführungin die Bioverfahrenstechnik [Bioprocess technology 1.Introduction tobioprocess technology] (Gustav Fischer Verlag, Stuttgart, 1991)) of Chmiel or in the textbook (Bioreaktoren und periphere Einrichtungen [Bioreactors and peripheralequipment] (Vieweg Verlag, Braunschweig / Wiesbaden, 1994)) of Storhas.

[0429] The culture medium used must be appropriately adapted to the requirements of the respective strain. Descriptions of culture media for various microorganisms are given in the manual "Manual of Methods for General Bacteriology" of the American Society for Bacteriology (Washington DC, USA, 1981).

[0430] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0431] The preferred carbon source is sugar, for example monose, disaccharide or polysaccharide. Good carbon source is for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar also can be added in the substratum by other by-products of complicated compound (for example molasses) or sugar refining. It is also favourable to add the mixture of different carbon sources. Other possible carbon sources are oil and fat, for example soybean oil, sunflower seed oil, peanut oil and coconut oil, fatty acid for example palmitic acid, stearic acid or linoleic acid, alcohol for example glycerol, methanol or ethanol, and organic acid, for example acetic acid or lactic acid.

[0432] The nitrogen source is generally an organic or inorganic nitrogen compound or a material containing such a compound. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or as a mixture.

[0433] Inorganic salt compounds that may be present in the culture medium include chloride, phosphorus or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0434] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and thiols, can be used as sulfur sources.

[0435] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0436] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0437] The fermentation medium used according to the present invention generally also includes other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, nicotinic acid, pantothenic acid and pyridoxine. Growth factors and salts are generally derived from the components of complex culture media, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors can be added to the culture medium. The exact composition of the compound in the culture medium depends to a large extent on the corresponding experiment and is determined respectively for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, A Practical Approach" (Ed. PM Rhodes, PF Stanbury, IRL Press (1997) p.53-73, ISBN 0 19 963577 3). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO) etc.

[0438] All components of the culture medium are sterilized by heating (1.5 bar and 121°C for 20 minutes) or by sterile filtration. These components can be sterilized together or separately as needed. All components of the culture medium can be given at the beginning of the culture or added continuously or in batches.

[0439] The culture temperature is usually between 15°C and 45°C, preferably 25°C to 40°C, and can be changed or kept constant during the experiment. The pH of the medium should be in the range of 5 to 8.5, preferably around 7.0. The pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammonia water) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable selective substances such as antibiotics can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is usually in the range of 20°C to 45°C. Continue to cultivate until the maximum amount of the desired product is formed. This goal will usually be achieved within 10 to 160 hours.

[0440] The fermentation broth is then processed further. Depending on requirements, the biomass can be completely or partially removed from the fermentation broth by separation techniques such as centrifugation, filtration, decantation or a combination of these methods, or can remain completely therein.

[0441] If the polypeptide is not secreted in the culture medium, the cells can also be lysed and the product can be obtained from the lysate by known methods for isolating proteins. The cells can alternatively be disrupted by high-frequency ultrasound, high pressure, for example in a high-pressure cell lyser (French press), by osmosis, by the action of detergents, lytic enzymes or organic solvents, by homogenizers or by a combination of several of the above methods.

[0442] The polypeptide can be purified by known chromatographic techniques, such as molecular sieve chromatography (gel filtration), e.g., Q-agarose chromatography, ion exchange chromatography and hydrophobic chromatography, and other conventional techniques, such as ultrafiltration, crystallization, salting out, dialysis and native gel electrophoresis. Suitable methods are described, for example, in Cooper, TG, Biochemische Arbeitsmethoden [Biochemical processes], Verlag Walter de Gruyter, Berlin, New York, or Scopes, R., Protein Purification, Springer Verlag, New York, Heidelberg, Berlin.

[0443] In order to separate recombinant proteins, it may be advantageous to use a carrier system or oligonucleotides that extend cDNA by the nucleotide sequence of limitation, and therefore encode a changed polypeptide or fusion protein, which is for example used for easier purification. Suitable modifications of this type are for example so-called "tags" serving as anchors, such as modifications (for example, described in Harlow, E. and Lane, D., 1988, Antibodies: A Laboratory Manual. Cold Spring Harbor (NY) Press) that are referred to as six histidine anchors or epi-positions of antibody antigens. These anchors can be used to connect proteins to solid supports, such as polymeric matrices, which can for example be used as the filler in a chromatographic column, or can be used on microtiter plates or other carriers.

[0444] At the same time, these anchors can also be used to identify proteins. In order to identify proteins, conventional labels such as fluorescent dyes, enzyme labels (which react with substrates to form detectable reaction products), or radioactive labels can also be used, alone or in combination with anchors to derivatize proteins.

[0445] Immobilization of peptides

[0446] Enzyme or polypeptide according to the present invention, or enzyme or polypeptide for the inventive method, can be used in free form or immobilized form in the methods described herein.Immobilized enzyme refers to an enzyme fixed on an inert carrier. Suitable carrier materials include, for example, clay, clay minerals (such as kaolinite, diatomaceous earth, perlite), silicon dioxide, aluminum oxide, sodium carbonate, calcium carbonate, cellulose powder, anion exchange materials, synthetic polymers (such as polystyrene, acrylic resin, phenolic resin, polyurethane) and polyolefins (such as polyethylene and polypropylene). In order to prepare loaded enzymes, carrier materials are usually in the form of finely dispersed particles, preferably porous forms. The particle diameter of the carrier material is usually no more than 5 mm, especially no more than 2 mm (particle size distribution curve). Similarly, when using dehydrogenase as a whole cell catalyst, free form or immobilized form can be selected. Carrier materials are, for example, calcium alginate and carrageenan. Enzyme and cells can also be directly cross-linked with glutaraldehyde (cross-linked with CLEAs). Corresponding and other immobilization techniques are described, for example, in J. Lalonde and A. Margolin "Immobilization of Enzymes" in K. Drauz and H. Waldmann, Enzyme Catalysis in Organic Synthesis 2002, Vol. III, 991-1032, Wiley-VCH, Weinheim. Further information on biotransformations and bioreactors for carrying out the process according to the invention is given in Rehm et al. (Ed.) Biotechnology, 2nd Edn, Vol 3, Chapter 17, VCH, Weinheim.

[0447] The present invention provides a method for preparing the compound of formula (I), comprising reacting a precursor compound of formula (Ia) described herein with an acyltransferase to form the compound of formula (I).

[0448] The methods of the present invention may be carried out as in vitro or in vivo reactions under conditions conducive to the production of the compounds of formula (I).

[0449] Reaction conditions of the biocatalytic production method of the present invention

[0450] The at least one acyltransferase present in the individual steps of the method of the invention or the multi-step method defined herein can be present in living cells, harvested cells, dead cells, permeabilized cells, crude cell extracts, purified extracts, or in substantially pure or completely pure form. The at least one enzyme can be present in solution or as an enzyme immobilized on a support or encapsulated. One or more enzymes can be present in both soluble and / or immobilized form.

[0451] The method according to the present invention can be carried out in common reactors known to those skilled in the art, and can be carried out in different scale ranges, for example, from laboratory scale (a few milliliters to tens of liters of reaction volume) to industrial scale (a few liters to thousands of cubic meters of reaction volume). If the polypeptide is used in the form of encapsulation by inanimate, optionally permeabilized cells, in the form of more or less purified cell extracts or in purified form, a chemical reactor can be used. Chemical reactors generally allow the control of the amount of at least one enzyme, the amount of at least one substrate, pH, temperature and the circulation of the reaction medium. When at least one polypeptide / enzyme is present in living cells, the process will be fermentation. In this case, biocatalytic production will be carried out in a bioreactor (fermenter), where the parameters necessary for the suitable living conditions of the living cells (e.g., culture medium with nutrients, temperature, ventilation, aerobic or anaerobic or other gases, antibiotics, etc.) can be controlled. The person skilled in the art is familiar with chemical reactors or bioreactors, for example using procedures for scaling up chemical or biotechnological processes from the laboratory scale to the industrial scale or for optimizing process parameters, which are also widely described in the literature (for biotechnological processes, see, for example, Crueger und Crueger, Biotechnologie—Lehrbuch der angewandten Mikrobiologie, 2. Ed., R. Oldenbourg Verlag, München, Wien, 1984).

[0452] The cells containing at least one enzyme can be permeabilized by physical or mechanical means such as ultrasound or radiofrequency pulses, high pressure cell lysers (French press) or chemical means such as the presence of a hypotonic medium, a lytic enzyme and a detergent in the culture medium or a combination of these methods. Examples of detergents include digitonin, n-dodecyl maltoside, octyl glucoside, X-100, 20, deoxycholate, CHAPS (3-[(3-cholamidopropyl)dimethylammonium]-1-propanesulfonate), P40 (ethylphenol poly (ethylene glycol ether)), etc.

[0453] Instead of living cells, non-living cell biomass containing the desired biocatalyst can also be used in the bioconversion reaction of the present invention.

[0454] If at least one enzyme is immobilized, it is attached to an inert support as described above.

[0455] The conversion reaction can be carried out batchwise, semi-batchwise or continuously. The reactants (and optionally nutrients) can be provided at the start of the reaction, or can be provided subsequently semi-continuously or continuously.

[0456] Depending on the particular reaction type, the reactions of the present invention can be carried out in aqueous, aqueous-organic or non-aqueous reaction media.

[0457] The aqueous or aqueous-organic medium may contain a suitable buffer to adjust the pH to 5 to 11, for example 6 to 10.

[0458] In aqueous-organic media, organic solvents that are miscible, partially miscible or immiscible with water can be used. Non-limiting examples of suitable organic solvents are listed below. Further examples are monobasic or polybasic, aromatic or aliphatic alcohols, particularly polybasic aliphatic alcohols, such as glycerol.

[0459] The non-aqueous medium may comprise substantially no water, ie, may comprise less than about 1% or 0.5% by weight water.

[0460] Biocatalytic methods can also be carried out in organic non-aqueous media. Suitable organic solvents can be selected from: for example, aliphatic hydrocarbons having 5 to 8 carbon atoms, such as pentane, cyclopentane, hexane, cyclohexane, heptane, octane or cyclooctane; chlorinated hydrocarbons; aromatic hydrocarbons, such as benzene, toluene, xylene, chlorobenzene or dichlorobenzene; esters, such as ethyl acetate, isopropyl myristate; ethers, such as diethyl ether, methyl tert-butyl ether, ethyl tert-butyl ether, dipropyl ether, diisopropyl ether, dibutyl ether, tetrahydrofuran or 2-methyltetrahydrofuran; ketones and alcohols. Other media include DMF, DMSO, deep eutectic solvents or ionic liquids. In the case of hydrolase-catalyzed ester synthesis, acyl donors such as ethyl acetate can also be used as media.

[0461] The concentrations of reactants / substrates can be adjusted to optimize reaction conditions, depending on the specific enzyme used. For example, the initial substrate concentration can be 0.001 to 1 M.

[0462] The reaction temperature can be adjusted according to the optimal reaction conditions, specifically depending on the specific circumstances of the enzyme used. For example, the reaction can be carried out at a temperature of 0 to 70°C (e.g., 20 to 50°C or 25 to 40°C). Examples of reaction temperatures include about 25°C, 28°C, 30°C, about 35°C, about 37°C, about 40°C, about 45°C, about 50°C, about 55°C, and about 60°C.

[0463] This process can be continued until substrate and product reach equilibrium, but also can be stopped in advance. Common process time is 10 minutes to 48 hours, particularly 1 hour to 24 hours, for example 1 hour to 4 hours. These parameters are only the non-limiting examples of suitable process conditions.

[0464] The method of the present invention may further include the step of reclaiming the final product or intermediate product, which is optionally a substantially pure form of a stereoisomer or enantiomer. The term "recovery" includes extracting, gathering in the crops, separating or purifying the compound from a culture medium or reaction medium. The recovery of the compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to: processing with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), processing with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, aluminum oxide, etc.), changing the pH value, solvent extraction (e.g., with conventional solvents, such as alcohol, ethyl acetate, hexane, etc.), processing, distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, freeze-drying, etc.

[0465] The identity and purity of the isolated product can be determined by known techniques, such as high performance liquid chromatography (HPLC), gas chromatography (GC), spectroscopy (e.g., IR, UV, NMR), staining methods, TLC, NIRS, enzyme or microbiological assays (see, for example: Patek et al. (1994) Appl. Environ. Microbiol. 60: 133-140; Malakhova et al. (1996) Biotekhnologiya 1127-32; and Schmidt et al. (1998) Bioprocess Engineer. 19: 67-70. Ullmann's Encyclopedia of Industrial Chemistry (1996) Bd. A27, VCH: Weinheim, S. 89-90, S. 521-540, S. 540-547, S. 559-566, 575-581 and S.581-587; Michal, G (1999) Biochemical Pathways: An Atlas of Biochemistry and Molecular Biology, John Wiley and Sons; Fallon, A. et al. (1987) Applications of HPLC in Biochemistry in: Laboratory Techniques in Biochemistry and Molecular Biology, Bd. 17.).

[0466] Fermentation production

[0467] The present invention also relates to a fermentation production method of the compound of formula (I).

[0468] Fermentation used according to the present invention can be carried out, for example, in stirred fermentor tanks, bubble columns and loop reactors. For a comprehensive overview of possible method types, including agitator types and geometric designs, see "Chmiel: Bioprozesstechnik: Einfuhrung in die Bioverfahrenstechnik, Band 1". In the method of the present invention, available typical variations are those known to those skilled in the art or, for example, explained in "Chmiel, Hammes and Bailey: Biochemical Engineering", such as batch, fed-batch, repeated fed-batch or continuous fermentation with or without biomass recovery. Depending on the production strain, injection of air, oxygen, carbon dioxide, hydrogen, nitrogen or a suitable gas mixture can be performed to achieve a good yield (YP / S).

[0469] The culture medium used must meet the requirements of the specific strain in an appropriate manner.

[0470] The culture media which can be used according to the invention generally comprise one or more carbon sources, nitrogen sources, inorganic salts, vitamins and / or trace elements.

[0471] The preferred carbon source is sugar, for example monose, disaccharide or polysaccharide. Very good carbon source is for example glucose, fructose, mannose, galactose, ribose, sorbose, ribulose, lactose, maltose, sucrose, raffinose, starch or cellulose. Sugar also can be added in the substratum by other by-products of complicated compound (for example molasses) or sugar refining. It is also favourable to add the mixture of various carbon sources. Other possible sources of carbon are oil and fat, for example soybean oil, sunflower seed oil, peanut oil and coconut oil, fatty acid for example palmitic acid, stearic acid or linoleic acid, alcohol for example glycerol, methanol or ethanol, and organic acid, for example acetic acid or lactic acid.

[0472] The nitrogen source is generally an organic or inorganic nitrogen compound or a material containing such a compound. Examples of nitrogen sources include ammonia gas or ammonium salts, such as ammonium sulfate, ammonium chloride, ammonium phosphate, ammonium carbonate or ammonium nitrate, nitrates, urea, amino acids or complex nitrogen sources, such as corn steep liquor, soy flour, soy protein, yeast extract, meat extract, etc. The nitrogen sources can be used alone or as a mixture.

[0473] Inorganic salt compounds that may be present in the culture medium include chloride, phosphate or sulfate salts of calcium, magnesium, sodium, cobalt, molybdenum, potassium, manganese, zinc, copper and iron.

[0474] Inorganic sulfur-containing compounds, such as sulfates, sulfites, dithionites, tetrathionates, thiosulfates, sulfides, and organic sulfur compounds, such as mercaptans and sulfhydryls, can be used as sulfur sources.

[0475] Phosphoric acid, potassium dihydrogen phosphate or dipotassium hydrogen phosphate or the corresponding sodium-containing salts can be used as phosphorus sources.

[0476] Chelating agents may be added to the culture medium to keep the metal ions in solution. Particularly suitable chelating agents include dihydroxyphenols, such as catechol or protocatechuate, or organic acids, such as citric acid.

[0477] The fermentation medium used according to the present invention may also contain other growth factors, such as vitamins or growth promoters, which include, for example, biotin, riboflavin, thiamine, folic acid, niacin, pantothenic acid and pyridoxine. Growth factors and salts are usually derived from complex components of the culture medium, such as yeast extract, molasses, corn steep liquor, etc. In addition, suitable precursors can be added to the culture medium. The precise composition of the compounds in the culture medium depends largely on the specific experiment and must be determined separately for each specific case. Information about culture medium optimization can be found in the textbook "Applied Microbiol. Physiology, APractical Approach" (1997). Growth medium can also be obtained from commercial suppliers, such as Standard 1 (Merck) or BHI (brain heart infusion, DIFCO), etc.

[0478] All components of the culture medium are sterilized by heating (1.5 bar and 121°C for 20 minutes) or by sterile filtration. These components can be sterilized together or separately as needed. All components of the culture medium can be given at the beginning of the culture or added continuously or in batches.

[0479] In some embodiments, the fermentation process may include the use of an overlay of an organic liquid phase that is immiscible with the aqueous phase.

[0480] In situ liquid-liquid extraction (biphasic fermentation) is a strategy that can be used according to the present invention to physically separate microbial products by partitioning from an aqueous culture phase into a water-immiscible organic liquid phase. The organic liquid phase or organic phase exists as an upper layer if its density is less than that of the aqueous phase, and as a lower layer if its density is greater than that of the aqueous phase.

[0481] In one embodiment, the organic phase immiscible with the aqueous phase (or simply the organic phase) comprises an alkane, an alcohol having more than 4 carbon atoms, an ester (e.g., isopropyl myristate), a triglyceride (including commercially available vegetable oils such as sunflower oil, soybean oil, or olive oil), a diester (e.g., dialkyl malonate), a ketone, or monoglyme. Other organic solvents immiscible with water or the aqueous phase used may be used. In another embodiment, the organic phase comprises isopropyl myristate. Suitable solvents include, but are not limited to, other esters, aromatic solvents, and the like. In one embodiment, the organic phase comprises an aromatic solvent. Non-limiting examples of aromatic hydrocarbon solvents include benzene, toluene, other alkylated benzenes, anisole, and the like, and mixtures thereof. In one embodiment, the organic phase comprises toluene. Further embodiments include hexane or dodecane.

[0482] The culture temperature is typically between 15°C and 45°C, preferably between 25°C and 40°C, and can be kept constant or varied during the experiment. In certain embodiments, if the cells are eukaryotic organisms, such as yeast, the temperature is preferably in the range of 28°C to 34°C. In certain embodiments, if the cells are prokaryotes, such as bacteria, the temperature is preferably in the range of 30°C to 40°C, for example, 37°C.

[0483] The pH value of the culture medium should be in the range of 4 to 8.5. In certain embodiments, the cell is a eukaryotic organism, such as yeast, and the pH value is preferably from about 4.0 to about 6.5. In certain embodiments, the cell is a prokaryotic organism, such as bacteria, and the pH value is from about 6.5 to about 7.5, for example, about 7.0. The pH value during growth can be controlled by adding alkaline compounds (such as sodium hydroxide, potassium hydroxide, ammonia or ammoniacal liquor) or acidic compounds (such as phosphoric acid or sulfuric acid). Defoaming agents such as fatty acid polyethylene glycol esters can be used to control foaming. In order to maintain the stability of the plasmid, suitable selective substances such as antibiotics can be added to the culture medium. In order to maintain aerobic conditions, oxygen or an oxygen-containing gas mixture (such as ambient air) is supplied to the culture. The temperature of the culture is generally in the range of 20° C. to 45° C. Continue to cultivate until the maximum amount of desired product is formed. This goal will usually be reached within 1 to 160 hours.

[0484] The method of the present invention may further comprise the step of recovering the compound of formula (I).

[0485] The term "recovery" includes extracting, harvesting, isolating or purifying the compound from the culture medium or reaction medium. The recovery of the compound can be carried out according to any conventional separation or purification method known in the art, including but not limited to: treatment with conventional resins (e.g., anion or cation exchange resins, nonionic adsorption resins, etc.), treatment with conventional adsorbents (e.g., activated carbon, silicic acid, silica gel, cellulose, aluminum oxide, etc.), changing the pH value, solvent extraction (e.g., with conventional solvents such as alcohol, ethyl acetate, hexane, etc.), distillation, dialysis, filtration, concentration, crystallization, recrystallization, pH adjustment, lyophilization, etc.

[0486] Before the separation of expectation, the biomass of fermented liquid can be removed.The method of removing biomass is well known to those skilled in the art, for example, filtration, sedimentation and flotation.Therefore, can for example, remove biomass by centrifuge, separator, decanter, filter or in flotation equipment.In order to reclaim valuable product to the greatest extent, it is usually recommended to wash biomass, for example, in the form of diafiltration.The selection of method depends on the content of biomass and the character of biomass in the fermented liquid, and the interaction of biomass and valuable product.

[0487] In one embodiment, the fermentation broth can be sterilized or pasteurized. In another embodiment, the fermentation broth is concentrated. This concentration can be performed batchwise or continuously, as desired. The pressure and temperature ranges should be selected to prevent product damage and minimize equipment and energy use. Skilled selection of pressure and temperature levels for multi-stage evaporation can particularly save energy.

[0488] Embodiments of the method of the present invention

[0489] A preferred embodiment of the method of the present invention is, wherein the compound of formula (Ia) is citronellin, and the compound of formula (I) is citronellin-3-O-acetate. Preferably, the enzyme is an acetyltransferase, and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is citronellin, which is a compound known in the art. The preferred IUPAC name is (2R, 3R)-3,5,7-trihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrobenzopyran-4-one.

[0490] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is taxifolin and the compound of formula (I) is taxifolin-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is taxifolin, which is a compound known in the art. The preferred IUPAC name is (2R,3R)-2-(3,4-dihydroxyphenyl)-3,5,7-trihydroxy-2,3-dihydrochromen-4-one.

[0491] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is dihydrotamaricin and the compound of formula (I) is dihydrotamaricin-3-O-acetate. Preferably, the enzyme is an acetyltransferase, and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68. In this embodiment, the substrate is dihydrotamaricin, which is a compound known in the art. The preferred chemical name is (2R, 3R)-3,5,7-trihydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0492] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is 3'-O-methyltaxifolin and the compound of formula (I) is 3'-O-methyltaxifolin-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is 3'-O-methyltaxifolin, which is a compound known in the art. The preferred chemical name is (2R,3R)-3,5,7-trihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0493] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is brevicornol and the compound of formula (I) is brevicornol-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is brevicornol, which is a compound known in the art. The preferred chemical name is (2R, 3R)-3,5,7-trihydroxy-2-phenyl-2,3-dihydrochromen-4-one.

[0494] Another preferred embodiment of the method of the present invention is, wherein the compound of formula (Ia) is 5-deoxy-orange pigment and the compound of formula (I) is 5-deoxy-orange pigment-3-O-acetate. Preferably, the enzyme is an acetyltransferase, and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is 5-deoxy-orange pigment, which is a compound known in the art. The preferred IUPAC name is (2R, 3R)-3,7-dihydroxy-2-(4-hydroxyphenyl)-2,3-dihydrobenzopyran-4-one.

[0495] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is 5-deoxytaxifolin and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is 5-deoxytaxifolin, which is a compound known in the art. The preferred IUPAC name is (2R, 3R)-3,7-dihydroxy-2-(3,4-dihydroxyphenyl)-2,3-dihydrochromen-4-one.

[0496] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is 5-deoxydihydrotamarixin and the compound of formula (I) is 5-deoxydihydrotamarixin-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68. In this embodiment, the substrate is 5-deoxydihydrotamarixin, which is a compound known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(3-hydroxy-4-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0497] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is 5-deoxy-3'-O-methyltaxifolin and the compound of formula (I) is 5-deoxy-3'-O-methyltaxifolin-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is 5-deoxy-3'-O-methyltaxifolin, which is a compound known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-(4-hydroxy-3-methoxyphenyl)-2,3-dihydrochromen-4-one.

[0498] Another preferred embodiment of the method of the present invention is wherein the compound of formula (Ia) is 5-deoxybreviol and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. Preferably, the enzyme is an acetyltransferase and the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity with any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68. In this embodiment, the substrate is 5-deoxybreviol, which is a compound known in the art. The preferred IUPAC name is (2R,3R)-3,7-dihydroxy-2-phenyl-2,3-dihydrochromen-4-one.

[0499] An embodiment of the present invention is wherein the method is performed in the presence of acyl-CoA. More preferably, the acyl-CoA is acetyl-CoA.

[0500] Acetyl-CoA is a common molecule involved in a variety of biochemical reactions. It is commonly available from many reagent suppliers, such as Sigma. If the method is an in vivo method, acetyl-CoA can be produced by cells.

[0501] Another aspect of the present invention provides a method for preparing a compound of formula (I), comprising reacting a precursor compound of formula (Ia) in the presence of a hydrolase. Preferably, the hydrolase is a lipase, as described herein.

[0502] When the enzyme is a hydrolase, suitable acetates for acetylation of formula (Ia) are selected from unsaturated, saturated or aromatic acetates, including but not limited to vinyl acetate, phenyl vinyl acetate, ethoxyvinyl acetate, isoprenyl acetate, isopropenyl acetate or ethyl acetate, preferably ethyl acetate, and the source of the acetate molecules of the method is ethyl acetate, vinyl acetate or other such molecules as will be appreciated by those skilled in the art.

[0503] Embodiments of this aspect of the invention include all of the embodiments listed above wherein the enzyme is an acetyltransferase.

[0504] For the avoidance of doubt, the processes of the invention set out herein may produce compounds of formula (I) as well as further reaction products which may be formed depending on the substrate materials and process conditions.

[0505] Multi-enzyme method of the present invention

[0506] The flavonoid biosynthesis pathway in plants is well known. The upstream shikimate pathway typically converts chorismate to phenylalanine and L-tyrosine via several well-characterized enzymes. Chorismate can be derived from sugars such as glucose.

[0507] The flavonoid biosynthetic pathway begins with the conversion of L-phenylalanine to trans-cinnamic acid through the nonoxidative deamination of phenylalanine ammonia lyase (PAL). Next, trans-cinnamic acid is hydroxylated in the para position by cinnamate-4-hydroxylase (C4H) to produce p-coumaric acid (4-hydroxycinnamic acid). C4H is a cytochrome P450 monooxygenase that benefits from regeneration by cytochrome P450 reductase (CPR). As an alternative pathway, the amino acid L-tyrosine can be converted to p-coumaric acid by tyrosine ammonia lyase (TAL). Subsequently, p-coumaric acid is activated to p-coumaryl-CoA by 4-coumarate-CoA ligase (4CL). From here, the biosynthetic pathway produces many compounds, such as flavonoids, stilbenes, curcuminoids, and lignins.

[0508] In the case of flavonoids, chalcone synthase (CHS) and chalcone isomerase (CHI) catalyze the condensation of p-coumaroyl-CoA with three molecules of malonyl-CoA to produce naringenin chalcone and, ultimately, naringenin. Alternatively, expression of a chalcone isomerase-like (CHIL) protein has a positive effect on CHS activity.

[0509] In this case, naringenin is 3-hydroxylated using flavanone-3-hydroxylase (F3H) to obtain naringenin (an example of a compound of formula (Ia)), which is then O-acetylated at this position using the acyltransferase method of the present invention to obtain naringenin-3-acetate (an example of a compound of formula (I)). The schematic diagram of this method is shown in FIG. Figure 1 shown.

[0510] In addition to the core enzymes described above, flavonoid 3'-hydroxylase (F3'H) is capable of adding a hydroxyl group to the 3' position of naringenin, which, in combination with F3'H and the acetyltransferase used in the methods of the present invention, produces taxifolin-3-O-acetate (a compound of formula (I)). F3'H is a P450 monooxygenase that benefits from the regeneration of cytochrome P450 reductase (CPR).

[0511] Alternatively, in addition to the core enzymes described above, flavonoid 3'-hydroxylase (F3'H) can add a hydroxyl group to the 3' position of citrus aurantifolia, which, in combination with the acetyltransferase used in the methods of the present invention, produces taxifolin-3-O-acetate (a compound of formula (I)). F3'H is a P450 monooxygenase that benefits from the regeneration of cytochrome P450 reductase (CPR).

[0512] Another possibility to obtain eriodictyol and taxifolin is to use caffeic acid as a starting molecule or to hydroxylate the 3-position of coumaric acid by using a 3-OH-specific hydroxylase (eg coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase).

[0513] In addition to the above enzymes, 4'-O-methyltransferase can add a methyl group to the 4'-OH of eriodictyol or taxifolin and, in combination with F3H and the acetyltransferase used in the methods of the present invention, or the acetyltransferase used in the methods of the present invention, respectively, to produce dihydrotamariscine-3-O-acetate (a compound of formula (I)). 4'-MT can be further engineered to improve its selectivity for the 4'-OH position. Like most methyltransferases, 4'-MT depends on the S-adenosylmethionine (SAM) cofactor and benefits from the regeneration of SAM, which can be provided in vivo through cellular metabolism (engineered for this purpose) or in vitro by adding various enzymes, such as SAH hydrolase (SAHH), methionine synthase (MS), methionine adenosyltransferase (MAT), adenosine kinase (ADK), and polyphosphate kinases (PPK2) I and II.

[0514] Another possibility for obtaining hesperetin and dihydrotamarind is to use isoferulic acid as a starting material, or to methylate the 4-OH position of caffeic acid by applying a 4-O-methyltransferase, or to methylate caffeoyl-CoA by a 4-OH-specific caffeoyl-O-methyltransferase. Alternatively, coumaric acid can be methylated at the 4-position to give 4-methoxycinnamic acid, which can then be hydroxylated at the 3-position by applying a 3-OH-specific hydroxylase (e.g., coumaric acid 3-hydroxylase or 4-hydroxybenzoic acid meta-hydroxylase). Another possibility is to use 4-methoxycinnamic acid as a starting material and to hydroxylate at the 3-position by applying a 3-OH-specific hydroxylase (e.g., coumaric acid 3-hydroxylase or 4-hydroxybenzoic acid meta-hydroxylase). 4-MT can be further engineered to increase its selectivity for the 4-OH position. 4-MT, like most methyltransferases, depends on the S-adenosylmethionine (SAM) cofactor and benefits from the regeneration of SAM, which can be provided in vivo by cellular metabolism (engineered for this purpose) or in vitro by the addition of various enzymes, such as SAH hydrolase (SAHH), methionine synthase (MS), methionine adenosyltransferase (MAT), adenosine kinase (ADK), and polyphosphate kinases (PPK2) I and II.

[0515] In addition to the above enzymes, 3'-O-methyltransferase can add a methyl group to the 3'-OH position of eriodictyol or taxifolin and be combined with F3H and the acetyltransferase used in the method of the present invention, or with the acetyltransferase used in the method of the present invention, respectively, to produce 3'-O-methyl-tachyphyllin-3-O-acetate (a compound of formula (I)). 3'-MT can be further engineered to increase its selectivity for the 3'-OH position. Like most methyltransferases, 3'-MT depends on the S-adenosylmethionine (SAM) cofactor and benefits from the regeneration of SAM, which can be provided in vivo through cellular metabolism (engineered for this purpose) or in vitro by adding various enzymes, such as SAH hydrolase (SAHH), methionine synthase (MS), methionine adenosyltransferase (MAT), adenosine kinase (ADK), and polyphosphate kinases (PPK2) I and II.

[0516] Another possibility for obtaining homoeriochoride and 3'-O-methyltaxifolin is to use ferulic acid as the starting material or to methylate the 3-OH position of caffeic acid using 3-O-methyltransferase caffeoyl-CoA via a 3-OH-specific caffeoyl-O-methyltransferase. 3-MT can be further engineered to improve its selectivity for the 3-OH position. Like most methyltransferases, 3-MT depends on the S-adenosylmethionine (SAM) cofactor and benefits from the regeneration of SAM, which can be provided in vivo through cellular metabolism (engineered for this purpose) or in vitro by adding various enzymes, such as SAH hydrolase (SAHH), methionine synthase (MS), methionine adenosyltransferase (MAT), adenosine kinase (ADK), and polyphosphate kinases (PPK2) I and II.

[0517] For 5-deoxyflavonoids, polyketide reductase (PKR) is added to the pathway. PKR couples with CHS to catalyze the reduction of a specific keto group in the tetraketide intermediate, generating 6'-deoxychalcone. Spontaneous or CHI-catalyzed ring closure generates 5-deoxyflavanones, such as liquiritigenin (5-deoxynaringenin) and / or 5-deoxypinusin. 3-Hydroxylation using flavanone-3-hydroxylase (F3H) is followed by O-acetylation at this position using the acyltransferase method of the present invention, yielding 5-deoxynaringenin-3-O-acetate and 5-deoxypinusin-3-O-acetate (an example of a compound of Formula (I)). To obtain 5-deoxytaxifolin-3-O-acetate, a hydroxylase is added to the above-described taxifolin-3-O-acetate pathway.

[0518] To obtain 5-deoxydihydrotamaricin-3-O-acetate and 5-deoxy-3'-O-methyltaxifolin-3-O-acetate, further methyltransferases were added to the pathway described for dihydrotamaricin-3-O-acetate and 3'-O-methyltaxifolin-3-O-acetate.

[0519] There are also P450 monooxygenases that can add OH groups to intermediate compounds to produce compounds of formula (I). In addition, various hydroxyl groups on the two aromatic rings can be modified by methyltransferases and glycosyltransferases to produce glycosylated derivatives of compounds of formula (I).

[0520] Therefore, it will be appreciated that by using the enzymes listed herein, phenylalanine, tyrosine, sugars and / or other carbon sources can be converted into compounds of formula (I), preferably citronellin-3-O-acetate, taxifolin-3-O-acetate, dihydrotacitratin-3-O-acetate, 3'-O-methyltacitratin-3-O-acetate, brevicornol-3-O-acetate, 5-deoxycitronellin-3-O-acetate, 5-deoxytacitratin-3-O-acetate, 5-deoxy-3'-O-methyltacitratin-3-O-acetate or 5-deoxybrevicornol-3-O-acetate. This can be achieved by a series of cascade enzyme reactions. Such methods can use phenylalanine and / or tyrosine as initial substrates, or, if appropriate upstream enzymes are used, carbon sources such as sugars and glycerol can be used.

[0521] For the avoidance of doubt, glycerol may also be used in place of, or in combination with, glucose or any other carbon source in the inventive methods disclosed herein.

[0522] Therefore, a preferred embodiment of the present invention is wherein the process for preparing the compound of formula (I) further comprises the use of one or more of the following enzymes:

[0523] (a) flavanone 3-hydroxylase (F3H),

[0524] (b) chalcone isomerase (CHI),

[0525] (c) chalcone synthase (CHS),

[0526] (d) 4-Coumarate CoA ligase (4CL),

[0527] (e) cytochrome P450 reductase (CPR),

[0528] (f) tyrosine ammonia lyase (TAL),

[0529] (g) chalcone isomerase-like (CHIL),

[0530] (h) cinnamate-4-hydroxylase (C4H),

[0531] (I) phenylalanine ammonia lyase (PAL),

[0532] (j) flavonoid 3'-hydroxylase (F3'H),

[0533] (k) 3'-O-methyltransferase (3'-MT),

[0534] (1) 4'-O-methyltransferase (4'-MT),

[0535] (m) 3-O-methyltransferase (3-MT),

[0536] (n) 4-O-methyltransferase (4-MT),

[0537] (o) 3-OH-specific P450 monooxygenases,

[0538] (p) glycosidase, and / or

[0539] (q) Polyketide reductase (PKR).

[0540] In addition to the enzymes listed above, those skilled in the art can also identify and employ other hydroxylases, such as P450 monooxygenases that specifically add hydroxyl groups to other positions, and methyltransferases that specifically methylate other positions, to modify the compounds useful in the methods of the present invention. Glycosyltransferases can be used to prepare glycosylated derivatives of the compounds of formula (I).

[0541] Depending on whether the method of the present invention is performed (i) in vitro or in vivo, (ii) in the case of in vivo, the background genetics of the recombinant host strain used, (iii) the starting materials for the method, and (iv) the preferred compound of formula (I) to be prepared, specific combinations of enzymes listed herein are preferred.

[0542] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method one.

[0543] In another embodiment of the present invention, the method is an in vivo method, the starting materials are glucose (or other carbon sources), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method two.

[0544] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method three in this article.

[0545] In another embodiment of the present invention, the method is an in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because the enzyme is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method four in this article.

[0546] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting materials are glucose (or other carbon sources), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method five in this article.

[0547] In another embodiment of the present invention, the method is an in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method six.

[0548] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method seven.

[0549] In another embodiment of the present invention, the method is an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as method eight.

[0550] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method nine in this article.

[0551] In another embodiment of the present invention, the method is an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because the enzyme is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method ten.

[0552] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method 11 in this article.

[0553] In another embodiment of the present invention, the method is an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 12.

[0554] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 13.

[0555] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 14.

[0556] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 15.

[0557] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 16.

[0558] In another embodiment of the present invention, the method is an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 17.

[0559] In one embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as method 18 in this article.

[0560] In another embodiment of the present invention, the method is an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 19.

[0561] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as method 20.

[0562] In another embodiment of the present invention, the method is an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 21.

[0563] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 22.

[0564] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 23.

[0565] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 24.

[0566] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 25.

[0567] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 26.

[0568] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. The CHIL enzyme and CHI enzyme are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 27.

[0569] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is naringenin, and the compound of formula (I) is naringenin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 28.

[0570] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is citronellin, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzyme: acetyltransferase. This embodiment is referred to herein as Method 29.

[0571] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as naringin, and the compound of formula (I) is naringin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step), a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as Method 30.

[0572] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as engeletin, and the compound of formula (I) is engeletin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 31.

[0573] In addition to preparing citrusin-3-O-acetate, the enzyme combination used in the methods No. 1 to 31 of the present invention can also be used in conjunction with an additional enzyme, flavonoid 3'-hydroxylase (F3'H), to prepare citrusin-3-O-acetate (a compound of formula (I)). Therefore, a further embodiment of the present invention provides a method for preparing citrusin-3-O-acetate, which comprises the enzymes listed in methods 1 to 31 and flavonoid 3'-hydroxylase (F3'H). It will be understood by those skilled in the art that adding F3'H to any one of methods 1 to 31 will produce an additional 31 methods. These methods are referred to herein as methods 32 to 62. Therefore, for example, method 32 is based on method 1 with the addition of F3'H, and so on.

[0574] Alternatively, the final 3'-hydroxylation can be achieved by hydroxylating the 3-position of phenylalanine, tyrosine, cinnamic acid, coumaric acid, or coumaroyl-CoA used as an intermediate or starting material in Methods 1 to 27 using the additional enzymes coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase in Methods 1 to 27. Using the additional enzymes coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase in any of Methods 1 to 27 results in 27 additional methods. These methods are referred to herein as Methods 63 to 89. Thus, for example, Method 63 is Method 1 with the addition of coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase, and so on.

[0575] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as Method 90.

[0576] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the practice of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 91.

[0577] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 92.

[0578] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is eriodictyol, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 93.

[0579] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is taxifolin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 94.

[0580] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as eriocitrin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step), a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as Method 95.

[0581] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as astilbin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 96.

[0582] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as a mixture of astilbin and astilbin in an extract of Engelhardia Roxburghiana, and the compounds of formula (I) are citronellin-3-O-acetate and taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 97.

[0583] In addition to preparing citrinin-3-O-acetate and taxifolin-3-O-acetate, the enzyme combination used in the methods No. 1 to 97 of the present invention can also be used in conjunction with an additional methyltransferase specific for the 4'-position (4'-MT) to prepare dihydrotamarixin-3-O-acetate (a compound of formula (I)). Therefore, a further embodiment of the present invention provides a method for preparing dihydrotamarixin-3-O-acetate, which comprises the enzymes listed in methods 1 to 97 and a methyltransferase specific for 4'-OH. Using a methyltransferase specific for the 4'-position (4'-MT) in any of methods 1 to 97 will produce an additional 97 methods. These methods are referred to herein as methods 98 to 194. Therefore, for example, method 98 is based on method 1 with the addition of a methyltransferase specific for the 4'-position (4'-MT), and so on.

[0584] Alternatively, the final 4'-O-methylation can be achieved by methylating the 4-position of tyrosine, coumaric acid, coumaroyl-CoA, caffeic acid, or caffeoyl-CoA used as an intermediate or starting material in Methods 1 to 27 and 63 to 89 using the additional enzyme 4-O-methyltransferase or 4-O-caffeoylmethyltransferase. The use of a methyltransferase specific for the 4-position (4-MT) in any of Methods 1 to 27 and 63 to 89 results in an additional 54 methods. These methods are referred to herein as Methods 195 to 249. Thus, for example, Method 195 is Method 1 with the addition of a 4-O-methyltransferase or 4-O-caffeoylmethyltransferase, and so on.

[0585] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as method 250.

[0586] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 251.

[0587] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 252.

[0588] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is hesperetin, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 253.

[0589] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is dihydrotamaricin, and the compound of formula (I) is dihydrotamaricin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzyme: acetyltransferase. This embodiment is referred to herein as Method 254.

[0590] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as hesperidin, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step), a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as Method 255.

[0591] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 256.

[0592] In addition to preparing agar-agar-acid ester and taxifolin-3-O-acetate, the enzyme combination used in the methods No. 1 to 97 of the present invention can also be used in conjunction with an additional methyltransferase specific for the 3'-position (3'-MT) to prepare 3'-O-methyl-tachyphyllin-3-O-acetate (a compound of formula (I)). Therefore, a further embodiment of the present invention provides a method for preparing 3'-O-methyl-tachyphyllin-3-O-acetate, which comprises the enzymes listed in methods 1 to 97 and a 3'-OH methyltransferase. Using a methyltransferase specific for the 3'-position (3'-MT) in any one of methods 1 to 97 will produce an additional 97 methods. These methods are referred to herein as methods 257 to 353. Therefore, for example, method 257 is based on method 1 with the addition of a methyltransferase specific for the 3'-position (3'-MT), and so on.

[0593] Alternatively, the final 3'-O-methylation can be achieved by methylating the 3-position of caffeic acid or caffeoyl-CoA used as an intermediate or starting material in Methods 63 to 89 using an additional 3-O-methyltransferase or 3-O-caffeoylmethyltransferase. Using a methyltransferase specific for position 3 (3-MT) in any of Steps 63 to 89 results in 27 additional methods. These methods are referred to herein as Methods 354 to 380. Thus, for example, Method 354 is Method 1 with the addition of a methyltransferase specific for position 3 (3-MT), and so on.

[0594] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 381.

[0595] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the practice of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 382.

[0596] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of this method. In this embodiment of the present invention, the method comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 383.

[0597] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is homoeriodictyol, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 384.

[0598] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 3'-O-methyl-taxifolin, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 385.

[0599] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as homoeriol-7-O-glucoside, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step), a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as Method 386.

[0600] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 387.

[0601] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting materials are glucose (or other carbon source) and / or phenylalanine, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the method comprises the same enzymes as methods 7, 9, and 11, but omits cinnamate 4-hydroxylase (C4H) and P450 reductase (CPR). These embodiments are referred to as methods 388, 389, and 390, respectively.

[0602] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is brevicornol-3-O-acetate. In this embodiment of the invention, the method comprises the same enzymes as methods 16, 18, and 20, but omits cinnamate 4-hydroxylase (C4H) and P450 reductase (CPR). These embodiments are referred to as methods 391, 392, and 393, respectively.

[0603] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is pinus, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 394.

[0604] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is brevicornol, and the compound of formula (I) is brevicornol-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 395.

[0605] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as pinus 7-glucoside, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step), a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as method 396.

[0606] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is a glycosylated precursor, such as brevicornin 5-galactosyl-(1-4)-glucoside, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase (or, if performed in vitro, a chemical hydrolysis step) and an acetyltransferase. This embodiment is referred to herein as Method 397.

[0607] In addition to preparing agaretin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamaricin-3-O-acetate, 3'-O-methyltamaricin-3-O-acetate or brevifolia viniferin-3-O-acetate, the present invention Nos. 1 to 27, 32 to 58, 63 to 92, 98 to 124, 129 to 155, 160 to 189, 195 to 252, 257 to 283, 288 to 314, 319 to 348 The enzyme combination used in the methods of 354-383, 388-393 can also be used in conjunction with an additional polyketide reductase (PKR) to prepare 5-deoxy-tangerin-3-O-acetate, 5-deoxy-taxifolin-3-O-acetate, 5-deoxy-dihydro-tamariscine-3-O-acetate, 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate or 5-deoxy-brevicornulin-3-O-acetate (compound of formula (I)). Therefore, a further embodiment of the present invention provides a method for preparing 5-deoxy-aurantidin-3-O-acetate, 5-deoxy-taxifolin-3-O-acetate, 5-deoxy-dihydro-tamariscine-3-O-acetate, 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate or 5-deoxy-brevicornulin-3-O-acetate, which comprises the enzymes listed in methods 1-27, 32-58, 63-92, 98-124, 129-155, 160-189, 195-252, 257-283, 288-314, 319-348, 354-383, 388-393 and a polyketide reductase. The use of a polyketide reductase in any of methods 1-27, 32-58, 63-92, 98-124, 129-155, 160-189, 195-252, 257-283, 288-314, 319-348, 354-383, 388-393 results in an additional 345 methods. These methods are referred to herein as methods 398 to 742. Thus, for example, method 398 is method 1 with the addition of a polyketide reductase, and so on.

[0608] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is isoliquiritigenin, and the compound of formula (I) is 5-deoxy-aurantigen-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 743.

[0609] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is liquiritigenin, and the compound of formula (I) is 5-deoxy-tangerin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 744.

[0610] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is 5-deoxy-orangerin, and the compound of formula (I) is 5-deoxy-orangerin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 745.

[0611] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is butein, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 746.

[0612] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxyeriodictyol, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 747.

[0613] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxytaxifolin, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 748.

[0614] A further embodiment of the present invention provides a method for preparing 5-deoxytaxifolin-3-O-acetate in vitro or in vivo, comprising the enzymes listed in Methods 743 to 745 and flavonoid 3'-hydroxylase (F3'H). It will be appreciated by those skilled in the art that the addition of F3'H to any of Methods 743 to 745 results in three additional methods. These methods are referred to herein as Methods 749 to 751. Thus, for example, Method 749 is Method 743 with the addition of F3'H, and so on.

[0615] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is 5-deoxyhesperetin chalcone, and the compound of formula (I) is 5-deoxydihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 752.

[0616] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is 5-deoxyhesperetin, and the compound of formula (I) is 5-deoxydihydrotamarixanthin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 753.

[0617] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is 5-deoxydihydrotamaricin, and the compound of formula (I) is 5-deoxydihydrotamaricin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 754.

[0618] A further embodiment of the present invention provides a method for preparing 5-deoxydihydrotamariscine-3-O-acetate in vitro or in vivo, comprising the enzymes listed in Methods 746 to 751 and 4'-O-methyltransferase (4'-MT). Those skilled in the art will appreciate that adding 4'-MT to any of Methods 746 to 751 will result in six additional methods. These methods are referred to herein as Methods 755 to 760. Thus, for example, Method 755 is based on Method 746 with the addition of 4'-MT, and so on.

[0619] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxyhomoeriochoride chalcone, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as Method 761.

[0620] In another embodiment of the present invention, the method is an in vitro or in vivo method, the starting material is 5-deoxyhomoeriodictyol, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the present invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 762.

[0621] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxy-3'-O-methyl-taxifolin, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 763.

[0622] A further embodiment of the present invention provides a method for preparing 5-deoxydihydrotamariscine-3-O-acetate in vitro or in vivo, comprising the enzymes listed in Methods 746 to 751 and a 3'-O-methyltransferase (3'-MT). Those skilled in the art will appreciate that adding 3'-MT to any of Methods 746 to 751 will result in six additional methods. These methods are referred to herein as Methods 764 to 769. Thus, for example, Method 764 is based on Method 746 with the addition of 3'-MT, and so on.

[0623] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxypinusin (7-hydroxyflavanone) chalcone, and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as method 770.

[0624] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxypinusin (7-hydroxyflavanone), and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as Method 771.

[0625] In another embodiment of the invention, the method is an in vitro or in vivo method, the starting material is 5-deoxybreviol and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. In this embodiment of the invention, the method comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as Method 772.

[0626] In addition, those skilled in the art will appreciate that the precursors of the starting materials of methods 743 to 772 may be glycosylated. Therefore, further embodiments of the present invention provide methods for preparing 5-deoxy-aurantidin-3-O-acetate, 5-deoxy-taxifolin-3-O-acetate, 5-deoxy-dihydro-tamariscine-3-O-acetate, 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate, or 5-deoxy-brevicornulin-3-O-acetate, the methods comprising the glycosylated precursors of the starting materials of methods 743 to 772, the enzymes listed in methods 743 to 772, and a glycosidase. Those skilled in the art will appreciate that using the glycosylated precursors of the starting materials and adding a glycosidase to any of methods 743 to 772 will result in an additional 30 methods. These methods are referred to herein as methods 773 to 802. Thus, for example, method 773 is based on method 743, using the glycosylated precursors of the starting materials and adding a glycosidase, and so on.

[0627] It will be apparent to those skilled in the art that further acetylated molecules with additional hydroxyl and methoxy groups can be prepared and used in the methods of the invention by using specific P450 monooxygenases and optionally P450 reductases, other types of hydroxylases and / or methyltransferases.

[0628] Conventional acyltransferases may be used to incorporate further acylated molecules into the methods of the invention.

[0629] Examples of such enzymes are well known in the art and can be readily used in the above methods.Each enzyme can also be codon optimized for expression in a specific host cell as described herein.

[0630] As described herein, the methods of the present invention may be in vitro or in vivo methods. If the method is an in vivo method, examples of suitable host cells are provided above in the description.

[0631] Furthermore, it will be appreciated by those skilled in the art that such host cells can be optimized for use in the methods of the present invention by up-regulating or down-regulating, exchanging and engineering certain genes, thereby increasing the metabolic flux of flavonoid precursors and / or reducing carbon losses caused by the production of undesirable products.

[0632] For example, by modifying the shikimate pathway, acetyl-CoA and / or malonyl-CoA biosynthesis, the metabolic flux of precursor molecules can be increased.

[0633] In one embodiment, cells engineered to produce a compound of formula (I) can be further engineered to increase the supply of the precursor malonyl-CoA. One strategy for increasing malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC) activity. In various embodiments, ACC, which is a large single-chain polypeptide in most eukaryotic organisms (including fungi) and a multi-subunit enzyme in plants and bacteria (e.g., E. coli), is overexpressed in the host strain.

[0634] In a further embodiment, an engineered host cell that overexpresses a gene encoding pyruvate dehydrogenase (PDH), an enzyme that converts pyruvate to acetyl-CoA, is also contemplated.

[0635] Alternatively, in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA through a mechanism independent of ACC activity may also be employed.

[0636] In some embodiments, cells engineered to produce a compound of Formula (I) are further engineered to increase the cell's malonyl-CoA supply and contain an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that can generate malonyl-CoA from malonate. Malonate can optionally be added to the culture medium containing cells engineered to express malonyl-CoA synthetase. Engineered cells containing an exogenous gene encoding a malonyl-CoA synthetase can also contain an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene.

[0637] In additional embodiments, a cell engineered to produce a compound of Formula (I) is further engineered to contain an exogenous nucleic acid sequence encoding a malonyl-CoA transferase that produces malonyl-CoA by transferring CoA directly from acetyl-CoA.

[0638] In some embodiments, cells engineered to produce a compound of Formula (I) are further engineered to increase the supply of coenzyme A (CoA), thereby increasing its availability for the production of acetyl-CoA, malonyl-CoA, and / or p-coumaroyl-CoA. Strategies for increasing the supply of CoA include upregulating endogenous pantothenate kinase (PanK) (EC 2.7.1.33), which produces CoA from pantothenate. Alternatively, or in addition, the host cell can be engineered to contain a nucleic acid sequence encoding a type III pantothenate kinase that is not subject to feedback inhibition by CoA.

[0639] Other strategies to increase the malonyl-CoA flux to the flavonoid pathway include mutating or lowering one or more genes involved in fatty acid biosynthesis. Without limiting the embodiment to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the β-ketoacyl-ACP synthase II gene can be destroyed to reduce fatty acid biosynthesis. Another example of a host cell fatty acid biosynthesis gene that can be mutated or lowered is the gene encoding malonyl-CoA-ACP transacylase. Other fatty acid biosynthesis genes that can be lowered in engineered host cells include β-ketoacyl-ACP synthase I enzyme and acyl carrier protein.

[0640] Additional genetic modifications that may be present in host cells engineered to produce compounds of Formula (I) include downregulation, disruption or deletion of genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetylphosphotransferase and acetate kinase.

[0641] Additionally, cells engineered to produce compounds of Formula (I) may have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of the precursors malonyl-CoA, acetyl-CoA, and / or p-coumaroyl-CoA.

[0642] In further embodiments, it is contemplated that the host cells engineered to produce the compounds of formula (I) are further engineered to increase the endogenous biosynthesis of the amino acids phenylalanine or tyrosine. Phenylalanine and tyrosine are precursors for flavonoid biosynthesis. Phenylalanine and tyrosine are derived from the shikimate pathway. Strategies for increasing phenylalanine and tyrosine production include, but are not limited to, transcriptional dysregulation, elimination of feedback inhibition, and overexpression of rate-limiting enzymes.

[0643] Alternatively, flux can be diverted towards tyrosine by deleting the L-phenylalanine branch of the aromatic acid biosynthetic pathway.

[0644] Alternatively or in addition, the coding genes of the tricarboxylic acid cycle (TCA) enzymes involved in the biosynthesis of α-ketoglutarate can be increased or overexpressed, while the α-ketoglutarate dehydrogenase that catalyzes the oxidative decarboxylation of α-ketoglutarate to succinyl-CoA in TCA can be destroyed or lowered to increase the supply of α-ketoglutarate that can serve as a cofactor for one or more flavonoid pathway enzymes. Succinate dehydrogenase can be modified to ensure flux to ketoglutarate and avoid accumulation of succinate. Alternatively, the transport of mitochondrial α-ketoglutarate to the cytoplasm can be improved by overexpressing the transporter responsible for transporting α-ketoglutarate from the mitochondria to the cytoplasm. α-ketoglutarate can be added to the culture medium as appropriate. Engineered cells can also include an α-ketoglutarate transporter.

[0645] Other TCA enzymes that can be modified include citrate synthase, which converts acetyl-CoA to citrate.

[0646] In a further embodiment, further engineering of cells engineered to produce compounds of formula (I) is contemplated to upregulate the endogenous biosynthesis of the cofactor heme. Cytochrome P450 (CYPs) is one of the exogenous genes in the engineered cells provided herein that contains heme as a cofactor. Improving the heme supply is an effective strategy to increase flavonoid biosynthesis. 5-Aminolevulinic acid (ALA) is the first precursor in the heme pathway. Strategies to increase heme supply include overexpression of genes that synthesize ALA precursors. In addition, one or more downstream genes that catalyze the synthesis of heme from ALA can be overexpressed to drive flux from ALA to heme production.

[0647] In further embodiments, it is contemplated that the host cells engineered to produce a compound of formula (I) are further engineered to regenerate S-adenosylmethionine by adding enzymes such as S-adenosylmethionine synthetase (SAM), SAH hydrolase (SAHH), methionine synthase (MS), methionine adenosyltransferase (MAT), serine hydroxymethyltransferase (SHM2), methylenetetrahydrofolate reductase (MTHFR), dihydrofolate reductase (DHFR), adenosine kinase (ADK), and polyphosphate kinase (PPK2) I and II. Alternatively, these enzymes can be added to the in vitro reaction.

[0648] In addition, the host cell may comprise deletions, downregulations, exchanges or engineering modifications of certain genes of the host organism to direct the flux of precursors and intermediates to the target pathway and avoid the formation of byproducts. For example, deletions, downregulations, exchanges or engineering modifications of one or more double bond reductases that reduce coumaroyl-CoA to 2,3-dihydrocoumaric acid, such as the enzyme called TSC13 in Saccharomyces cerevisiae. Also included are host cell strains that exchange or engineer one or more aromatic aminotransferases to shift the equilibrium toward aromatic amino acids, such as Aro8 in Saccharomyces cerevisiae. Also included are host cells that comprise deletions, downregulations, exchanges or engineering modifications of one or more 2-oxoacid decarboxylases, such as Aro10, PDC1, PDC5 and PDC6 in Saccharomyces cerevisiae, to prevent the decarboxylation of phenylpyruvate. Also included is the deletion, downregulation, exchange or engineering of one or more phenylacrylic acid decarboxylases and / or ferulic acid decarboxylases, such as PAD1 and FDC1 in S. cerevisiae and UbiX and UbiD in E. coli, to prevent the decarboxylation of cinnamic and coumaric acids (or also caffeic, ferulic and isoferulic acids, if they are used as starting materials).

[0649] In order to avoid accumulation of the final product in the cell, suitable transporters can also be used to optimize the export. For example, any transporter capable of binding and exporting flavonoids from the cell can be used for this purpose. As will be appreciated by those skilled in the art, examples of such transporters can be found in plants.

[0650] For the avoidance of doubt, as stated above, the methods of the present invention may use a hydrolase enzyme instead of an acyltransferase.

[0651] Thus, in each of the methods of the present invention listed as methods 1 to 802 provided above, the acetyltransferase can be replaced by a hydrolase.

[0652] Recombinant cells of the present invention

[0653] The present invention provides, for the first time, a recombinant cell comprising a compound of formula (I). The recombinant cell is particularly useful for preparing specific compounds of formula (I), particularly citronellin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltamarixanthin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxycitronellin-3-O-acetate, 5-deoxytamarixanthin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, 5-deoxy-3'-O-methyltamarixanthin-3-O-acetate, or 5-deoxybrevicornin-3-O-acetate. Prior to the present invention, no one had been able to prepare these compounds using in vivo methods involving recombinant cells.

[0654] By using recombinant cell technology to synthesize the compound of formula (I), the compound can be produced without synthetic chemistry, thereby providing a source of the compound of formula (I) that is more easily accepted by users for consumption, in particular, a source of citronellol-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltaxifolin-3-O-acetate, brevifoliain-3-O-acetate, 5-deoxycitronellol-3-O-acetate, 5-deoxytaxifolin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, 5-deoxy-3'-O-methyltaxifolin-3-O-acetate or 5-deoxybrevifoliain-3-O-acetate.

[0655] In some embodiments, the compound of Formula (I) or Formula (Ia) is produced by whole-cell biotransformation. In whole-cell biotransformation, host cells expressing one or more enzymes involved in the biosynthetic pathway take up and modify the precursor within the cell; after modification in vivo, the compound of Formula (I) or Formula (Ia) remains within the cell and / or is secreted into the culture medium.

[0656] Therefore, one aspect of the present invention provides a recombinant cell comprising a compound of formula (I). Preferably, the compound of formula (I) is citronellin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamarixanthin-3-O-acetate, 3'-O-methyltamarixanthin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxycitronellin-3-O-acetate, 5-deoxytaxifolin-3-O-acetate, 5-deoxydihydrotamarixanthin-3-O-acetate, 5-deoxy-3'-O-methyltamarixanthin-3-O-acetate, or 5-deoxybrevicornin-3-O-acetate.

[0657] One embodiment of this aspect of the invention is wherein the recombinant cell further comprises a polypeptide capable of synthesizing the compound of formula (I) from the compound of formula (Ia). Preferably, the polypeptide capable of synthesizing the compound of formula (I) from the compound of formula (Ia) is an acyltransferase or a hydrolase.

[0658] A preferred embodiment of the present invention is wherein the recombinant cell comprises an acetyltransferase comprising the amino acid sequence HXXXD (SEQ ID NO: 29) and / or the amino acid sequence [DN]FGxG (SEQ ID NO: 30). Preferably, the acetyltransferase further comprises the amino acid sequence [ST]S[WL] (SEQ ID NO: 94). Preferably, the acetyltransferase is a recombinant acetyltransferase. More preferably, the acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36, and 55 to 68. A further preferred embodiment of the present invention is wherein the recombinant cell heterologously expresses or overexpresses the acetyltransferase.

[0659] A preferred embodiment of the present invention is wherein the recombinant cell comprises a recombinant nucleic acid sequence encoding an acyltransferase, preferably an acetyltransferase. More preferably, the recombinant nucleic acid sequence encoding an acetyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or comprises a nucleotide sequence of any one of SEQ ID NOs: 8 to 28, 37 to 54 and 69 to 93, or a reverse complement thereof.

[0660] In some embodiments, the recombinant cells of this form of the invention further comprise one or more of the following enzymes:

[0661] (a) flavanone 3-hydroxylase (F3H),

[0662] (b) chalcone isomerase (CHI),

[0663] (c) chalcone synthase (CHS),

[0664] (d) 4-Coumarate CoA ligase (4CL),

[0665] (e) cytochrome P450 reductase (CPR),

[0666] (f) tyrosine ammonia lyase (TAL),

[0667] (g) chalcone isomerase-like (CHIL),

[0668] (h) cinnamate-4-hydroxylase (C4H),

[0669] (I) phenylalanine ammonia lyase (PAL),

[0670] (j) flavonoid 3'-hydroxylase (F3'H),

[0671] (k) 3'-O-methyltransferase (3'-MT),

[0672] (1) 4'-O-methyltransferase (4'-MT),

[0673] (m) 3-O-methyltransferase (3-MT),

[0674] (n) 4-O-methyltransferase (4-MT),

[0675] (o) 3-OH-specific P450 monooxygenases,

[0676] (p) glycosidase, and / or

[0677] (q) Polyketide reductase (PKR).

[0678] In one embodiment of the invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell one.

[0679] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell two.

[0680] In another embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as recombinant cell three in this article.

[0681] In another embodiment of the present invention, the recombinant cell is used for an in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because the enzyme is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell four.

[0682] In another embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as recombinant cell five in this article.

[0683] In another embodiment of the present invention, the recombinant cell is used for an in vivo method, the starting material is glucose (or other carbon source), phenylalanine and / or tyrosine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell six.

[0684] In one embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell seven.

[0685] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell eight.

[0686] In another embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumaric acid-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as recombinant cell nine in this article.

[0687] In another embodiment of the present invention, the recombinant cell is used for an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell ten.

[0688] In another embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is glucose (or other carbon sources) and / or phenylalanine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as recombinant cell 11 in this article.

[0689] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: phenylalanine ammonia lyase (PAL), cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 12.

[0690] In one embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 13.

[0691] In one embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 14.

[0692] In one embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or tyrosine, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: tyrosine ammonia lyase (TAL), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 15.

[0693] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 16.

[0694] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellin-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 17.

[0695] In one embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to as recombinant cell 18 in this article.

[0696] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 19.

[0697] In another embodiment of the present invention, the recombinant cell is used for an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is citronellal-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 20.

[0698] In another embodiment of the present invention, the recombinant cell is used in an in vivo method, the starting material is cinnamic acid, the compound of formula (I) is citronellal-3-O-acetate, and the microbial host cell comprises a functional CPR, such as a yeast cell. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: cinnamate-4-hydroxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 21.

[0699] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 22.

[0700] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 23.

[0701] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaric acid, and the compound of formula (I) is citronellin-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of this method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 24.

[0702] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 25.

[0703] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 26.

[0704] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is coumaroyl-CoA, and the compound of formula (I) is citronellin-3-O-acetate. CHIL and CHI enzymes are omitted here because these enzymes are not necessary for the implementation of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 27.

[0705] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is naringenin, and the compound of formula (I) is naringenin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 28.

[0706] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is citronellin, and the compound of formula (I) is citronellin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 29.

[0707] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as naringin, and the compound of formula (I) is naringin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: a glycosidase, a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 30.

[0708] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as citronellol, and the compound of formula (I) is citronellol-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: a glycosidase and an acetyltransferase. This embodiment is referred to herein as recombinant cell 31.

[0709] In addition to preparing citrusin-3-O-acetate, the enzyme combinations numbered 1 to 31 in the method of the present invention can also be added with an additional enzyme, flavonoid 3'-hydroxylase (F3'H), for the preparation of citrusin-3-O-acetate (a compound of formula (I)). Therefore, a further embodiment of the present invention provides a recombinant cell for the preparation of citrusin-3-O-acetate, which comprises the enzymes and flavonoid 3'-hydroxylase (F3'H) listed in recombinant cells 1 to 31. It will be understood by those skilled in the art that the addition of F3'H to any one of recombinant cells 1 to 31 will produce an additional 31 recombinant cells. These recombinant cells are referred to herein as recombinant cells 32 to 62. Therefore, for example, recombinant cell 32 is based on recombinant cell 1 with the addition of F3'H, and so on.

[0710] Alternatively, the final 3'-hydroxylation can be achieved by hydroxylating the 3-position of phenylalanine, tyrosine, cinnamic acid, coumaric acid, or coumaroyl-CoA used as an intermediate or starting material in methods 1 to 27 by adding the additional enzyme coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase to recombinant cells 1 to 27. Adding the additional enzyme coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase to any of recombinant cells 1 to 27 will produce 27 additional recombinant cells. These recombinant cells are referred to herein as recombinant cells 63 to 89. Thus, for example, recombinant cell 63 is recombinant cell 1 with the addition of coumarate 3-hydroxylase or 4-hydroxybenzoate meta-hydroxylase, and so on.

[0711] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 90.

[0712] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the practice of this method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 91.

[0713] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is caffeic acid, and the compound of formula (I) is taxifolin-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of the method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 92.

[0714] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is eriodictyol, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 93.

[0715] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is taxifolin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 94.

[0716] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as eriocitrin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase, a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 95.

[0717] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as astilbin, and the compound of formula (I) is taxifolin-3-O-acetate. In this embodiment of the invention, the method comprises the following enzymes: a glycosidase and an acetyltransferase. This embodiment is referred to herein as recombinant cell 96.

[0718] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as a mixture of astilbin and astilbin in an extract of Engelhardia Roxburghiana, and the compounds of formula (I) are citrinin-3-O-acetate and taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase and an acetyltransferase. This embodiment is referred to herein as recombinant cell 97.

[0719] In addition to preparing citrinin-3-O-acetate and taxifolin-3-O-acetate, the enzyme combination used in the recombinant cells numbered 1 to 97 of the present invention can also be used to prepare dihydrotamarixin-3-O-acetate (a compound of formula (I)) by adding a methyltransferase specific for the 4'-position (4'-MT). Therefore, a further embodiment of the present invention provides a recombinant cell for preparing dihydrotamarixin-3-O-acetate, which comprises the enzymes listed in recombinant cells 1 to 97 and a methyltransferase specific for 4'-OH. Adding a methyltransferase specific for the 4'-position (4'-MT) to any of recombinant cells 1 to 97 will produce an additional 97 recombinant cells. These recombinant cells are referred to herein as recombinant cells 98 to 194. Therefore, for example, recombinant cell 98 is based on recombinant cell 1 with the addition of a methyltransferase specific for the 4'-position (4'-MT), and so on.

[0720] Alternatively, the final 4'-O-methylation can be achieved by using recombinant cells 1 to 27 and 63 to 89 and adding an additional 4-O-methyltransferase or 4-O-caffeoyl methyltransferase to recombinant cells 1 to 27 and 63 to 89 to methylate the 4-position of tyrosine, coumaric acid, coumaroyl-CoA, caffeic acid, or caffeoyl-CoA as an intermediate or starting material. Adding a methyltransferase specific for position 4 (4-MT) to any of recombinant cells 1 to 27 and 63 to 89 will produce an additional 54 recombinant cells. These recombinant cells are referred to herein as recombinant cells 195 to 249. Thus, for example, recombinant cell 195 is based on method 1 with the addition of a 4-O-methyltransferase or 4-O-caffeoyl methyltransferase, and so on.

[0721] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamaricin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 250.

[0722] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. The CHIL enzyme is omitted here because it is not required for the practice of this method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 251.

[0723] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is isoferulic acid, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of this method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 252.

[0724] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is hesperetin, and the compound of formula (I) is dihydrotamarixin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 253.

[0725] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is dihydrotamaricin, and the compound of formula (I) is dihydrotamaricin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 254.

[0726] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as hesperidin, and the compound of formula (I) is dihydrotamarixin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: a glycosidase, a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 255.

[0727] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, and the compound of formula (I) is dihydrotamariscine-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: a glycosidase (if performed in vitro) and an acetyltransferase. This embodiment is referred to herein as recombinant cell 256.

[0728] In addition to preparing citrusin-3-O-acetate and taxifolin-3-O-acetate, the enzyme combination used in the recombinant cells numbered 1 to 97 of the present invention can also be used to prepare 3'-O-methyl-taxifolin-3-O-acetate (a compound of formula (I)) by adding a methyltransferase specific for the 3'-position (3'-MT). Therefore, a further embodiment of the present invention provides a recombinant cell for preparing 3'-O-methyl-taxifolin-3-O-acetate, which comprises the enzymes listed in recombinant cells 1 to 97 and a 3'-OH methyltransferase. Adding a methyltransferase specific for the 3'-position (3'-MT) to any one of recombinant cells 1 to 97 will produce an additional 97 recombinant cells. These recombinant cells are referred to herein as recombinant cells 257 to 353. Therefore, for example, recombinant cell 257 is based on method 1 and a methyltransferase specific for the 3'-position (3'-MT) is added, and so on.

[0729] Alternatively, the final 3'-O-methylation can be achieved by methylating the 3-position of caffeic acid or caffeoyl-CoA used as an intermediate or starting material in methods 63 to 89 by adding an additional 3-O-methyltransferase or 3-O-caffeoylmethyltransferase to recombinant cells 63 to 89. Adding a methyltransferase specific for position 3 (3-MT) to any of recombinant cells 63 to 89 results in 27 additional recombinant cells. These recombinant cells are referred to herein as recombinant cells 354 to 380. Thus, for example, recombinant cell 354 is recombinant cell 1 with the addition of a methyltransferase specific for position 3 (3-MT), and so on.

[0730] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-dihydroquinone-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), chalcone isomerase-like protein (CHIL), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 381.

[0731] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-dihydroquinone-3-O-acetate. The CHIL enzyme is omitted here because it is not necessary for the practice of this method. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 382.

[0732] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is ferulic acid, and the compound of formula (I) is 3'-O-methyl-dihydroquinone-3-O-acetate. CHIL and CHI enzymes are omitted here because they are not necessary for the practice of this method. In this embodiment of the present invention, the recombinant cell contains the following enzymes: 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 383.

[0733] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is homoeriodictyol, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 384.

[0734] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 3'-O-methyl-taxifolin, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 385.

[0735] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as homoeriol-7-O-glucoside, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase, a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 386.

[0736] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, and the compound of formula (I) is 3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase and an acetyltransferase. This embodiment is referred to herein as recombinant cell 387.

[0737] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is glucose (or other carbon source) and / or phenylalanine, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the same enzymes as recombinant cells 7, 9, and 11, but omits cinnamate 4-hydroxylase (C4H) and P450 reductase (CPR). These embodiments are referred to as recombinant cells 388, 389, and 390.

[0738] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is cinnamic acid, and the compound of formula (I) is brevicornol-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the same enzymes as recombinant cells 16, 18, and 20, but omits cinnamate 4-hydroxylase (C4H) and P450 reductase (CPR). These embodiments are referred to as recombinant cells 391, 392, and 393.

[0739] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is pinocybin, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 394.

[0740] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is brevicornin, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 395.

[0741] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylated precursor, such as pinus 7-glucoside, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase, a flavanone-3-hydroxylase (F3H), and an acetyltransferase. This embodiment is referred to herein as recombinant cell 396.

[0742] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is a glycosylation precursor, such as brevicornin-5-galactosyl-(1-4)-glucoside, and the compound of formula (I) is brevicornin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: a glycosidase and an acetyltransferase. This embodiment is referred to herein as recombinant cell 397.

[0743] In addition to preparing agaretin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamaricin-3-O-acetate, 3'-O-methyltamaricin-3-O-acetate or brevifolia viniferin-3-O-acetate, the present invention Nos. 1 to 27, 32 to 58, 63 to 92, 98 to 124, 129 to 155, 160 to 189, 195 to 252, 257 to 283, 288 to 314, 319 to 348, The enzyme combination used in the recombinant cells of 354-383 and 388-393 can also be used in conjunction with an additional polyketide reductase (PKR) to prepare 5-deoxy-aurantidin-3-O-acetate, 5-deoxy-taxifolin-3-O-acetate, 5-deoxy-dihydro-tamariscine-3-O-acetate, 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate or 5-deoxy-brevicornulin-3-O-acetate (compounds of formula (I)). Therefore, a further embodiment of the present invention provides a recombinant cell for preparing 5-deoxy-aurantidin-3-O-acetate, 5-deoxy-taxifolin-3-O-acetate, 5-deoxy-dihydro-tamariscin-3-O-acetate, 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate or 5-deoxy-brevicornulin-3-O-acetate, comprising an enzyme listed in any one of recombinant cells 1-27, 32-58, 63-92, 98-124, 129-155, 160-189, 195-252, 257-283, 288-314, 319-348, 354-383, 388-393 and a polyketide reductase. The use of a polyketide reductase in any of recombinant cells 1-27, 32-58, 63-92, 98-124, 129-155, 160-189, 195-252, 257-283, 288-314, 319-348, 354-383, and 388-393 results in an additional 345 recombinant cells. These recombinant cells are referred to herein as recombinant cells 398 to 742. Thus, for example, recombinant cell 398 is recombinant cell 1 with the addition of a polyketide reductase, and so on.

[0744] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is isoliquiritigenin, and the compound of formula (I) is 5-deoxy-tangerin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 743.

[0745] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is liquiritigenin, and the compound of formula (I) is 5-deoxy-tangerin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 744.

[0746] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxy-orangerin, and the compound of formula (I) is 5-deoxy-orangerin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 745.

[0747] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is butein, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 746.

[0748] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxyeriodictyol, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the present invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 747.

[0749] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxytaxifolin, and the compound of formula (I) is 5-deoxytaxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 748.

[0750] A further embodiment of the present invention provides a recombinant cell for producing 5-deoxytaxifolin-3-O-acetate in vitro or in vivo, comprising the enzymes listed in recombinant cells 743 to 745 and flavonoid 3'-hydroxylase (F3'H). It will be appreciated by those skilled in the art that adding F3'H to any of recombinant cells 743 to 745 will result in three additional recombinant cells. These recombinant cells are referred to herein as recombinant cells 749 to 751. Thus, for example, recombinant cell 749 is recombinant cell 743 with the addition of F3'H, and so on.

[0751] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxyhesperidin chalcone, and the compound of formula (I) is 5-deoxydihydrotamariscine-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 752.

[0752] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxyhesperetin, and the compound of formula (I) is 5-deoxydihydrotamarixanthin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 753.

[0753] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxydihydrotamaricin, and the compound of formula (I) is 5-deoxydihydrotamaricin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 754.

[0754] A further embodiment of the present invention provides a recombinant cell for producing 5-deoxydihydrotamariscine-3-O-acetate in vitro or in vivo, comprising the enzymes listed in recombinant cells 746 to 751 and 4'-O-methyltransferase (4'-MT). Those skilled in the art will appreciate that adding 4'-MT to any of recombinant cells 746 to 751 will produce six additional recombinant cells. These recombinant cells are referred to herein as recombinant cells 755 to 760. Thus, for example, recombinant cell 755 is recombinant cell 746 with the addition of 4'-MT, and so on.

[0755] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxyhomoeriochoride chalcone, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 761.

[0756] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxyhomoeriodictyol, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: flavanone-3-hydroxylase (F3H) and acetyltransferase. This embodiment is referred to herein as recombinant cell 762.

[0757] In another embodiment of the present invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxy-3'-O-methyl-taxifolin, and the compound of formula (I) is 5-deoxy-3'-O-methyl-taxifolin-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzyme: an acetyltransferase. This embodiment is referred to herein as recombinant cell 763.

[0758] Other embodiments of the present invention provide recombinant cells for producing 5-deoxydihydrotamariscine-3-O-acetate in vitro or in vivo, comprising the enzymes listed in recombinant cells 746 to 751 and a 3'-O-methyltransferase (3'-MT). Those skilled in the art will appreciate that adding 3'-MT to any of recombinant cells 746 to 751 will produce six additional recombinant cells. These recombinant cells are referred to herein as recombinant cells 764 to 769. Thus, for example, recombinant cell 764 is recombinant cell 746 with the addition of 3'-MT, and so on.

[0759] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxypinosyl (7-hydroxyflavanone) chalcone, and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. In this embodiment of the invention, the recombinant cell comprises the following enzymes: chalcone isomerase (CHI), flavanone-3-hydroxylase (F3H), and acetyltransferase. This embodiment is referred to herein as recombinant cell 770.

[0760] In another embodiment of the invention, the recombinant cell is used in an in vitro or in vivo method, the starting material is 5-deoxypinusin (7-hydroxyflavanone), and the compound of formula (I) is 5-deoxybreviol-3-O-acetate. In this embodiment of the invention, the ...

Claims

1. A method for preparing a compound of formula (I): in: R 1 A hydrogen atom, -OH or -OR 1A ; R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 2 A hydrogen atom, -OH or -OR 2A ; R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 3 A hydrogen atom, -OH or -OR 3A ; R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 4 A hydrogen atom, -OH or -OR 4A ; R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 5 -OC(O)-(C 1-24 alkyl); R 6 and R 7 Independently from C 1-6 Alkyl, -OH, C 1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group; m is 0, 1, or 2; and n is 0, 1, 2, or 3; The method comprises reacting a precursor compound of formula (Ia) with an acyltransferase to form a compound of formula (I): in: R 1 A hydrogen atom, -OH or -OR 1A ; R 1A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 2 A hydrogen atom, -OH or -OR 2A ; R 2A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 3 A hydrogen atom, -OH or -OR 3A ; R 3A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 4 A hydrogen atom, -OH or -OR 4A ; R 4A C 1-6 Alkyl, optionally selected from -OH and C 1-6 The substituents of the alkoxy group are substituted one or more times; R 6 and R 7 Independently from C 1-6 Alkyl, -OH, C 1-6 Alkoxy and -O-(C 1-6 Alkylene)-O-(C 1-6 alkyl) group; m is 0, 1, or 2; and n is 0, 1, 2 or 3.

2. The method according to claim 1, wherein the method is carried out in the presence of acyl-CoA.

3. The method according to claim 1 or 2, wherein the acyltransferase is an acetyltransferase.

4. The method according to any one of claims 1 to 3, wherein the acyltransferase comprises the amino acid sequence HXXXD (SEQ ID NO: 29) and / or the amino acid sequence [DN]FGxG (SEQ ID NO: 30).

5. The method according to any one of claims 1 to 4, wherein the acyltransferase comprises the amino acid sequence [ST]S[WL] (SEQ ID NO: 94).

6. The method of any one of claims 1 to 5, wherein the acyltransferase has at least 50%, 55%, 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

7. The method according to any one of the preceding claims, wherein the compound of formula (Ia) is aurantin, taxifolin, dihydrotamarixanthin, 3'-O-methyltamarixanthin, brevicornin, 5-deoxyaurantin, 5-deoxytaxifolin, 5-deoxydihydrotamarixanthin, 5-deoxy-3'-O-methyltamarixanthin or 5-deoxybrevicornin.

8. The method according to any one of the preceding claims, wherein the compound of formula (I) is agaretin-3-O-acetate, taxifolin-3-O-acetate, dihydrotamaricin-3-O-acetate, or 3'-O-methyltamaricin-3-O-acetate, brevicornin-3-O-acetate, 5-deoxyagaretin-3-O-acetate, 5-deoxytaxifolin-3-O-acetate, 5-deoxydihydrotamaricin-3-O-acetate, 5-deoxy-3'-O-methyltamaricin-3-O-acetate or 5-deoxybrevicornin-3-O-acetate.

9. The method according to any one of the preceding claims, wherein the method is performed in vivo.

10. A recombinant polypeptide having acyltransferase activity, comprising an amino acid sequence having at least 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98% or 99% sequence identity to any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68, or comprising the amino acid sequence of any one of SEQ ID NOs: 1 to 7, 31 to 36 and 55 to 68.

11. A recombinant cell comprising a compound of formula (I).

12. The recombinant cell according to claim 11, further comprising an acyltransferase, preferably an acetyltransferase.

13. The recombinant cell according to claim 11 or 12, further comprising a recombinant nucleic acid sequence encoding an acyltransferase, preferably an acetyltransferase.

14. The recombinant cell according to any one of claims 11 to 13, wherein the cell further comprises the following enzyme: (a) flavanone 3-hydroxylase (F3H), (b) chalcone isomerase (CHI), (c) chalcone synthase (CHS), (d) 4-Coumarate CoA ligase (4CL), (e) cytochrome P450 reductase (CPR), (f) tyrosine ammonia lyase (TAL), (g) chalcone isomerase-like (CHIL), (h) cinnamate-4-hydroxylase (C4H), (I) phenylalanine ammonia lyase (PAL), (j) flavonoid 3'-hydroxylase (F3'H), (k) 3'-O-methyltransferase (3'-MT), (1) 4'-O-methyltransferase (4'-MT), (m) 3-O-methyltransferase (3-MT), (n) 4-O-methyltransferase (4-MT), (o) 3-OH-specific P450 monooxygenases, (p) glycosidase, and / or (q) Polyketide reductase (PKR).

15. The recombinant cell according to any one of claims 11 to 14, wherein the recombinant cell is a bacterium, an archaeon, a fungus such as a yeast, an algae cell or a plant cell.

16. A growth medium comprising the recombinant cell of any one of claims 11 to 15 and a compound of formula (I).

17. A method for preparing a compound of formula (I), comprising growing the recombinant cell of any one of claims 11 to 15 under growth conditions suitable for production of the compound of formula (I).

18. A compound of formula (I) obtained or obtainable by the process of any one of claims 1 to 9.

19. Use of a compound of formula (I) obtained or obtainable by the process of any one of claims 1 to 9 for (a) enhancing the sweetness of an ingestible composition, (b) reducing the bitterness of an ingestible composition, or (c) reducing the sourness of an ingestible composition.

20. A method for a) enhancing the sweetness of an ingestible composition of a product, (b) reducing the bitterness of an ingestible composition of a product and / or (c) reducing the sourness of an ingestible composition of a product, the method comprising introducing into the product a compound of formula (I) obtained or obtainable by the method of any one of claims 1 to 9.

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