Recombinant cells, extracts, consumable products and methods for production of bioactive plant metabolites

Abstract

Disclosed herein are recombinant host cells and methods capable of producing tyramine-containing hydroxycinnamic amide compounds, derivatives, and extracts.SOLUTION: Some embodiments provided include, for example, consumable products containing tyramine-containing hydroxycinnamic acid amides produced by recombinant host cells. Some embodiments provided herein are methods for producing tyramine-containing hydroxycinnamic amide or hydroxycinnamic amide derivatives.SELECTED DRAWING: None

Description

[Technical Field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This patent application claims the benefit of priority to U.S. Provisional Application No. 62 / 925,941, filed October 25, 2019, which is hereby fully incorporated by reference in its entirety for all purposes. [Background technology]

[0002] N-Hydroxycinnamic acid amides (HCAAs) are synthesized by the condensation of hydroxycinnamoyl-CoA thioesters containing cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA from cinnamic acid by a series of enzymes including cinnamate 4-hydroxylase, coumarate 3-hydroxylase, caffeate O-methyltransferase, ferulate 5-hydroxylase, and hydroxycinnamate:CoA ligase (Douglas (1996) Trends Plant Sci I: pp. 171-178).

[0003] Tyramine-derived HCAAs are commonly associated with the cell walls of tissues near areas of pathogen infection or wound healing. Furthermore, feruloyl-tyramine and feruloyl-octopamine are covalently bound cell wall components of both native and wounded periderms of potato (Solanum tuberosum) tubers and are putative components of the aromatic domain of suberin. HCAAs are thought to create a barrier to pathogens by reducing cell wall digestibility. HCAAs are formed by the condensation of hydroxycinnamoyl-CoA thioesters with phenylethylamines such as tyramine or polyamines such as putrescine. The final step in tyramine-derived HCAAs biosynthesis is catalyzed by hydroxycinnamoyl-CoA:tyramine N-(hydroxycinnamoyl)transferase.

[0004] Plant-specific feruloyltyramine, p-coumaroyltyramine, and caffeoyltyramine have been produced in Escherichia coli by heterologous expression of two biosynthetic genes, encoding p-coumarate:coenzyme A ligase and tyramine N-hydroxycinnamoyltransferase, cloned from Arabidopsis thaliana and pepper, respectively (Kang et al. (2009) Biotechnol. Lett. 31(9):1469-75). In addition, transgenic rice seeds expressing tyramine N-hydroxycinnamoyltransferase and tyrosine decarboxylase from a single self-processing polypeptide have been described (Park et al. (2009) Biotechnol. Lett. 31(6):911-5). Furthermore, the metabolic pathway for the synthesis of N-hydroxycinnamoylphenethylamine and tyramine was reproduced in E. coli by expressing several genes, including 4-coumarate-CoA ligase, tyramine N-hydroxycinnamoyltransferase or phenethylamine N-hydroxycinnamoyltransferase, phenylalanine decarboxylase or tyrosine decarboxylase, and tyrosine ammonia-lyase, and manipulating the shikimate metabolic pathway to increase the endogenous tyrosine concentration in E. coli (Sim et al. (2015) Microbial Cell Fact. 14:162). [Prior art documents] [Patent documents]

[0005] [Patent Document 1] U.S. Patent No. 6,368,837 [Patent Document 2] U.S. Patent No. 6,521,748 [Patent Document 3] U.S. Patent No. 5,605,793 [Patent Document 4] U.S. Patent No. 5,811,238 [Patent Document 5] U.S. Patent No. 5,830,721

Patent document 6

Patent document 7

Patent document 8

Non-licensed literature

[0006] [Non-licensed document 1] Douglas (1996) Trends Plant Sci l: pages 171~178 [Non-licensed document 2] Kangら(2009) Biotechnol. Lett. 31(9):1469~75 pages [Non-licensed document 3] Parkら(2009) Biotechnol. Lett. 31(6):911~5 pages

Non-licensed Document 4

Non-licensed Document 5

Non-licensed Document 6

Non-licensed Document 7

Non-licensed literature 9

Non-licensed literature 10

[0007] The disclosure provides recombinant eukaryotic host cells capable of producing tyramine-containing hydroxycinnamic acid amides, the recombinant eukaryotic host cells overproducing L-tyrosine or L-phenylalanine and harboring one or more nucleic acid molecules encoding one or more enzymes of the phenylpropanoid CoA pathway to produce hydroxycinnamoyl-CoA esters, a nucleic acid molecule encoding a tyrosine decarboxylase (EC 4.1.1.25), and a nucleic acid molecule encoding a tyramine N-hydroxycinnamoyltransferase (EC 2.3.1.110). In some embodiments, the tyramine-containing hydroxycinnamic acid amide is N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, p-coumaroyltyramine, cinnamoyltyramine, or sinapoyltyramine. In other embodiments, the one or more nucleic acid molecules encoding one or more enzymes of a phenylpropanoid CoA pathway for producing hydroxycinnamoyl-CoA esters include phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamate 4-hydroxylase, coumarate 3-hydroxylase, caffeoyl-CoA O-methyltransferase, ferulate 5-hydroxylase, caffeate / 5-hydroxyferulate O-methyltransferase, tyrosine ammonia-lyase, or a combination thereof. In further embodiments, the host cell overproduces S-adenosyl-methionine. Methods for producing tyramine-containing hydroxycinnamic acid amides using recombinant eukaryotic host cells, as well as extracts and consumable products containing tyramine-containing hydroxycinnamic acid amides, are also provided. [Brief explanation of the drawings]

[0008] [Figure 1]

[0023] Figure 1 shows a schematic pathway for the biosynthesis of tyramine-containing hydroxycinnamic acid amides from hydroxycinnamoyl-CoA esters and tyramine. However, for clarity, cofactors and cosubstrates are not shown. Enzymes in the phenylpropanoid pathway include phenylalanine ammonia-lyase (PAL, EC 4.3.1.24), cinnamate 4-hydroxylase (C4H, EC 1.14.14.91), p-coumaroyl-CoA ligase (4CL, EC 6.2.1.12), coumarate 3-hydroxylase (C3H, EC 1.14.13.-), coumaroyl-CoA 3-hydroxylase (CCoA3H or 5-O-(4-coumaroyl)-D-quinate 3'-monooxygenase, EC 1.14.14.96), caffeoyl-CoA ligase (CCoA3H, EC 1.14.14.96), and caffeoyl-CoA ligase (CCoA3H). O-methyltransferase (CCoAOMT, EC 2.1.1.104), ferulate 5-hydroxylase (F5H, EC 1.14.-.-), and caffeic acid / 5-hydroxyferulate O-methyltransferase (COMT, EC 2.1.1.68). Additional enzymes in the biosynthesis of tyramine-containing hydroxycinnamic acid amides include hydroxycinnamoyl-CoA:tyramine hydroxycinnamoyltransferase (THT, EC 2.3.1.110), tyrosine ammonia lyase (TAL, EC 4.3.1.23), phenylalanine hydroxylase (PAH, EC 1.14.16.1), and tyrosine decarboxylase (TYDC, EC 4.1.1.25). [Figure 2]FIG. 1 is a schematic diagram of engineered pathways in S. cerevisiae and E. coli for the overproduction of phenylalanine and / or tyrosine. Erythrose 4-phosphate (E4P), phosphoenolpyruvate (PEP), 3-deoxy-D-arabino-heptulosonic acid 7-phosphate (DAHP), 3-dehydroquinic acid (DHQ), 3-dehydroshikimic acid (DHS), shikimic acid (SHIK), shikimic acid-3-phosphate (SHP), 5-enolpyruvylshikimic acid-3-phosphate (EP3P), prephenic acid (PPA), phenylpyruvate (PPY), para-hydroxyphenylpyruvate (HPP), phenylacetaldehyde (PAC), para-hydroxyacetaldehyde (p-PAC), L-phenylalanine (L-PHE), L-tyrosine (L-TYR), and p-coumaric acid (p-CA). "*" indicates overexpressed enzymes, Aro10 and Pdc5 are knockout in the boxes, and "fbr" indicates feedback resistance. DETAILED DESCRIPTION OF THE INVENTION

[0009] A class of tyramine-containing hydroxycinnamic acid amides has now been shown to exhibit agonist activity against the global nuclear transcription factor HNF4α (hepatocyte nuclear factor 4α), which regulates the expression of genes involved in maintaining balanced metabolism (homeostasis). By agonizing HNF4α activity, plant-specific tyramine derivatives can be used to alleviate the harmful effects of free fatty acids, modulate metabolism, improve digestive health, and address the underlying etiology of metabolic disorders such as non-alcoholic fatty liver disease, non-alcoholic steatohepatitis, and type II diabetes. Accordingly, the present disclosure provides recombinant host cells, extracts, food products, and recombinant production methods for these bioactive plant metabolites.

[0010] As used herein, the bioactive plant metabolites of the present disclosure have the formula (I): [ka] It is a tyramine-containing hydroxycinnamic acid amide having the structure:

[0011] In some embodiments, R 1 , R 2 , R 3 , R 4 , R 5 , R 6 , R 7 , R 8 , and R 9 are each independently hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6 Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl.

[0012] In some embodiments, R 1 , R 2 , R 3 , and R 8are each independently hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6 Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl; R 4 , R 5 , R 6 , R 7 , and R 9 are each independently hydrogen, deuterium, hydroxyl, or halogen.

[0013] In some embodiments, R 1 , R 2 , and R 8 are each independently hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl; R 3 , R 4 , R 5 , R 6 , R 7 , and R 9 are each independently hydrogen, deuterium, hydroxyl, or halogen.

[0014] In some embodiments, the dashed bond may or may not be present.

[0015] In some embodiments, X is CH2 or O.

[0016] In some embodiments, Z is CHR a , N.R. a , or O.

[0017] In some embodiments, R a is hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl.

[0018] In some embodiments, the compound of Formula (I) is (E)-3-(3,4-dihydroxyphenyl)-N-(4-ethoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-methoxyethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(methylsulfonyl)ethoxy)phenethyl)acrylamide, (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetic acid, ethyl ... (3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetate, (E)-N-(4-(cyclopropylmethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3,3-trifluoropropoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-4-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl) (E)-N-(4-((4-fluorobenzyl)oxy)phenethyl)acrylamide, (E)-N-(4-(cyanomethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-2-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(dimethylamino)ethoxy)phenethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-isobutoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-4-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-methoxybenzyl)oxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(oxetan-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydrofuran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(thiophen-2-yloxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3-dimethylbutoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-hydroxyethoxy)phenethyl)acrylamide, (E)-N-(4-((1H-tetrazol-5-yl)methoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((1-methylpyrrolidin-2-yl)methoxy)phenethyl)acrylamide, (E)-2-hydroxy-5-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenyl hydrogen carbonate, (E)-3-(4-hydroxy-3-(pyridin-4-yloxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-hydroxy-3-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3 -(4-fluorophenoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-(cyanomethoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-2-(2-hydroxy-4-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid, (E)-3-(3-hydroxy-4-(pyridin-4-ylmethoxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-( (4-Fluorobenzyl)oxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-hydroxy-4-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-(cyanomethoxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-N-(3-(3,4-dihydroxyphenyl)acryloyl)-N-(4-hydroxyphenethyl)glycine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxphenethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-(pyridin-4-ylmethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-isobutylacrylamide, (E)-N-(cyanomethyl)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, 3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)propanamide, 3-(3,4-dihydroxyphenyl)-N-(4-(methylsulfonamido)phenethyl)propanamide.

[0019] In some embodiments, the bioactive plant metabolite of the present disclosure has formula (II): [ka] The resulting tyramine-containing hydroxycinnamic acid amide has the structure:

[0020] In some embodiments, R 1 , R 2 , R 3 , and R 4 are each independently hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6 Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl.

[0021] In some embodiments, the dashed bond may or may not be present.

[0022] In some embodiments, Z is CHRa , N.R. a , or O.

[0023] In some embodiments, R a is hydrogen, deuterium, hydroxyl, halogen, cyano, nitro, optionally substituted amino, optionally substituted C-amido, optionally substituted N-amido, optionally substituted ester, optionally substituted —(O)C 1~6 Alkyl, optionally substituted -(O)C 1~6 Alkenyl, optionally substituted -(O)C 1~6 Alkynyl, optionally substituted -(O)C 4~12 Cycloalkyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Cycloalkyl, optionally substituted -(O)C 4~12 Heterocyclyl, optionally substituted -(O)C 1~6 Alkyl C 4~12 Heterocyclyl, optionally substituted -(O)C 4~12 Aryl, optionally substituted -(O)C 1~6 Alkyl C 5~12 Aryl, optionally substituted -(O)C 1~12 Heteroaryl and optionally substituted -(O)C 1~6 Alkyl C 1~12 heteroaryl.

[0024] In some embodiments, the compound of Formula (II) is (E)-3-(3,4-dihydroxyphenyl)-N-(4-ethoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-methoxyethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(methylsulfonyl)ethoxy)phenethyl)acrylamide, (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetic acid, ethyl ... -(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetate, (E)-N-(4-(cyclopropylmethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3,3-trifluoropropoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-4-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-4-yl)methoxy)phenethyl)acrylamide (E)-N-(4-(cyanomethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-2-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(dimethylamino)ethoxy)ethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-isobutoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-4-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-methoxybenzyl)oxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(oxetan-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydrofuran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(thiophen-2-yloxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3-dimethylbutoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-hydroxyethoxy)phenethyl)acrylamide, (E)-N-(4-((1H-tetrazol-5-yl)methoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((1-methylpyrrolidin-2-yl)methoxy)phenethyl)acrylamide, (E)-2-hydroxy-5-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenyl hydrogen carbonate, (E)-3-(4-hydroxy-3-(pyridin-4-yloxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-hydroxy-3-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3 -(4-fluorophenoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-(cyanomethoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-2-(2-hydroxy-4-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid, (E)-3-(3-hydroxy-4-(pyridin-4-ylmethoxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-( (4-Fluorobenzyl)oxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-hydroxy-4-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-(cyanomethoxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-N-(3-(3,4-dihydroxyphenyl)acryloyl)-N-(4-hydroxyphenethyl)glycine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxphenethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-(pyridin-4-ylmethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-isobutylacrylamide, (E)-N-(cyanomethyl)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, 3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)propanamide, or 3-(3,4-dihydroxyphenyl)-N-(4-(methylsulfonamido)phenethyl)propanamide.

[0025] In some embodiments, the bioactive plant metabolites of the present disclosure include tyramine-containing hydroxycinnamic acid amides having the structure of formula (III): [ka] During the ceremony, Each occurrence of X may independently be C or N; Z is -CR 6 - or -SO2-, R 1 -OH, -OCH2CH2R 7 , or -NHR 8 or R 1 is R 5 may be taken together with R to form a 6-membered substituted heterocycloalkyl ring; 2 and R 3 are independently hydrogen or -CH2CH2R 7 or R 2 and R 3 together form a 5- or 6-membered heterocycloalkyl ring; R 4 is hydrogen or -CH2CH2R 7 may be a group; R 5 is optionally present and, when present, is a substituent for one or more ring atoms and is independently for each occurrence halo, hydroxy, alkyl, substituted alkyl, alkoxy, substituted sulfonyl, carboxyl ester, amino, substituted amino, cyano, aryl, substituted aryl, cycloalkyl, heteroaryl, substituted heteroaryl; R 6 may be H, oxo, substituted alkyl, spirocycloalkyl, or spiroheterocycloalkyl; R 7 is hydrogen, hydroxy, alkyl, substituted alkyl, alkoxy, substituted sulfonyl, carboxyl ester, amino, substituted amino, cyano, aryl, substituted aryl, cycloalkyl, heteroaryl, substituted heteroaryl; R 8 may be a substituted sulfonyl, substituted alkyl, carboxyl ester, or aminocarbonyl; the dashed bond may be present or absent.

[0026] In some embodiments, the bioactive plant metabolites of the present disclosure include tyramine-containing hydroxycinnamic acid amides having the structure of formula (IV): [ka] During the ceremony, R l is present or absent and, if present, is a substituent at one or more ring atoms (e.g., at positions 2, 3, and / or 4), which is independently for each ring atom a hydroxy group, a halo group, a substituted or unsubstituted lower alkyl group, or a substituted or unsubstituted lower alkoxy group; Dashed bonds are either present or absent. In accordance with this disclosure, tyramine-containing hydroxycinnamic acid amides include both cis and trans isomers.

[0027] For groups herein, the parenthetical subscripts below further define the group as follows: n )" defines the exact number of carbon atoms (n) in the group. For example, "C1-C6-alkyl" represents an alkyl group having from 1 to 6 carbon atoms (e.g., 1, 2, 3, 4, 5, or 6, or any range derivable therein, e.g., 3 to 6 carbon atoms).

[0028] The term "lower alkyl" is intended to mean a branched or unbranched saturated monovalent hydrocarbon radical containing one to six carbon atoms (i.e., C1-C6-alkyl), such as methyl, ethyl, propyl, isopropyl, tert-butyl, butyl, n-hexyl, and the like.

[0029] Similarly, a lower alkoxy group is a C1-C6-alkoxy group having the structure -OR, where R is "alkyl" as further defined above. Particular alkoxy groups include, by way of example, methoxy, ethoxy, n-propoxy, isopropoxy, n-butoxy, tert-butoxy, iso-butoxy, sec-butoxy, n-pentoxy, 1,2-dimethylbutoxy, and the like.

[0030] The term "halo" is used herein to refer to chloro (Cl), fluoro (F), bromo (Br), and iodo (I) groups. In certain embodiments, the halo group is a fluoro group.

[0031] In any of the groups described herein, a substituted group (e.g., a substituted lower alkyl group or a substituted lower alkoxy group) means that an available hydrogen is replaced with an alkyl, alkenyl, alkynyl, aryl, heteroaryl, aralkyl, alkylaryl, heteroaralkyl, heteroarylalkenyl, heteroarylalkynyl, alkylheteroaryl, hydroxy, hydroxyalkyl, alkoxy, aryloxy, aralkoxy, alkoxyalkoxy, acyl, halo, nitro, cyano, carboxy, aralkoxycarbonyl, heteroarylsulfonyl, alkoxycarbonyl, alkylsulfonyl, alkylthio, arylthio, aryloxycarbonyl, arylsulfonyl, heteroarylthio, aralkylthio, heteroaralkylthio, cycloalkyl, heterocyclyl, or glycosyl group.

[0032] Any undefined valency of an atom of a structure shown in this application implicitly represents a hydrogen atom bonded to that atom.

[0033] In some embodiments, the tyramine-containing hydroxycinnamic acid amide has the structure of formula (V): [ka] During the ceremony, R 2 is present or absent and, if present, is a hydroxy or methoxy group; R 3 is present or absent, and if present is a hydroxy group; R 4 is either present or absent and, if present, is a hydroxy or methoxy group.

[0034] "Isomers" refers in particular to optical isomers (e.g., essentially pure enantiomers, essentially pure diastereomers, and mixtures thereof) as well as conformational isomers (i.e., isomers that differ only in the angle of at least one of their chemical bonds), positional isomers (especially tautomers), and geometric isomers (e.g., cis-trans isomers).

[0035] In certain embodiments, the tyramine-containing hydroxycinnamic acid amide of formula (I)-(V) is [ka] is selected from.

[0036] The tyramine-containing hydroxycinnamic acid amides of this disclosure have been found in several plant genera, including Solanum species (e.g., tomato, potato, nettle, pepper, and eggplant), Allium species (e.g., garlic, onion, and chive), Tribulus species (e.g., sea anemone), and Annona species (e.g., cherimoya, custard apple, and sugar apple). Generally, the biosynthetic approach of this disclosure can be carried out as depicted in Scheme 1. [ka]

[0037] More specifically, the biosynthetic pathway for tyramine-containing hydroxycinnamic acid amides of this disclosure is presented in Figure 1. As shown in Figure 1, a recombinant host cell capable of producing tyramine-containing hydroxycinnamic acid amides is provided, wherein the host cell overproduces L-tyrosine and / or L-phenylalanine and comprises one or more nucleic acid molecules encoding one or more enzymes of a phenylpropanoid CoA pathway to produce a hydroxycinnamoyl-CoA ester, a nucleic acid molecule encoding a tyrosine decarboxylase (EC 4.1.1.25), and an exogenous nucleic acid molecule encoding a tyramine N-hydroxycinnamoyltransferase (EC 2.3.1.110).

[0038] Tyrosine and phenylalanine overproduction A host cell exhibiting "overproduction of L-tyrosine or L-phenylalanine" refers to a cell that has been genetically modified to produce increased amounts of L-tyrosine, L-phenylalanine, or both L-tyrosine and L-phenylalanine compared to a wild-type cell. As used herein, the terms "phenylalanine," "L-phenylalanine," "Phe," and "L-Phe" are used interchangeably. Similarly, the terms "tyrosine," "L-tyrosine," "Tyr," and "L-Tyr" are used interchangeably.

[0039] Many bacteria are natural producers of aromatic compounds via the shikimate pathway (Bongaerts et al. (2001) Metab. Eng. 3:289-300; Ikeda et al. (2006) Appl. Microbial. Biotechnol. 69:615-626; Sprenger et al. (2007) Appl. Microbial. Biotechnol. 75:739-749). In this pathway, phosphoenolpyruvate (PEP) and erythrose 4-phosphate (E4P), converted from glucose through the central metabolic pathway, first combine to form 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), which is then converted to chorismate. From chorismate, the pathway branches to form various aromatic end products, including phenylalanine (Phe), tyrosine (Tyr), and tryptophan (Trp) (Figure 2). Genetically modified strains have been created to enhance the productivity of aromatic compounds. For example, in Phe production, the most important steps are the first and last steps of the shikimate pathway. However, the enzymes involved in these steps, DAHP synthase (encoded by aroG) and chorismate mutase / prephenate dehydratase (encoded by pheA), are strongly inhibited by Phe. Therefore, feedback-resistant mutants (fbr) have been studied and utilized for Phe production (Kikuchi et al. (1997) Appl. Environ. Microbiol. 63:761-762; Nelms et al. (1992) Appl. Environ. Microbiol. 58:2592-2598). In addition, the expression levels of both aroG and pheA are controlled by the transcriptional repressor TyrR, and therefore deletion of tyrR is also efficient for Phe production (Berry (1996) Trends Biotechnol. 14:250-256).To enhance the availability of the DAHP precursors PEP and E4P, the transketolase (tktA) and PEP synthase (pps) genes were overexpressed (Patnaik & Liao (1994) Appl. Environ. Microbiol. 60:3903-3908), the PEP carboxylase gene (ppc) was deleted (Miller et al. (1987) J. Ind. Microbiol. 2:143-149), and the carbon storage regulator genes (csrA or csrB) were overexpressed or deleted (Tatarko & Romeo (2001) Curr. Microbiol. 43:26-32; Yakandawala et al. (2008) Appl. Microbiol. Biotechnol. 78:283-291), and the glucose transport system has been modified from the PEP-dependent sugar phosphotransferase system (PTS) to the galactose permease (GalP)-glucokinase (Glk) system (Baez-Viveros et al. (2004) Biotechnol. Bioeng. 87:516-524; Yi et al. (2003) Biotechnol. Prog. 19:1450-1459) or the Zymomonas mobilis glucose facilitator (Glf)-Glk system (Patnaik & Liao (1994) Appl. Environ. Microbiol. 60:3903-3908). These modifications have been used in appropriate combinations to enhance the production of aromatic compounds. To shift synthesis to L-tyrosine, the pheA gene encoding chorismate mutase / prephenate dehydratase was deleted and tyrA encoding chorismate mutase / prephenate dehydrogenase was inserted with a strong trc promoter, achieving an L-Tyr titer of 55 g / L in 48 hours (Olsen et al. (2007) Appl. Microbiol. Biotechnol. 74(5):1031-40).

[0040] Exemplary bacterial strains for the overproduction of tyrosine and / or phenylalanine include, but are not limited to, the strains set forth in Table 1.

[0041] [Table 1A]

[0042] [Table 1B]

[0043] In S. cerevisiae, aromatic compounds are synthesized via the aromatic amino acid biosynthetic pathway (AAP) (Braus (1991) Microbiol Rev. 55:349-70). This highly regulated pathway is a central node in yeast metabolism and feeds into several other pathways (e.g., the quinone, folate, and Ehrlich pathways; Figure 2). Using flux balance analysis, it has been shown that increased availability of erythrose-4-phosphate (E4P) can be achieved by deleting ZWF1 and overexpressing the transketolase-encoding TKL1 to reverse the flux from the glycolytic intermediates fructose-6-phosphate (F6P) and glyceraldehyde-3-phosphate (G3P) to E4P and xylulose-5-phosphate (X5P). This resulted in an up to seven-fold increase in flux to the aromatic amino acid pathway (APP; Deaner & Alper (2017) Metab. Eng. 40:14-22). Another approach to improve flux to AAP is the overexpression of transaldolase (Tall) and enolase 2 (Eno2) (Mao et al. (2017) Biotechnol. Lett. 39(7):977-982). Tall supports the conversion of sedoheptulose-7-phosphate (S7P) and G3P to E4P and F6P, while Eno2 converts 2-phosphoglycerate to phosphoenolpyruvate (PEP).

[0044] The first enzymatic step of the shikimate pathway is catalyzed by DAHP synthase, which condenses E4P and PEP to 3-deoxy-D-arabinoheptulosonic acid 7-phosphate (DAHP; Figure 2). In yeast, AR03 and AR04 encode two DAHP synthase isoforms. Deletion of AR03 and Aro4 K229L and AR07 FBR The combination of overexpression of Aro4 (to avoid feedback inhibition) increases flux through the aromatic amino acid pathway (Luttik et al. (2008) Metab. Eng. 10:141-153). Furthermore, the tyrosine-insensitive mutant Aro4 G226S improved the production of tyrosine-derived naringenin (Koopman et al. (2012) Microb. Cell Fact. 11:155) and has been used to produce tyrosine-derived opioids (Galanie et al. (2015) Science 349:1095-100). The final step in the shikimate pathway, the conversion of EPSP to chorismate by chorismate synthase (Aro2), has been shown to be a bottleneck in AAP. Accordingly, overexpression of Aro2 has been shown to result in a two-fold improvement in the level of p-coumarate from tyrosine (Mao et al. (2017) Biotechnol. Lett. 39(7):977-982).

[0045] Further downstream from chorismate to the tyrosine and phenylalanine branch, a common enzymatic step catalyzed by Aro7 converts chorismate to prephenate (Figure 2). Aro7 is the third feedback-regulatable enzyme in the aromatic amino acid biosynthetic pathway. Mutations in Aro7 (e.g., Aro7 G141S or Aro7 T226I ) has been shown to relieve feedback regulation in S. cerevisiae and improve titers of tyrosine and phenylalanine pathway intermediates when compared to similarly engineered strains overexpressing the wild-type isoform of Aro7 (Luttik et al. (2008) Metab. Eng. 10:141-153; Trenchard et al. (2015) Metab. Eng. 31:74-83).

[0046] The next step is the conversion of prephenate to phenylpyruvate (PPY), a precursor of phenylalanine, or hydroxyphenylpyruvate (4-HPP), a precursor of tyrosine. Tyr1 catalyzes the conversion to 4-HPP, and overexpression of Tyr1 in combination with modifications of the above pathway has been shown to increase the production of tyrosine-derived p-coumaric acid (Mao et al. (2017) Biotechnol. Lett. 39(7):977-982).

[0047] Aro10 catalyzes the entry reaction into the Ehrlich pathway, which is involved in amino acid catabolism. Deletion of ARO10, PDC5, and PDC6 results in a 22-fold reduction in the titer of the Ehrlich pathway intermediate phenylethanol in strains producing the flavonoid naringenin (Koopman et al. (2012) Microb. Cell Fact. 11:155).

[0048] Exemplary eukaryotic host cells for the overproduction of tyrosine and / or phenylalanine include, but are not limited to, the strains set forth in Table 2.

[0049] [Table 2]

[0050] Phenylepropanoid CoA pathway According to the present disclosure, one or more nucleic acid molecules encoding one or more enzymes of the phenylpropanoid-CoA pathway are engineered into a recombinant host cell to produce hydroxycinnamoyl-CoA esters from phenylalanine and / or tyrosine. As used herein, the term "phenylpropanoid-CoA pathway" refers to the enzymatic pathway within a cell required for the production of hydroxycinnamoyl-CoA esters (i.e., p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA), preferably from phenylalanine and / or tyrosine. As shown in Figure 1, the enzymes in the phenylpropanoid-CoA pathway include phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamate 4-hydroxylase, coumarate 3-hydroxylase, coumaroyl-CoA 3-hydroxylase, caffeoyl-CoA O-methyltransferase, ferulate 5-hydroxylase, caffeate / 5-hydroxyferulate O-methyltransferase, and tyrosine ammonia-lyase. More specifically, phenylalanine is converted to cinnamate by expressing phenylalanine ammonia-lyase (PAL; EC 4.3.1.24). Cinnamic acid (also known as trans-cinnamic acid, cinnamic acid, or trans-cinnamate) is then converted to p-coumaric acid (also known as para-hydroxycinnamic acid, p-hydroxycinnamic acid, 4-hydroxycinnamic acid, or 4-hydroxycinnamate) by expressing the P450 enzyme cinnamate 4-hydroxylase (C4H; EC 1.14.14.91). Coumaroyl-CoA ligase (4CL; EC 6.2.1.12) converts p-coumaric acid (and other substituted cinnamic acids) to the corresponding CoA thiol ester (i.e., p-coumaroyl-CoA), which is used in the biosynthesis of flavonoids, isoflavonoids, lignin, suberin, and coumarin (Ehlting et al. (1999) Plant J. 19(1):9-20).

[0051] Phenylanine ammonia-lyase is widely distributed in plants (Koukol et al. (1961) J. Biol. Chem. 236:2692-2698), fungi (Bandoni et al. (1968) Phytochemistry 7:205-207), yeast (Ogata et al. (1967) Agric. Biol. Chem. 31:200-206), and Streptomyces (Emes et al. (1970) Can. J. Biochem. 48:613-622), but has not been found in E. coli or mammalian cells (Hanson & Havir, In: The Enzymes (3rd ed.) Boyer, ed., Academic: New York, 1967; pp. 75-167). The PAL enzyme converts phenylalanine to cinnamic acid, which can be further converted to p-coumaric acid by cinnamate-4-hydroxylase (C4H, EC 1.14.14.91). Additionally, because C4H is a cytochrome P450 enzyme, cytochrome P450 reductase (CPR) can also be co-expressed. Thus, in some embodiments, the host cells of the present disclosure express the PAL enzyme in combination with the C4H enzyme. In other embodiments, the host cells of the present disclosure express the PAL enzyme in combination with the C4H and CPR enzymes.

[0052] Phenylanine ammonia lyase also accepts tyrosine as a substrate to some extent, converting it directly to p-coumaric acid. For example, PAL enzymes isolated from parsley (Appert et al. (1994) Eur. J. Biochem. 225:491) or corn (Havir et al. (1971) Plant Physiol. 48:130) demonstrate the ability to use tyrosine as a substrate. Similarly, the PAL enzyme isolated from Rhodosporidium (Hodgins (1971) J. Biol. Chem. 246:2977) can also use L-tyrosine as a substrate. Such enzymes are referred to herein as "PAL / TAL" enzymes (EC 4.3.1.25; Rosier et al. (1997) Plant Physiol. 113:175-179). Thus, PAL enzymes, particularly those having a PAL / TAL activity ratio of at least 0.1, can also be expressed by the host cells of this disclosure. When it is desired to create a recombinant organism that expresses wild-type genes encoding PAL / TAL activity, genes can be isolated from corn, wheat, parsley, Rhizoctonia solani, Rhodosporidium, Sporobolomyces pararoseus, Rhodosporidium, and Phanerochaete chrysosporium (see Hanson & Havir (1981) Biochem. Plants 7:577-625). As an illustration, using the aromatic amino acid overproducing strain of P. putida S12, the pal gene encoding the bifunctional PAL / TAL enzyme from Rhodosporidium toruloides has been shown to increase the production of cinnamic acid (Nijkamp et al. (2005) Appl. Microbiol. Biotechnol. 69:170-77) and p-coumaric acid (Nijkamp et al. (2007) Appl. Microbiol. Biotechnol. 74:617-624).Similarly, PALs from Arabidopsis thaliana (AtPa11 or AtPa12) have been used in S. cerevisiae to convert phenylalanine to cinnamic acid (Koopman et al. (2012) Microb. Cell Fact. 11:155).

[0053] Another biosynthetic pathway leading to the production of p-coumaric acid is based on the use of an enzyme with TAL activity (EC 4.3.1.23). Instead of the two enzymatic reactions used to convert phenylalanine to p-coumaric acid, TAL converts L-tyrosine directly to p-coumaric acid. Thus, in some embodiments, the host cells of the present disclosure express a TAL enzyme.

[0054] The classification of PAL and TAL enzymes is primarily determined by the enzyme's activity on each substrate, with classifications assigned based on preferred substrates. TAL enzymes are defined as preferentially using L-tyrosine as a substrate, while PAL enzymes are defined as preferentially using L-phenylalanine as a substrate. However, these enzymes typically accept both L-tyrosine and L-phenylalanine as substrates to varying degrees. Thus, in some embodiments, PAL and TAL enzymes are generally referred to as "PAL / TAL enzymes."

[0055] In some embodiments, specificity for one substrate over another can be achieved, for example, by mutating a naturally occurring PAL gene to one that encodes an enzyme that preferentially uses L-tyrosine as a substrate (see U.S. Pat. No. 6,368,837 or U.S. Pat. No. 6,521,748). Various techniques can be used to mutagenesis PAL / TAL enzymes. Suitable techniques for mutagenesis include error-prone PCR (Leung et al. (1989) Techniques 1:11-15; Zhou et al. (1991) Nucleic Acids Res. 19:6052-6052; Spee et al. (1993) Nucl. Acids Res. 21:777-778), in vitro mutagenesis, and in vivo mutagenesis. Protein engineering can be achieved by a method commonly known as "gene shuffling" (U.S. Patent Nos. 5,605,793; 5,811,238; 5,830,721; and 5,837,458), or by rational design based on three-dimensional structure and classical protein chemistry.

[0056] The sources of PAL, TAL, or PAL / TAL enzymes and C4H enzymes in this disclosure can be obtained or derived from any naturally occurring source. Examples of suitable PAL, TAL, PAL / TAL, and C4H enzymes useful in this disclosure are listed in Table 3.

[0057] [Table 3A]

[0058] [Table 3B]

[0059] In another aspect, L-phenylalanine is converted to L-tyrosine using an enzyme having phenylalanine hydroxylase (PAH, EC 1.14.16.1) activity. The L-tyrosine produced using phenylalanine hydroxylase is then converted to p-coumaric acid using an enzyme having TAL activity. Thus, in some embodiments, the host cells of the present disclosure express a PAH enzyme in combination with a TAL enzyme. The PAH activity can be endogenous or can be introduced into the host cell to increase tyrosine production. PAH enzymes are well known in the art and have been reported in Proteobacteria (Zhao et al. (1994) Proc. Natl. Acad. Sci. USA. 91:1366). For example, Pseudomonas aeruginosa possesses a multigene operon that includes phenylalanine hydroxylase (Zhao et al. (1994) Proc. Natl. Acad. Sci. USA. 91:1366). The enzymatic conversion of L-phenylalanine to L-tyrosine is also known in eukaryotes. Human phenylalanine hydroxylase is specifically expressed in the liver and converts L-phenylalanine to L-tyrosine (Wang et al. (1994) J. Biol. Chem. 269(12):9137-46). The source of the PAH enzymes in this disclosure can be obtained or derived from any naturally occurring source. Examples of suitable PAH enzymes useful in this disclosure are listed in Table 4.

[0060] [Table 4]

[0061] According to some embodiments, host cells are engineered to recombinantly express nucleic acids encoding enzymes required to convert a portion of the aromatic amino acids overproduced by the host cell (L-phenylalanine and / or L-tyrosine) to p-coumaric acid by recombinantly expressing nucleic acids encoding (i) PAL and C4H, (ii) PAL, C4H and CPR, (iii) PAL / TAL and C4H, (iv) PAL / TAL, C4H and CPR, (v) TAL, and / or (vi) PAH and TAL of the phenylpropanoid pathway.

[0062] p-Coumaric acid produced by a recombinant host cell is converted to p-coumaroyl-CoA by expressing an enzyme with coumaroyl-CoA ligase activity. Coumaroyl-CoA ligase (4CL, EC 6.2.1.12) is used in the context of the present disclosure to catalyze the conversion of p-coumaric acid and other substituted cinnamic acids (e.g., cinnamic acid, caffeic acid, ferulic acid, and sinapic acid) to the corresponding CoA thiol esters (i.e., p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA). Coumaroyl-CoA ligases are well known in the art. Coumaroyl-CoA ligases can be endogenous or exogenous to the host cell. In certain embodiments, coumaroyl-CoA ligase is overexpressed in the host cell to increase p-coumaroyl-CoA production. A non-limiting list of publicly available coumaroyl-CoA ligases useful in this disclosure is provided in Table 5.

[0063] [Table 5]

[0064] In one embodiment, a coumaroyl-CoA ligase is selected based on its ability to convert p-coumaric acid to p-coumaroyl-CoA, hi another embodiment, multiple coumaroyl-CoA ligases are co-expressed to increase production of tyramine-containing hydroxycinnamic acid amides.

[0065] For the production of caffeic acid or caffeoyl-CoA from p-coumaric acid or p-coumaroyl-CoA, respectively, the recombinant host cell may further contain and express a nucleic acid encoding coumarate 3-hydroxylase (C3H, EC 1.14.13.-) or coumaroyl-CoA 3-hydroxylase (CCoA3H, EC 1.14.14.96). Similarly, for the production of ferulic acid or feruloyl-CoA from p-coumaric acid or p-coumaroyl-CoA, respectively, the recombinant host cell can further contain and express nucleic acids encoding coumarate 3-hydroxylase (C3H, EC 1.14.13.-) or coumaroyl-CoA 3-hydroxylase (CCoA3H, EC 1.14.14.96), and caffeic acid / 5-hydroxyferulic acid O-methyltransferase (COMT, EC 2.1.1.68) or caffeoyl-CoA O-methyltransferase (CCoAOMT, EC 2.1.1.104). To accelerate the conversion of caffeoyl-CoA to feruloyl-CoA, thereby increasing the production rate of the final product, host cells can be supplemented with S-adenosyl-methionine (AdoMet), selected for AdoMet overproduction (Choi et al. (2009) Korean J. Chem. Eng. 26(1):156-9), or optionally engineered to overproduce AdoMet. Illustratively, a yeast strain expressing a chimeric protein consisting of the yeast Met13p N-terminal catalytic domain and the Arabidopsis thaliana MTHFR (AtMTHFR-1) C-terminal regulatory domain was found to accumulate over 100-fold more AdoMet than wild-type (Roje et al. (2002) J. Biol. Chem. 277:4056-4061). Thus, in certain embodiments, recombinant host cells overproduce AdoMet.Additionally, to synthesize sinapoyl-CoA, recombinant host cells can express nucleic acids encoding coumarate 3-hydroxylase (C3H, EC 1.14.13.-) or coumaroyl-CoA 3-hydroxylase (CCoA3H, EC 1.14.14.96), caffeic acid / 5-hydroxyferulate O-methyltransferase (COMT, EC 2.1.1.68) or caffeoyl-CoA O-methyltransferase (CCoAOMT, EC 2.1.1.104), and ferulate 5-hydroxylase (F5H, EC 1.14.-.-). A non-limiting list of publicly available enzymes for producing these hydroxycinnamoyl-CoA esters is provided in Table 6.

[0066] [Table 6A]

[0067] [Table 6B]

[0068] Production of tyramine-containing hydroxycinnamic acid amides To convert hydroxycinnamoyl-CoA esters (i.e., p-coumaroyl-CoA, cinnamoyl-CoA, caffeoyl-CoA, feruloyl-CoA, and sinapoyl-CoA) to the corresponding tyramine-containing hydroxycinnamic acid amides, the host cell also harbors and expresses a nucleic acid molecule encoding tyramine N-hydroxycinnamoyltransferase (THT, EC 2.3.1.110). Tyramine N-hydroxycinnamoyltransferase is used in the context of the present disclosure to conjugate hydroxycinnamoyl-CoA esters to tyramine to produce tyramine-containing hydroxycinnamic acid amides (i.e., N-caffeoyltyramine, N-feruloyltyramine, p-coumaroyltyramine, cinnamoyltyramine, or sinapoyltyramine). THT is well known in the art and can be endogenous or exogenous to the host cell. In certain embodiments, THT is overexpressed in the host cell. A non-limiting list of publicly available THT enzymes useful in this disclosure is provided in Table 7.

[0069] [Table 7]

[0070] To provide a source of tyramine, the host cell further comprises a nucleic acid molecule encoding tyrosine decarboxylase (TYDC, EC 4.1.1.25). Tyrosine decarboxylases useful in the context of the present disclosure convert tyrosine to tyramine. TYDC can be endogenous or exogenous to the host cell, and is preferably overexpressed in the host cell. A non-limiting list of publicly available TYDC enzymes useful in this disclosure is provided in Table 8.

[0071] [Table 8]

[0072] As used herein, the terms "recombinant host," "recombinant host cell," or "host cell" are intended to refer to a host whose genome has been augmented by at least one integrated DNA sequence. Such DNA sequences include, but are not limited to, non-naturally occurring genes, DNA sequences that are not normally transcribed into RNA or translated into protein ("expressed"), and other genes or DNA sequences that one desires to introduce into a non-recombinant host. It is recognized that typically, the genome of a recombinant host cell described herein is augmented by stable introduction of one or more recombinant genes. However, autonomous or replicating plasmids or vectors may also be used within the scope of this disclosure. Additionally, the present disclosure may be practiced using low copy number, e.g., single copy, or high copy number (as exemplified herein) plasmids or vectors.

[0073] Generally, the introduced DNA is not originally native to the host that is the recipient of that DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host and then introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of a gene product or alter the expression pattern of a gene. In some cases, the introduced DNA modifies or even replaces an endogenous gene or DNA sequence, e.g., by homologous recombination or site-directed mutagenesis.

[0074] The term "recombinant gene" or "recombinant nucleic acid molecule" refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or similar gene or DNA sequence may already be present in such host. "Introduced" or "propagated" in this context is known in the art to mean introduced or propagated by the hand of man. Thus, a recombinant gene may be a DNA sequence from another species, or it may be a DNA sequence derived from or present in the same species, but incorporated into a host by recombinant methods to form a recombinant host. It is recognized that a recombinant gene introduced into a host may be identical to a DNA sequence normally present in the host being transformed, or it may be introduced to provide one or more additional copies of DNA, thereby allowing for overexpression or altered expression of the gene product of that DNA.

[0075] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region in those microorganisms, if desired. A coding sequence and a regulatory region are considered to be operably linked when they are positioned so that the regulatory region is effective to regulate the transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned 1 to about 50 nucleotides downstream of the regulatory region of a monocistronic gene.

[0076] In many cases, the coding sequence of a polypeptide described herein is specified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. As used herein, the term "heterologous nucleic acid" refers to a nucleic acid introduced into a recombinant host that does not naturally occur in said host. Thus, if the recombinant host is a microorganism, the coding sequence may be from another prokaryotic or eukaryotic microorganism, plant, or animal. In some cases, however, the coding sequence is a sequence that originated in the host and has been reintroduced into that organism. A native sequence can often be distinguished from a naturally occurring sequence by the presence of non-native sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking the native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids are typically integrated at a location other than the location where the native sequence is found.

[0077] "Regulatory region" or "regulatory sequence" refers to a nucleotide sequence that influences transcription or translation initiation and rate, as well as the stability and / or mobility of the transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5' and 3' untranslated regions (UTRs), transcription initiation sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. Regulatory regions typically include at least a core (basal) promoter. Regulatory regions may also include at least one control element, such as an enhancer sequence, upstream element, or upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective to regulate the transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned 1 to about 50 nucleotides downstream of the promoter. However, the regulatory region can be located as much as about 5,000 nucleotides upstream of the translation start site, or as much as about 2,000 nucleotides upstream of the transcription start site.

[0078] The choice of regulatory region(s) to be included will depend on several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for those skilled in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It is understood that more than one regulatory region may be present, such as introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.

[0079] Numerous promoters are useful for driving expression of relevant genes in desired host cells and are well known to those of skill in the art. Expression in host cells can be achieved in a transient or stable manner. Transient expression can be achieved by inducing the activity of a regulatable promoter operably linked to the gene of interest. Stable expression can be achieved by using a constitutive promoter operably linked to the gene of interest. In fact, any promoter capable of driving these genes is suitable for the present disclosure, including, but not limited to, FBAIN, FBAINm, EXP, FBA1, GPAT, CYC1, HIS3, GAL1, GAL10, ADH1, PGK, PROS, GAPDH, ADCI, TRP1, URA3, LEU2, ENO, TPI; AOX1 (particularly useful for expression in Pichia); and lac, trp, IPL, IPRR, T7, tac, and trc (particularly useful for expression in E. coli).

[0080] When the host cell is yeast, transcription and translation regions functional in yeast cells are provided specifically by the host species (see, e.g., WO 2004 / 101757). Promoters can be obtained, for example, from genes in the glycolytic pathway, such as alcohol dehydrogenase, glyceraldehyde-3-phosphate dehydrogenase, glyceraldehyde-3-phosphate O-acyltransferase, phosphoglycerate mutase, fructose-bisphosphate aldolase, phosphoglucose isomerase, phosphoglycerate kinase, and the like; or from regulatable genes, such as acid phosphatase, lactase, metallothionein, glucoamylase, translation elongation factor EFl-cx (TEF) protein (U.S. Pat. No. 6,265,185), ribosomal protein S7 (U.S. Pat. No. 6,265,185), and the like. Any one of several regulatory sequences can be used, depending on whether constitutive or inducible transcription is desired, the efficiency of the promoter in expressing the open reading frame of interest, ease of construction, and the like.

[0081] The nucleotide sequence surrounding the translation initiation codon "ATG" has been found to affect expression in yeast cells. If the desired polypeptide is poorly expressed in yeast, the exogenous nucleotide sequence of the gene can be modified to include an efficient yeast translation initiation sequence to obtain optimal gene expression. For expression in yeast, this can be achieved by site-directed mutagenesis of an inefficiently expressed gene by fusing it in-frame with an endogenous yeast gene, preferably a highly expressed gene.

[0082] Termination control regions may also be derived from various genes from preferred hosts. Optionally, a termination site may not be necessary, but is most preferred if included. The termination region may be derived from the 3' region of the gene from which the initiation region was obtained or from a different gene. Numerous termination regions are known and function well in various hosts (when utilized in both the same and different genera and species from which they are derived). Termination regions are usually selected for convenience rather than for any particular characteristics. Preferably, the termination region is derived from a yeast gene, particularly Saccharomyces, Schizosaccharomyces, Candida, Yarrowia, or Kluyveromyces. The 3' regions of mammalian genes encoding γ-interferon and α-2 interferon are also known to function in yeast. Termination control regions may also be derived from various genes from preferred hosts. Optionally, a termination site may not be necessary, but is most preferred if included. In one embodiment, the terminator is selected from the group consisting of LIP2, PEX20, and XPR2.

[0083] One or more genes, e.g., one or more heterologous nucleic acids, can be combined in a recombinant nucleic acid construct into a "module" useful for producing tyramine-containing hydroxycinnamic acid amides. Combining multiple genes or heterologous nucleic acids in a module facilitates the use of the module in various species. For example, genes involved in the biosynthesis of L-tyrosine and / or L-phenylalanine, hydroxycinnamoyl-CoA esters, tyramine, and tyramine-containing hydroxycinnamic acid amides can be combined such that each coding sequence is operably linked to a separate regulatory region to form a tyramine-containing hydroxycinnamic acid amide module for production in eukaryotes. Alternatively, the modules can express a polycistronic message for the production of tyramine-containing hydroxycinnamic acid amides in prokaryotic hosts such as Rhodobacter, Escherichia coli, Bacillus, or Lactobacillus. In addition to the genes useful for tyramine-containing hydroxycinnamic acid amide production, the recombinant construct typically also contains an origin of replication and one or more selectable markers for maintenance of the construct in an appropriate species.

[0084] Due to the degeneracy of the genetic code, it is recognized that several nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as a codon for that amino acid. Thus, the codons in the coding sequence for a given polypeptide can be modified using an appropriate codon bias table for that host (e.g., a microorganism) to obtain optimal expression in that host. As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into the assembly of modules of constructs.

[0085] Standard recombinant DNA and molecular cloning techniques can be used to prepare the constructs and recombinant host cells of this disclosure. See, e.g., Sambrook et al. (1989) Molecular Cloning: A Laboratory Manual, 2nd ed., Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; Silhavy et al. (1984) Experiments with Gene Fusions, Cold Spring Harbor Laboratory, Cold Spring Harbor, NY; and Ausubel et al. (1987) In Current Protocols in Molecular Biology, Wiley-Interscience.

[0086] The present disclosure provides a tyramine-containing hydroxycinnamic acid amide-producing recombinant host cell harboring a nucleic acid encoding L-tyrosine and / or L-phenylalanine overproduction, enzymes for the biosynthesis of hydroxycinnamoyl-CoA esters and tyramine precursors, and a tyramine N-hydroxycinnamoyltransferase for producing tyramine-containing hydroxycinnamic acid amides in eukaryotic host cells. Prokaryotic cells contemplated for use with the present disclosure include both single cells and cells in cell culture, e.g., cell lines. Examples of suitable cells include bacterial host cells, e.g., Escherichia coli or Bacillus species; yeast host cells, e.g., Saccharomyces cerevisiae; insect host cells, e.g., Spodoptera frugiperda; or human host cells, e.g., HeLa and Jurkat cells. Preferred eukaryotic host cells are haploid cells from the genera Candida, Pichia, and Saccharomyces. While bacterial host cells can be used, the present disclosure preferably employs eukaryotic host cells, particularly yeast host cells of the genera Saccharomyces, Kluyveromyces, Pichia, Hansenula, Schizosaccharomyces, Kluyveromyces, Yarrowia, and Candida. S. cerevisiae possesses several attractive features as a metabolic engineering platform for the production of the compounds of this disclosure. In addition to its excellent accessibility to molecular and synthetic biology techniques, its eukaryotic nature facilitates the functional expression of plant-derived biosynthetic genes. For example, S. cerevisiae can functionally express cytochrome P450-containing enzymes, and its intracellular compartments are comparable to those of plant cells. Finally, its GRAS (Generally Recognized As Safe) status facilitates its subsequent application in the production of compounds for use in mammals. Thus, in certain embodiments, the host cell is preferably a eukaryotic host cell, most preferably S. cerevisiae.

[0087] Microbial expression systems and expression vectors containing regulatory sequences directing high-level expression of foreign proteins are well known to those skilled in the art. Any of these can be used to construct chimeric genes for the production of tyramine-containing hydroxycinnamic acid amides in host cells. These chimeric genes can then be introduced into appropriate microorganisms by transformation to allow high-level expression of the enzyme.

[0088] Once an appropriate expression construct has been prepared for expression in a host cell, it is placed into a plasmid vector capable of autonomous replication in the host cell, or it is directly integrated into the genome of the host cell. Integration of the expression cassette can occur randomly within the host genome, or can be targeted by using a construct containing sufficient regions of homology with the host genome to target recombination with the host locus. When the construct is targeted to an endogenous locus, all or part of the transcriptional and translational regulatory regions can be provided by the endogenous locus.

[0089] When two or more genes are expressed from separate replicating vectors, it is desirable that each vector have a different selection means and should lack homology to other constructs to maintain stable expression and prevent reassortment of elements between the constructs. Judicious selection of regulatory regions, selection means, and propagation methods for the introduced constructs can be determined empirically so that all introduced genes are expressed at the required levels, resulting in the synthesis of the desired product.

[0090] Constructs carrying the coding region of interest can be introduced into host cells by any standard technique, including transformation (e.g., lithium acetate transformation [Guthrie, C., Methods in Enzymology, 194:186-187 (1991)]), protoplast fusion, biolistic bombardment, electroporation, microinjection, or any other method for introducing a gene of interest into host cells.

[0091] For convenience, host cells that have been engineered by any method to incorporate a DNA sequence (e.g., an expression cassette) are referred to herein as "transformed" or "recombinant." A transformed host will have at least one copy of the expression construct, and may have two or more, depending on whether the gene is integrated into the genome, amplified, or present on an extrachromosomal element with multiple copies. Transformed host cells can be identified by selection for a marker contained on the introduced construct.

[0092] Alternatively, because many transformation techniques introduce many DNA molecules into host cells, a separate marker construct can be co-transformed with the desired construct. Typically, transformed hosts are selected for their ability to grow on selective media. The selective media may incorporate an antibiotic or may lack a factor, such as a nutrient or growth factor, necessary for the growth of the untransformed host. The introduced marker gene can confer antibiotic resistance or encode an essential growth factor or enzyme, which, when expressed in the transformed host, allows growth on selective media. Selection of transformed hosts can also occur when the expressed marker protein can be detected directly or indirectly. The marker protein can be expressed alone or as a fusion with another protein. Marker proteins can be detected by their enzymatic activity (e.g., β-galactosidase can convert the substrate X-gal [5-bromo-4-chloro-3-indolyl-β-D-galactopyranoside] into a colored product, and luciferase can convert luciferin into a light-emitting product); or by their light-producing or light-altering properties (e.g., the fluorescence of green fluorescent protein in Aequorea Victoria when illuminated with blue light). Alternatively, antibodies can be used to detect marker proteins or molecular tags on proteins of interest, for example. Cells expressing the marker protein or tag can be selected, for example, visually or by techniques such as FACS or antibody-based panning. For selection of yeast transformants, any marker that functions in yeast can be used. Resistance to kanamycin, hygromycin, and the aminoglycoside G418, as well as the ability to grow on media lacking uracil or leucine, are preferred for use herein.

[0093] In addition to recombinant host cells, this disclosure also includes methods for producing tyramine-containing hydroxycinnamic acid amides using recombinant host cells. According to the disclosed methods, recombinant eukaryotic host cells capable of producing tyramine-containing hydroxycinnamic acid amides are provided, and the recombinant eukaryotic host cells are cultured for a time sufficient to produce the tyramine-containing hydroxycinnamic acid amide. Once produced, the tyramine-containing hydroxycinnamic acid amide is isolated from the recombinant eukaryotic host cells or culture supernatant. Generally, medium conditions that can be optimized for high-level expression of a particular coding region of interest include the type and amount of carbon source, the type and amount of nitrogen source, the carbon-to-nitrogen ratio, the oxygen level, the growth temperature, the pH, the length of the biomass production phase, and the time of cell harvest. A target microorganism, such as yeast, is grown in a complex medium (e.g., yeast extract-peptone-dextrose broth (YPD)) or a defined minimal medium (e.g., Yeast Nitrogen Base (DIFCO Laboratories, Detroit, MI)) that lacks components necessary for growth, thereby forcing selection for the desired expression cassette.

[0094] The fermentation or culture medium of the present disclosure must contain a suitable carbon source for the production of tyramine-containing hydroxycinnamic acid amides. Suitable carbon sources may include, but are not limited to, monosaccharides (e.g., glucose, fructose), disaccharides (e.g., lactose, sucrose), oligosaccharides, polysaccharides (e.g., starch, cellulose, or mixtures thereof), sugar alcohols (e.g., glycerol), or mixtures of renewable raw materials (e.g., whey permeate, corn steep liquor, sugar beet molasses, barley malt). In addition, carbon sources may include various commercially available sources of fatty acids, including alkanes, fatty acids, esters of fatty acids, monoglycerides, diglycerides, triglycerides, phospholipids, and vegetable oils (e.g., soybean oil) and animal fats. In addition, carbon sources may include one-carbon sources (e.g., carbon dioxide, methanol, formaldehyde, formic acid, carbon-containing amines) for which metabolic conversion to key biochemical intermediates has been demonstrated. Thus, it is contemplated that the carbon source utilized in the present disclosure can encompass a wide variety of carbon-containing sources and is limited only by the choice of host organism. While all of the above carbon sources and mixtures thereof are expected to be suitable in the present disclosure, preferred carbon sources are sugars and / or fatty acids. Glucose and / or fatty acids containing 10-22 carbons are most preferred.

[0095] Nitrogen can be supplied from inorganic (e.g., (NH4)2SO4) or organic sources (e.g., urea or glutamic acid). In addition to suitable carbon and nitrogen sources, the fermentation medium must also contain appropriate minerals, salts, cofactors, buffers, vitamins, and other components known to those skilled in the art suitable for microbial growth.

[0096] Alternatively or additionally to producing tyramine-containing hydroxycinnamic acid amides from a carbon source (e.g., glucose or molasses), this disclosure also provides for exogenous feeding of one or more intermediate substrates in the biosynthetic pathway for producing tyramine-containing hydroxycinnamic acid amides to the fermenter medium. Thus, in a further aspect, L-phenylalanine, L-tyrosine, cinnamic acid, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, and / or S-adenyl-L-methionine can be exogenously fed to the recombinant host cells of this disclosure. Those skilled in the art will recognize that the carbon flow from aromatic amino acid production to tyramine-containing hydroxycinnamic acid amide production must be balanced so that a decrease in the concentration of free aromatic amino acids is not detrimental to the viability or health of the recombinant host cells. Thus, in some embodiments, L-phenylalanine and / or L-tyrosine can be exogenously fed to the culture medium to increase production of tyramine-containing hydroxycinnamic acid amides.

[0097] The recombinant host cells of this disclosure can be cultured using methods known in the art. For example, the cells can be cultured by small- or large-scale fermentation in shake flask cultures, laboratory or industrial fermenters, in an appropriate medium under conditions that allow expression of the coding region of interest. If commercial production of tyramine-containing hydroxycinnamic acid amides is desired, various fermentation methods can be applied. For example, large-scale production of a specific gene product overexpressed from a recombinant host can be produced by batch, fed-batch, or continuous fermentation processes.

[0098] A batch fermentation process is a closed system in which the medium composition is fixed at the beginning of the process and receives no further additions other than those necessary to maintain pH and oxygen levels throughout the process. Thus, at the beginning of the cultivation process, the medium is inoculated with the desired organism, allowing growth or metabolic activity to occur without adding additional sources (i.e., carbon and nitrogen sources) to the medium. In a batch process, the metabolic product and biomass composition of the system changes constantly until the time the culture is terminated. In a typical batch process, cells progress through a stationary lag phase, a high-growth logarithmic phase, and finally a stationary phase, where growth rate slows or stops. If left untreated, cells in the stationary phase will eventually die. A variation of the standard batch process is the fed-batch process, in which a feedstock is continuously added to a fermenter over the course of the fermentation process. Fed-batch processes are also suitable in the present disclosure. Fed-batch processes are useful when catabolite repression tends to inhibit cellular metabolism or when it is desirable to have a limited amount of a feedstock in the medium at any one time. Measuring feedstock concentrations in fed-batch systems is difficult and therefore must be estimated based on changes in measurable factors such as pH, dissolved oxygen, and the partial pressure of waste gases (e.g., CO2). Batch and fed-batch culture methods are common and well known in the art; examples can be found in Biotechnology: A Textbook of Industrial Microbiology, 2nd Edition, (1989) Thomas D. Brock, Sinauer Associates, Sunderland, Mass.; or Mukund V. Deshpande, (1992). Appl. Biochem. Biotechnol., 36:227.

[0099] Commercial production of tyramine-containing hydroxycinnamic acid amides can also be achieved by a continuous fermentation process, in which a defined medium is continuously added to a bioreactor while an equal amount of culture volume is simultaneously removed for product harvest. Continuous cultures generally maintain cells in logarithmic growth phase at a constant cell density. Continuous or semi-continuous culture methods allow for modulation of one factor or any number of factors that affect cell growth or end-product concentration. For example, one approach may limit the carbon source and allow all other parameters to moderate metabolism. In other systems, several factors affecting growth may be continuously varied while maintaining constant cell concentration, as measured by medium turbidity. Continuous systems aim to maintain steady-state growth; therefore, cell growth rate must be balanced against cell loss due to medium being removed from the culture. Methods for modulating nutrients and growth factors for continuous culture processes, as well as techniques for maximizing product formation rates, are well known in the art of industrial microbiology.

[0100] Tyramine-containing hydroxycinnamic acid amides can be extracted from host cells or culture supernatants by solvent extraction (e.g., partitioning) or precipitation, treatment with activated carbon, evaporation, filtration, chromatographic fractionation, or a combination thereof. Solvent extraction can be carried out using, for example, n-pentane, hexane, butane, chloroform, dichloromethane, diethyl ether, acetonitrile, water, butanol, isopropanol, ethanol, methanol, glacial acetic acid, acetone, norflurane (HFA134a), ethyl acetate, dimethyl sulfoxide, heptafluoropropane (HFA227), and subcritical or supercritical fluids, such as liquid carbon dioxide and water, or any combination thereof in any proportion. When solvents such as those listed above are used, the resulting extract typically contains nonspecific lipid-soluble substances, which can be removed by various processes, including cooling to a specific temperature, typically -20°C, followed by filtration or centrifugation to remove waxy ballast, extraction with subcritical or supercritical carbon dioxide or a nonpolar solvent (e.g., hexane), and "dewaxing" involving distillation.

[0101] The extract enriched in tyramine-containing hydroxycinnamic acid amides is ideally obtained by chromatographic fractionation. Chromatographic fractionation typically involves column chromatography and can be based on molecular size, charge, solubility, and / or polarity. Depending on the type of chromatographic method, column chromatography may be performed on a matrix material such as dextran, agar, polyacrylamide, or silica, and may contain solvents such as dimethyl sulfoxide, pyridine, water, dimethylformamide, methanol, saline, ethylene dichloride, chloroform, propanol, ethanol, isobutanol, formamide, methylene dichloride, butanol, acetonitrile, isopropanol, tetrahydrofuran, dioxane, chloroform / dichloromethane, etc.

[0102] Typically, the product of the chromatography step is collected in multiple fractions, which can then be tested for the presence of the desired compound using any suitable analytical technique (e.g., thin layer chromatography, mass spectrometry), and fractions enriched in the desired compound can then be selected for further purification.

[0103] Alternatively, or in conjunction with chromatography, crystallization can be performed to obtain highly pure tyramine-containing hydroxycinnamic amides. The solubility of the tyramine-containing hydroxycinnamic amides is adjusted by changing the temperature and / or composition of the solution, e.g., by removing ethanol, and / or adjusting the pH to promote precipitation, followed by filtration or centrifugation of the precipitated crystals or oil.

[0104] For example, an extract containing N-trans-caffeoyltyramine can be obtained by subjecting host cells or culture supernatant to 80% ethanol at room temperature, filtering and concentrating the 80% ethanol extract, resuspending the concentrated extract in water, partitioning the aqueous solution with hexane, adding chloroform to the aqueous layer, and subjecting the chloroform layer to liquid chromatography on silica gel. See, e.g., Ko et al. (2015) Internatl. J. Mol. Med. 36(4):1042-8.

[0105] Extracts containing tyramine-containing hydroxycinnamic acid amides can be standardized using conventional techniques such as high-performance liquid chromatography (HPLC) or high-performance thin-layer chromatography (HPTLC). The term "standardized extract" refers to an extract that has been standardized by identifying characteristic components or bioactive markers present in the extract. Characterization can be by analyzing spectral data, such as mass spectrometry (MS), infrared spectrometry (IR), and nuclear magnetic resonance (NMR) spectroscopy data.

[0106] The substantially pure tyramine-containing hydroxycinnamic acid amide or extract containing tyramine-containing hydroxycinnamic acid amide can be combined with a carrier and provided in any suitable form for consumption by or administration to a subject. Suitable consumable forms include, but are not limited to, a dietary supplement, a food ingredient or additive, a food product (e.g., a functional food), a medical food, a nutraceutical, or a pharmaceutical composition.

[0107] A food ingredient or additive is an edible substance (including any substance intended for use in the production, manufacturing, packing, processing, preparation, treatment, packaging, transport, or containment of food) that is intended, directly or indirectly, to be a component of or otherwise affect the characteristics of any food. Food products, particularly functional foods, are foods that have been fortified or concentrated during processing to contain additional, supplemental nutrients and / or beneficial ingredients. Food products according to this disclosure can be, for example, butter, margarine, sweet or tangy spreads, biscuits, health bars, bread, cakes, cereals, candy, confectionery, yogurt or fermented dairy products, juice-based and vegetable-based beverages, shakes, flavored waters, fermented beverages (e.g., kombucha or fermented yerba mate), convenience snacks, such as baked or fried vegetable chips or other extruded snack products, or any other suitable food form.

[0108] A dietary supplement is a product taken orally containing the compounds or extracts of the present disclosure and intended to supplement the diet. A nutraceutical is a product derived from food sources that provides additional health benefits in addition to the basic nutritional value found in food. A pharmaceutical composition is defined as any component of a drug intended to produce pharmacological activity or other direct effect in the diagnosis, cure, mitigation, treatment, or prevention of disease, or to affect the structure or any function of the human or other animal body. Dietary supplements, nutraceuticals, and pharmaceutical compositions can be found in many forms, such as tablets, coated tablets, pills, capsules, pellets, granules, softgels, gel capsules, liquids, powders, emulsions, suspensions, elixirs, syrups, and any other form suitable for use.

[0109] As used herein, the phrase "carrier" refers to a substance, composition, or vehicle, such as a liquid or solid filler, diluent, excipient, manufacturing aid (e.g., lubricant, talc, magnesium, calcium, or zinc stearate, or stearic acid), or solvent encapsulating material, that is involved in carrying or transporting a compound of interest from one organ or part of the body to another. Each carrier should be compatible with the other ingredients of the formulation and not deleterious to the subject. Some examples of substances which can serve as carriers include: (1) sugars, such as lactose, glucose, and sucrose; (2) starches, such as corn starch and potato starch; (3) cellulose and its derivatives, such as sodium carboxymethylcellulose, ethyl cellulose, cellulose acetate, and hydroxypropyl methylcellulose; (4) powdered tragacanth; (5) malt; (6) gelatin; (7) talc; (8) excipients, such as cocoa butter and suppository wax; (9) oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil, and soybean oil; (10) glycols, such as propylene glycol, ... (11) polyols, such as glycerin, sorbitol, mannitol, and polyethylene glycol; (12) esters, such as ethyl oleate and ethyl laurate; (13) agar; (14) buffers, such as magnesium hydroxide and aluminum hydroxide; (15) alginic acid; (16) pyrogen-free water; (17) isotonic saline; (18) Ringer's solution; (19) ethyl alcohol; (20) pH buffer solutions; (21) polyesters, polycarbonates, and / or polyanhydrides; (22) phospholipids and phospholipid derivatives; and (23) other non-toxic, compatible materials employed in conventional formulations.

[0110] To prepare solid compositions such as tablets or capsules, the compound or extract is mixed with carriers (e.g., conventional tableting ingredients such as corn starch, lactose, sucrose, sorbitol, talc, stearic acid, magnesium stearate, dicalcium phosphate, or gums) and other diluents (e.g., water) to form a solid composition. This solid composition is then subdivided into unit dosage forms containing an effective amount of a compound of the present disclosure. Tablets or pills containing the compound or extract can be coated or otherwise compounded to provide a dosage form offering the advantages of prolonged action and / or potentially enhanced absorption.

[0111] The liquid forms into which the compounds or extracts of the present disclosure are incorporated for oral or parenteral administration include aqueous solutions, suitably flavored syrups, aqueous or oily suspensions, and flavored emulsions and elixirs using edible oils and similar vehicles.Suitable dispersing or suspending agents for aqueous suspensions include synthetic natural gums, such as tragacanth, gum arabic, alginate, dextran, sodium carboxymethylcellulose, methylcellulose, polyvinylpyrrolidone or gelatin.Liquid preparations for oral administration may take the form of, for example, solutions, syrups or suspensions, or may be presented as a dry product that is reconstituted with water or other suitable vehicle before use. Such liquid preparations may be prepared by conventional means with acceptable additives such as suspending agents (e.g., sorbitol syrup, methylcellulose, or hydrogenated edible fats); emulsifying agents (e.g., lecithin or acacia); non-aqueous vehicles (e.g., almond oil, oily esters, or ethyl alcohol); preservatives (e.g., methyl or propyl p-hydroxybenzoate or sorbic acid); and artificial or natural colorants and / or sweeteners.

[0112] The method for preparing the formulation or composition of this disclosure includes the step of bringing the compound or extract of this disclosure into association with a carrier and, optionally, one or more auxiliary and / or active ingredients. Generally, the formulation is prepared by uniformly and intimately bringing the compound or extract of this disclosure into association with a liquid carrier, or a finely divided solid carrier, or both, and then, if necessary, shaping the product. Thus, the disclosed formulation can consist of, or consist essentially of, the compound or extract described herein in combination with a suitable carrier.

[0113] When the compounds or extracts of the present disclosure are administered to humans and animals as pharmaceuticals, nutritional supplements, or dietary supplements, they may be given on their own or in combination with an acceptable carrier, for example, as a composition containing 0.1 to 99% (more preferably 10 to 30%) of the active ingredient.

[0114] While it is contemplated that individual tyramine-containing hydroxycinnamic acid amides may be used in the consumables of this disclosure, it is further contemplated that two or more of the compounds or extracts may be combined in any relative amounts to produce a custom combination of ingredients containing two or more tyramine-containing hydroxycinnamic acid amides in a desired ratio to enhance the efficacy of the product, improve the organoleptic properties, or any other measure of quality important to the end use of the product. [Example]

[0115] Recombinant yeast strains for producing tyramine-containing hydroxycinnamic acid amides Because tyramine-containing hydroxycinnamic acid amides are not endogenous metabolites, a synthetic production pathway must be replicated in yeast. Synthesis, like all phenylpropanoids, begins with phenylalanine and / or tyrosine, which are endogenously produced by the cell. Genes for introduction and overexpression in the recombinant yeast strains of the present disclosure are listed in Table 9.

[0116] [Table 9A]

[0117] [Table 9B]

[0118] The Saccharomyces cerevisiae strain used is an isogenic haploid. The starting yeast strain contains knockouts of auxotrophic (-ura3, -leu2, his3) marker genes. Clones are enriched and grown in YPD liquid culture medium (10 g / l BACTO-yeast extract, 20 g / l BACTO-peptone, and 2% dextrose) at 30°C. Recombinants are selected on dropout agar plates lacking uracil, leucine, or histidine (YNB+CSM). Gene deletions in the uracil, histidine, and leucine biosynthetic pathways result in auxotrophy. For homologous recombination, mismatch-deficient strains are used. Open reading frames are synthesized and / or amplified by PCR.

[0119] The constructs are introduced into yeast using conventional cloning and yeast transformation protocols, and the cells are grown in medium containing glucose as the sole carbon source. If additional substrates are required (e.g., phenylalanine, tyrosine, or cinnamic acid), they are added 24 hours after the start of culture. The supernatants are then analyzed by high-performance liquid chromatography (HPLC) to identify the appropriate products.

[0120] In certain embodiments, the yeast cells overproduce one or both of phenylalanine and tyrosine. In certain embodiments, phenylalanine and tyrosine are produced in approximately equal proportions by the recombinant host cells. To avoid the production of aromatic alcohols and redirect pathway flux toward aromatic amino acids, a double knockout of ARO10 (phenylpyruvate decarboxylase) and PDC5 (pyruvate decarboxylase) is introduced into the strain. Yeast strains and growth media supplements for producing tyramine-containing hydroxycinnamic acid amides are provided in Table 10.

[0121] [Table 10A]

[0122] [Table 10B]

[0123] [Table 10C]

[0124] Strains exhibiting high levels of tyramine-containing hydroxycinnamic acid amide production are used to produce extracts and consumables containing tyramine-containing hydroxycinnamic acid amide. The production strain is grown in a bioreactor for a time sufficient to produce tyramine-containing hydroxycinnamic acid amide. After fermentation is complete, the cell mass is removed from the supernatant by centrifugation or filtration. The tyramine-containing hydroxycinnamic acid amide is then recovered from the supernatant by extraction with a suitable solvent, such as aqueous alcohol or ethyl acetate. The tyramine-containing hydroxycinnamic acid amide can then be further purified by solvent partitioning and / or chromatography and crystallized by modifying the solvent, e.g., adjusting the solution temperature and / or composition. The tyramine-containing hydroxycinnamic acid amide can also be recovered directly from the cell mass by adding ethanol or another suitable solvent, such as ethyl acetate, by adding the solvent directly to the cell culture, followed by filtration or centrifugation. After removal of the solvent from the supernatant, the crystals (or other desolvated form, such as an oil or precipitate) are collected. This material is then further purified, for example, by solvent partitioning and / or chromatography, and crystallization by altering the temperature and / or composition of the solvent to obtain highly pure material, which is then recovered, washed, and dried to obtain a purified (>90%) source of tyramine-containing hydroxycinnamic acid amide.

Claims

1. 1. A recombinant eukaryotic host cell capable of producing a tyramine-containing hydroxycinnamic acid amide, comprising: (a) one or more nucleic acid molecules encoding one or more enzymes capable of overproducing L-tyrosine or L-phenylalanine; (b) one or more nucleic acid molecules encoding one or more enzymes of a phenylpropanoid CoA pathway for producing a hydroxycinnamoyl-CoA ester; (c) a nucleic acid molecule encoding a tyrosine decarboxylase (EC 4.1.1.25), and (d) a nucleic acid molecule encoding tyramine N-hydroxycinnamoyltransferase (EC 2.3.1.110) A recombinant eukaryotic host cell comprising:

2. 2. The recombinant eukaryotic host cell of claim 1, wherein the one or more nucleic acid molecules encoding one or more enzymes of a phenylpropanoid CoA pathway to produce hydroxycinnamoyl-CoA esters include phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamate 4-hydroxylase, coumarate 3-hydroxylase, coumaroyl-CoA 3-hydroxylase, caffeoyl-CoA O-methyltransferase, ferulate 5-hydroxylase, caffeate / 5-hydroxyferulate O-methyltransferase, tyrosine ammonia-lyase, or a combination thereof.

3. 3. The recombinant eukaryotic host cell of claim 1 or 2, which further overproduces methionine.

4. 3. The recombinant eukaryotic host cell of claim 1 or 2, which is a recombinant yeast strain.

5. The yeast strain Table 1A Table 1B 5. The recombinant eukaryotic host cell of claim 4, wherein the host cell is selected from the group consisting of:

6. Tyramine-containing hydroxycinnamic acid amides include N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, cinnamoyltyramine, sinapoyltyramine, p-coumaroyltyramine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-ethoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-methoxyethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(methylsulfonyl) )ethoxy)phenethyl)acrylamide, (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetic acid, ethyl (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetate, (E)-N-(4-(cyclopropylmethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3,3-trifluoropropoxy)phenethyl ) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-4-yl)methoxy)phenethyl) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-fluorobenzyl)oxy)phenethyl) acrylamide, (E)-N-(4-(cyanomethoxy)phenethyl)-3-(3,4-dihydroxyphenyl) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-3-ylmethoxy)phenethyl) acrylamide Acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-2-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(dimethylamino)ethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-isobutoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-4-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-methoxybenzyl)oxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(oxetan-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydrofuran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(thiophen-2-yloxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3-dimethylbutoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-hydroxyethoxy)phenethyl)acrylamide, (E)-N-(4-((1H-tetrazol-5-yl)methoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((1-methylpyrrolidin-2-yl)methoxy)phenethyl)acrylamide, (E)-2-hydroxy-5-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenyl hydrogen carbonate, (E)-3-(4-hydroxy-3-(pyridin-4-yloxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-hydroxy-3-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acryl acrylamide, (E)-3-(3-(4-fluorophenoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-(cyanomethoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-2-(2-hydroxy-4-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid, (E)-3-(3-hydroxy-4-(pyridin-4-ylmethoxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-((4-fluorobenzyl)oxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-hydroxy-4-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-(cyanomethoxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-N-(3-(3,4-dihydroxyphenyl)acryloyl)-N-(4-hydroxyphenethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)glycine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-(pyridin-4-ylmethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-isobutylacrylamide, (E)-N-(cyanomethyl)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, 3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)propanamide, or 3-(3,3. The recombinant eukaryotic host cell according to claim 1 or 2, wherein the nucleotide sequence is selected from at least one of: N-(4-(methylsulfonamido)phenethyl)propanamide; N-(4-dihydroxyphenyl)-N-(4-(methylsulfonamido)phenethyl)propanamide;

7. 7. The recombinant eukaryotic host cell of claim 6, wherein the tyramine-containing hydroxycinnamic acid amide is N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, cinnamoyltyramine, sinapoyltyramine, or p-coumaroyltyramine.

8. 1. A method for producing a tyramine-containing hydroxycinnamic acid amide or a tyramine-containing hydroxycinnamic acid derivative, comprising: (a) providing a recombinant eukaryotic host cell capable of producing a tyramine-containing hydroxycinnamic acid amide, said recombinant eukaryotic host cell comprising: A recombinant eukaryotic host cell that overproduces L-tyrosine or L-phenylalanine, (i) one or more nucleic acid molecules encoding one or more enzymes for producing hydroxycinnamoyl-CoA esters of tyramine-containing hydroxycinnamic acid amides; (ii) a nucleic acid molecule encoding a tyrosine decarboxylase (EC 4.1.1.25), and (iii) further comprising a nucleic acid molecule encoding a tyramine N-hydroxycinnamoyltransferase (EC 2.3.1.110); (b) culturing the recombinant eukaryotic host cells for a period of time sufficient for the recombinant eukaryotic host cells to produce the tyramine-containing hydroxycinnamic acid amide; (c) isolating the tyramine-containing hydroxycinnamic acid amide from the recombinant eukaryotic host cells or culture supernatant, thereby producing the tyramine-containing hydroxycinnamic acid amide. A method comprising:

9. 9. The method of claim 8, wherein the one or more nucleic acid molecules encoding one or more enzymes in a phenylpropanoid CoA pathway to produce hydroxycinnamoyl-CoA esters include phenylalanine ammonia-lyase, 4-coumarate-CoA ligase, cinnamate 4-hydroxylase, coumarate 3-hydroxylase, caffeoyl-CoA O-methyltransferase, 5-hydroxylase, caffeic acid / 5-hydroxyferulic acid O-methyltransferase, tyrosine ammonia-lyase, or a combination thereof.

10. 10. The method of claim 8 or 9, wherein the recombinant eukaryotic host cell further overproduces S-adenosylmethionine.

11. 10. A recombinant eukaryotic host cell as defined in claim 8 or 9, which is a recombinant yeast strain.

12. The yeast strain Table 2A Table 2B 12. The recombinant eukaryotic host cell of claim 11, selected from:

13. Tyramine-containing hydroxycinnamic acid amides include N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, p-coumaroyltyramine, cinnamoyltyramine, sinapoyltyramine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-ethoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-methoxyethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(methylsulfonyl) )ethoxy)phenethyl)acrylamide, (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetic acid, ethyl (E)-2-(4-(2-(3-(3,4-dihydroxyphenyl)acrylamido)ethyl)phenoxy)acetate, (E)-N-(4-(cyclopropylmethoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3,3-trifluoropropoxy)phenethyl ) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-4-yl)methoxy)phenethyl) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-fluorobenzyl)oxy)phenethyl) acrylamide, (E)-N-(4-(cyanomethoxy)phenethyl)-3-(3,4-dihydroxyphenyl) acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-3-ylmethoxy)phenethyl) acrylamide Acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-2-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-(dimethylamino)ethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-isobutoxyphenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(pyridin-4-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((4-methoxybenzyl)oxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(oxetan-3-ylmethoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydro-2H-pyran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((tetrahydrofuran-2-yl)methoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(thiophen-2-yloxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(3,3-dimethylbutoxy)phenethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-(2-hydroxyethoxy)phenethyl)acrylamide, (E)-N-(4-((1H-tetrazol-5-yl)methoxy)phenethyl)-3-(3,4-dihydroxyphenyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-((1-methylpyrrolidin-2-yl)methoxy)phenethyl)acrylamide, (E)-2-hydroxy-5-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenyl hydrogen carbonate, (E)-3-(4-hydroxy-3-(pyridin-4-yloxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-hydroxy-3-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acryl acrylamide, (E)-3-(3-(4-fluorophenoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-(cyanomethoxy)-4-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-2-(2-hydroxy-4-(3-((4-hydroxyphenethyl)amino)-3-oxoprop-1-en-1-yl)phenoxy)acetic acid, (E)-3-(3-hydroxy-4-(pyridin-4-ylmethoxy)phenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-((4-fluorobenzyl)oxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(3-hydroxy-4-isobutoxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-3-(4-(cyanomethoxy)-3-hydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, (E)-N-(3-(3,4-dihydroxyphenyl)acryloyl)-N-(4-hydroxyphenethyl)acrylamide (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)glycine, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-(pyridin-4-ylmethyl)acrylamide, (E)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)-N-isobutylacrylamide, (E)-N-(cyanomethyl)-3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)acrylamide, 3-(3,4-dihydroxyphenyl)-N-(4-hydroxyphenethyl)propanamide, or 3-(3,10. The method according to claim 8 or 9, wherein the aryl group is selected from at least one of 4-dihydroxyphenyl-N-(4-(methylsulfonamido)phenethyl)propanamide,

14. 14. The method of claim 13, wherein the tyramine-containing hydroxycinnamic acid amide is selected from at least one of N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, p-coumaroyltyramine, cinnamoyltyramine, and sinapoyltyramine.

15. An extract comprising tyramine-containing hydroxycinnamic acid amide produced by the method according to any one of claims 1 to 14.

16. 16. A consumable product comprising the extract of claim 15.

17. 17. The consumable product of claim 16, which is a dietary supplement, a food ingredient or additive, a food product, a medical food, a nutraceutical, or a pharmaceutical composition.

18. 1. A method for culturing a recombinant eukaryotic host cell comprising one or more nucleic acid molecules capable of producing a tyramine-containing hydroxycinnamic acid amide or a tyramine-containing hydroxycinnamic acid derivative, the method comprising: a) inoculating recombinant eukaryotic host cells into a growth medium; b) culturing the host cells to a desired cell density; c) harvesting the host cells from the culture medium; A method comprising:

19. 19. The method of claim 18, wherein the growth medium is selected from glucose, p-coumaric acid, caffeic acid, ferulic acid, sinapic acid, cinnamic acid, phenylalanine, tyrosine, or combinations thereof.

20. 20. A recombinant eukaryotic host cell as defined in claim 18 or 19, which is a recombinant yeast strain.

21. The yeast strain Table 3A Table 3B 21. The recombinant eukaryotic host cell of claim 20, selected from:

22. 19. The method of claim 18, wherein the tyramine-containing hydroxycinnamic acid amide produced is selected from at least one of N-caffeoyltyramine, N-feruloyltyramine, 5-hydroxyferuloyltyramine, p-coumaroyltyramine, cinnamoyltyramine, and sinapoyltyramine.

Images