Bioproduction of tetrahydrocurcumin and tetrahydrodemethoxycurcumin

Abstract

The invention is related to materials and methods for production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin from substrates. The invention provides methods and materials for cell-free and host cell-based bioproduction of tetrahydrocurcumin and tetrahydrodemethoxycurcumin.

Description

[0001] Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0002] BTOPRODUCTTON OF TETRAHYDROCURCUMIN AND TETRAHYDRODEMETHOXYCURCUMIN

[0003] SEQUENCE LISTING

[0004] This application incorporates by reference a sequence listing submitted as a text file entitled “DEBU-037-01WO-Seq-Listing.xml” created October 28, 2025 and having a size of 44 kilobytes.

[0005] BACKGROUND

[0006] Curcuminoids are a class of natural compounds widely known for their pronounced yellow coloring that are derived from turmeric, a plant in the ginger family, that have antiinflammatory, antioxidant, and other properties. Curcuminoids have been used in traditional Asian medicine for centuries. They are also used as a food additive and coloring agent. Curcuminoids have anti-inflammatory, antioxidant, antibacterial, antiviral, and neuroprotective properties.

[0007] Two of the major reduction metabolites of curcumin are tetrahydrocurcumin (THCC) and tetrahydrodemethoxycurcumin (THDCC). THCC is colorless and more hydrophilic and stable than curcumin due to its non-planar form and benzene rings located at the ends of the heptane chain. THCC also exhibits higher antioxidant activity than curcumin and possesses various biological properties, including anti-inflammatory, chemopreventive, antibacterial, antidyslipidemic, antiviral, cytotoxic, antiangiogenic, neurological, antihistamine, immunological, and anti-aging properties. THDCC is structurally similar to THCC, but it lacks one methoxy group. THDCC has potential antioxidant and therapeutic properties. It is also a minor constituent of Curcuma zeodaria, a Thai plant known for its anti-inflammatory activity. Conventional methods of production of THCC and THDCC include chemical hydrogenation of curcuminoids extracted from plant material such as the rhizome of Curcuma longa.

[0008] SUMMARY

[0009] The present invention provides, in various embodiments, novel methods for bioproduction of tetrahydrocurcumin and the related compound tetrahydrodemethoxycurcumin. The methods of the invention are economic and reliable as compared to conventional methods of production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin. In addition, the methods Attorney Docket No. DEBU-037 / 01WO 37396 / 185 provided in the invention provide higher titer and higher purity values of tetrahydrocurcumin and tetrahydrodem ethoxy curcumin from these processes as compared to existing methods of production.

[0010] Production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin

[0011] In certain aspects, the invention provides a method for production of tetrahydrocurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin through one or more intermediates. In certain aspects, the substrate is ferulic acid. In certain aspects, the substrate is glucose or glycerol. In certain aspects, the substrate is tyrosine. In some embodiments, the substrate ferulic acid is produced endogenously by the engineered host cell from glucose, glycerol, or tyrosine. In some embodiments, the substrate ferulic acid is produced endogenously by the engineered host cell through overexpression of the tyrosine ammonia lyase (TAL), p-coumarate 3 -hydroxylase (C3H), and caffeic acid O-methyltransferase (COMT) enzymes.

[0012] In certain aspects, the invention provides a method for production of tetrahydrodemethoxycurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrodemethoxycurcumin through one or more intermediates. In certain aspects, the substrate is ferulic acid. In certain aspects, the substrate is coumaric acid. In certain aspects, the substrate is a combination of ferulic acid and coumaric acid. In certain aspects, the substrate is glucose or glycerol. In certain aspects, the substrate is tyrosine.

[0013] In some embodiments, the substrates ferulic acid and coumaric acid are produced endogenously by the engineered host cell from glucose, glycerol, or tyrosine. In some embodiments, the substrate ferulic acid is produced endogenously by the engineered host cell through overexpression of the tyrosine ammonia lyase (TAL), p-coumarate 3 -hydroxylase (C3H), and caffeic acid O-methyltransferase (COMT) enzymes.

[0014] In one aspect, the invention provides a method for production of tetrahydrocurcumin, wherein the method comprises providing an enzyme in a reaction medium, wherein the enzyme results in transformation of a substrate to tetrahydrocurcumin. In certain embodiments, the substrate is curcumin. In certain embodiments, the enzyme is a curcumin reductase. In certain Attorney Docket No. DEBU-037 / 01WO 37396 / 185 embodiments, the curcumin reductase results in transformation of curcumin to tetrahydrocurcumin.

[0015] In addition, the invention provides a method to produce tetrahydrodemethoxy curcumin, wherein the method comprises providing an enzyme in a reaction medium, wherein the enzyme results in transformation of a substrate to tetrahydrodemethoxycurcumin. In certain embodiments, the substrate is demethoxycurcumin. In certain embodiments, the enzyme is a curcumin reductase. In certain embodiments, the curcumin reductase results in transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin.

[0016] Thus, the methods of the invention provide that tetrahydrocurcumin or tetrahydrodemethoxycurcumin may be produced in an engineered host cell or in a cell-free medium.

[0017] In some embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxycurcumin has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0018] In other embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxycurcumin has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

[0019] An overview of the cell-free and cell-based production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin is provided in FIGS. 1-11.

[0020] Cell-free production of tetrahydrocurcumin from curcumin and tetrahydrodemethoxycurcumin from demethoxycurcumin

[0021] In certain aspects, the invention provides methods for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin, wherein the method comprises providing an enzyme in a cell-free medium, wherein the enzyme results in transformation of one or more substrates to tetrahydrocurcumin or tetrahydrodemethoxycurcumin.

[0022] In certain embodiments, the enzyme is a curcumin reductase resulting in transformation of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin. In Attorney Docket No. DEBU-037 / 01WO 37396 / 185 certain embodiments, the curcumin reductase is engineered for efficient transformation of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin.

[0023] In certain embodiments, the methods of the invention provide that tetrahydrocurcumin is produced in a cell-free medium. In certain embodiments, the invention provides methods for cell-free transformation of one or more substrates to tetrahydrocurcumin in a cell-free medium. In certain embodiments, the invention provides cell-free enzymatic transformation of one or more substrates to tetrahydrocurcumin. In certain embodiments, the invention provides cell-free transformation of curcumin to tetrahydrocurcumin.

[0024] In certain embodiments, the methods of the invention provide that tetrahydrodemethoxycurcumin is produced in a cell-free medium. In certain embodiments, the invention provides methods for cell-free transformation of one or more substrates to tetrahydrodemethoxycurcumin in a cell-free medium. In certain embodiments, the invention provides cell-free enzymatic transformation of one or more substrates to tetrahydrodemethoxycurcumin. In certain embodiments, the invention provides cell-free transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin.

[0025] In certain aspects, the invention a cell-free production method of tetrahydrocurcumin or tetrahydrodemethoxycurcumin with high titer values and higher purity of tetrahydrocurcumin and tetrahydrodemethoxycurcumin as compared to other conventional methods. The methods of the invention have high efficiency in conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin in a cell-free medium. The curcumin reductase (CurA) enzyme disclosed herein provides a high-efficiency cell-free and enzymatic conversion of curcumin to tetrahydrocurcumin and demethoxycurcumin to tetrahydrodemethoxycurcumin. Thus, the curcumin reductase enzyme (CurA) disclosed herein produces tetrahydrocurcumin and tetrahydrodemethoxycurcumin with high efficiency, high titer, and high purity.

[0026] In certain embodiments, the curcumin reductase enzyme used herein is listed Table 1.

[0027] Table 1

[0028] Curcumin reductase (CurA) Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0029] In certain embodiments, the curcumin reductase enzyme (CurA) is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase enzyme enzyme is selected from the enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase enzyme enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase enzyme is the enzyme provided in SEQ ID NO: 1.

[0030] In certain embodiments, the curcumin reductase enzyme used in cell-free conversion of curcumin to tetrahydrocurcumin or dem ethoxy curcumin to tetrahydrodem ethoxy curcumin is a purified enzyme. In certain embodiments, the curcumin reductase enzyme (CurA) for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin is immobilized and used in batch or in packed-bed reactors.

[0031] In certain embodiments, the curcumin reductase enzyme (CurA) for the cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin is generated from lysing a host cell overexpressing the curcumin reductase enzyme. In certain embodiments, the lysate generated from lysing said host cell overexpressing the curcumin reductase enzyme is utilized for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin. In certain embodiments, the curcumin reductase enzyme utilized for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin is purified from the lysate of hosts expressing the curcumin reductase enzyme.

[0032] In certain aspects, the curcumin reductase enzyme (CurA) required for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is expressed in a host organism. In certain embodiments, the host organism is selected from a group consisting of: bacteria, yeast, and / or fungal cells. In certain embodiments, the curcumin reductase enzyme (CurA) is introduced in the host organism by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the host organism. In certain embodiments, host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the enzyme. The predetermined biomass is calculated based on the quantity Attorney Docket No. DEBU-037 / 01WO 37396 / 185 of the enzyme required for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxy curcumin. In certain embodiments, the culture comprising host organisms expressing the curcumin reductase enzyme (CurA) is lysed and used as the reaction medium for the methods provided in the invention. In certain other embodiments, once the culture comprising host organisms is lysed, the cell-debris is removed from the lysed matter to prepare the reaction medium for the methods of the invention.

[0033] In certain embodiments, the cell-free medium further comprises NADPH and / or NADH. In certain embodiments, the cell-free medium further comprises an NADPH regeneration system. In certain embodiments, the NADPH regeneration system comprises glucose and glucose dehydrogenase (GDH). In certain embodiments, one or more enzymes are produced in an engineered host cell. In certain embodiments, the engineered host cell is selected from a group consisting of bacteria, yeast, and / or fungal cells. In certain embodiments, the curcumin reductase enzyme is introduced in the host cell by integration into genome of the host cell or on a plasmid. In certain embodiments, the engineered host cell expressing the curcumin reductase enzyme is cultured until a pre-determined biomass is achieved to produce the requisite quantity of the curcumin reductase enzyme. In certain embodiments, the engineered host cells are lysed followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin.

[0034] In certain embodiments, the curcumin reductase enzyme is purified from the cell lysate for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the curcumin reductase enzyme is immobilized on a solid support for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the curcumin reductase enzyme is in solution for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the cell-free medium further comprises a buffer, curcumin or demethoxycurcumin, cell lysate, glucose, GDH, and / or water. In certain embodiments, the buffer is a phosphate buffer. In certain embodiments, the reaction is conducted in a bubble column reactor, wherein the curcumin reductase enzyme is in solution. In certain embodiments, the reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

[0035] In certain embodiments, the cell-free medium for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin from curcumin or demethoxycurcumin, respectively, may further Attorney Docket No. DEBU-037 / 01WO 37396 / 185 comprise any other additional ingredients required for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin.

[0036] In certain embodiments, the cell-free medium comprises buffer, cell lysate, glucose, an organic solvent, a buffer, a detergent, and water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for enzymatic conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin. In certain embodiments, the detergent is Tween-80. In certain embodiments, the detergent is Brij-35. In certain embodiments, the buffer maintains the pH of about 4 to about 9 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 4.5 to about 9.5 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 4.5 to about 9 in the reaction mixture. In certain embodiments, the buffer maintains the pH of about 6 to about 8 in the reaction mixture. In certain embodiments, the buffer maintains the pH in the range of 6 to 8 in the reaction mixture. In certain embodiments, the buffer is a phosphate buffer. In certain embodiments, the buffer is a citrate buffer. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 600 mM. In certain embodiments, the buffer is present at a concentration of about 10 mM to about 500 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 100 mM.

[0037] In certain embodiments, the cell-free reaction medium comprises glucose at a concentration of about 1 mM to about 500 mM. In certain embodiments, the cell-free reaction medium comprises an organic solvent. In certain embodiments, the organic solvent in the cell- free reaction medium is selected from DMSO, isopropanol, ethanol, THF, MeTHF, and combinations thereof. In certain embodiments, the organic solvent in the cell-free reaction medium is present at a concentration of 0-50 vol%. In certain embodiments, sucrose is present at a concentration of about 20 mM to about 800 mM in the cell-free reaction medium. In certain embodiments, sucrose is present at a concentration of about 50 mM to about 600 mM in the cell- free reaction medium.

[0038] In certain embodiments, the quantity of the curcumin reductase enzyme for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is dependent on the target quantity of tetrahydrocurcumin or tetrahydrodemethoxycurcumin to be produced and / or the concentration of other ingredients present in the reaction mixture. In certain embodiments, the curcumin reductase enzyme is present at a concentration of about 0.01 - 200 pM. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0039] In certain embodiments, the reaction for production of tetrahydrocurcumin or tetrahydrodemethoxy curcumin is conducted for a duration of time until the desired quantity of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is obtained. In certain embodiments, the reaction for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is carried out from about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is carried out from about 1 hour to about 48 hours.

[0040] In certain embodiments, the temperature of the reaction mixture for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is varied to obtain optimal results to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the temperature of the reaction mixture for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is from about 15 °C to about 50 °C. The temperature of the reaction mixture for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin may influence the rate of production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to obtain optimal yield of tetrahydrocurcumin or tetrahydrodemethoxycurcumin in cell-free production tetrahydrocurcumin or tetrahydrodemethoxycurcumin.

[0041] In certain aspects, the methods provided in the invention may be carried out in any reactor suitable for carrying out the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the reaction for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is conducted in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column reactor / bioreactor, the curcumin reductase enzyme involved in cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is in solution. In certain embodiments, the reaction for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is conducted in a bubble column reactor / bioreactor containing the lysate from the host organism. In certain embodiments, it is advantageous to use the bubble column reactor / bioreactor for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin when the reaction mixture involves the lysate (or lysate with cellular debris removed) from the host cell organisms in which the curcumin reductase enzyme responsible for cell-free production of tetrahydrocurcumin or Attorney Docket No. DEBU-037 / 01WO 37396 / 185 tetrahydrodemeth oxy curcumin were utilized. In certain embodiments, the reaction for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxy curcumin is conducted in a packed bed reactor / bioreactor. In certain embodiments, the curcumin reductase enzyme is immobilized in the packed bed reactor / bioreactor. The packed bed react ors / bioreactors are preferred for the purified enzyme playing a role in cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the curcumin reductase enzyme may be immobilized in a single reactor / bioreactor. In certain other embodiments, the curcumin reductase enzyme may be immobilized in different reactors / bioreactors, wherein these reactors / bioreactors are linked sequentially.

[0042] Production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin in an engineered host cell

[0043] In certain aspects, the invention is a production method in a host cell of tetrahydrocurcumin or tetrahydrodemethoxycurcumin with high titer and high purity values of tetrahydrocurcumin and tetrahydrodemethoxycurcumin as compared to other conventional methods. The methods of the invention have high efficiency in conversion of curcumin to tetrahydrocurcumin or dem ethoxy curcumin to tetrahydrodemethoxycurcumin in a host cell.

[0044] In certain aspects, the invention provides a method for production of tetrahydrocurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin through one or more intermediates.

[0045] In certain aspects, the invention provides a method for production of tetrahydrodemethoxycurcumin in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrodemethoxycurcumin through one or more intermediates.

[0046] In certain embodiments, the substrate is curcumin. In certain embodiments, the substrate is demethoxycurcumin. In certain embodiments, the one or more genetic modifications comprise expression and / or overexpression of a curcumin reductase enzyme. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is cultured in a medium comprising curcumin. In certain embodiments, the engineered host cell comprises further genetic modifications for production of curcumin. In certain embodiments, one or more genetic modifications comprise overexpression of a curcumin reductase enzyme. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0047] In certain embodiments, the substrate is ferulic acid. In certain embodiments, the substrate is coumaric acid. In certain embodiments, the substrate is a combination of ferulic acid and coumaric acid. In certain embodiments, the substrate is a feedstock such as glucose or glycerol.

[0048] In certain aspects, the invention provides methods of production of tetrahydrocurcumin from curcumin in an engineered host cell. In certain embodiments, curcumin is transformed to tetrahydrocurcumin by an enzyme. In certain aspects, the invention provides methods of production of tetrahydrodemethoxycurcumin from demethoxycurcumin. In certain embodiments, the enzyme is a curcumin reductase (CurA). In certain embodiments, the curcumin reductase enzyme is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO 1. In certain embodiments, the curcumin reductase enzyme is selected from enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO 1. In certain embodiments, the curcumin reductase enzyme is selected from enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO 1. In certain embodiments, the curcumin reductase enzyme is the enzymes provided in SEQ ID NO 1

[0049] In certain embodiments, the engineered host cell is selected from the group consisting of bacteria, yeast, and / or fungal cells. In certain embodiments, the one or more enzymes are introduced in the engineered host cell by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the engineered host cell.

[0050] In certain embodiments, the engineered host cells are cultured in a medium. In certain embodiments, the medium in which engineered host cells are cultured may include one or more ingredients beneficial to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the medium may comprise tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In various aspects, host cells may be engineered for enhanced production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or Attorney Docket No. DEBU-037 / 01WO 37396 / 185 improving production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin can be increasing yield, titer, purity, or rate of production.

[0051] In certain embodiments, the curcumin for production of tetrahydrocurcumin or the demeth oxy curcumin for production of tetrahydrodemethoxycurcumin is produced in the engineered host cell. The pathways for generation of curcumin and demethoxycurcumin are also disclosed herein.

[0052] In certain aspects, the invention provides a method for production of tetrahydrocurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin through one or more intermediates.

[0053] In certain aspects, the invention provides a method for production of tetrahydrodemethoxycurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrodemethoxycurcumin through one or more intermediates.

[0054] In certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to precursors or cofactors to produce tetrahydrocurcumin. In certain embodiments, the one or more intermediates are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, and curcumin. In certain embodiments, one or more genetic modifications are selected from overexpression of a ligase (e.g. feruloyl-CoA synthase), a diketide-CoA synthase, a curcumin synthase, or a curcumin reductase.

[0055] In certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to precursors or cofactors to produce tetrahydrodemethoxycurcumin. In certain embodiments, the one or more intermediates are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, coumaric acid, coumaroyl-CoA, coumaroyl-diketide- CoA, and demethoxycurcumin. In certain embodiments, one or more genetic modifications are selected from overexpression of a ligase (e.g. feruloyl-CoA synthetase), a diketide-CoA synthase, a curcumin synthase, or a curcumin reductase.

[0056] In certain embodiments, a feedstock is transformed to ferulic acid by enzymatic transformation through one or more enzymes (e.g. shikimate pathway). In certain embodiments, ferulic acid is transformed to feruloyl-CoA. In certain embodiments, the transformation of ferulic acid to feruloyl-CoAis mediated by a ligase (e.g. feruloyl-CoA synthetase). In certain Attorney Docket No. DEBU-037 / 01WO 37396 / 185 embodiments, the feruloyl-CoA is transformed to feruloyl-diketide-CoA. In certain embodiments, the transformation of feruloyl-CoAto feruloyl-diketide-CoAis mediated by a diketide-CoA synthase. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin. In certain embodiments, the transformation of feruloyl-diketide-CoA to curcumin is mediated by a curcumin synthase. In certain embodiments, curcumin is transformed to tetrahydrocurcumin. In certain embodiments, the transformation of curcumin to tetrahydrocurcumin is mediated by a curcumin reductase. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts.

[0057] In certain embodiments, a feedstock is transformed to ferulic acid by enzymatic transformation through one or more enzymes (e.g. shikimate pathway). In certain embodiments, ferulic acid is transformed to feruloyl-CoA. In certain embodiments, the transformation of ferulic acid to feruloyl-CoA is mediated by a ligase (e g. feruloyl-CoA synthetase). In certain embodiments, the feruloyl-CoA is transformed to feruloyl-diketide-CoA. In certain embodiments, the transformation of feruloyl-CoA to feruloyl-diketide-CoAis mediated by a diketide-CoA synthase.

[0058] In certain embodiments, a feedstock is transformed to coumaric acid by enzymatic transformation through one or more enzymes (e.g. shikimate pathway). In certain embodiments, coumaric acid is transformed to coumaroyl-CoA. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoAis mediated by a ligase (e.g. 4-coumarate-CoA ligase). In certain embodiments, the coumaroyl-CoA is transformed to coumaroyl-diketide-CoA. In certain embodiments, the transformation of coumaroyl-CoA to coumaroyl-diketide-CoAis mediated by a diketide-CoA synthase.

[0059] In certain embodiments, feruloyl-diketide-CoA and coumaroyl-diketide-CoA are transformed to demethoxycurcumin. In certain embodiments, the transformation of feruloyl- diketide-CoA and coumaroyl-diketide-CoA to demethoxycurcumin is mediated by a curcumin synthase. In certain embodiments, demethoxycurcumin is transformed to tetrahydrodemethoxycurcumin. In certain embodiments, the transformation of demethoxycurcumin to tetrahydrodem ethoxytcurcumin is mediated by a curcumin reductase. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0060] In certain embodiments, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of tetrahydrocurcumin and / or tetrahydrodemethoxy curcumin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of tetrahydrocurcumin and / or tetrahydrodemethoxy curcumin is through enzymatic transformation. In certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to precursors or cofactors for production of tetrahydrocurcumin and / or tetrahydrodemethoxycurcumin. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more nonnative genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is E. coll.

[0061] In certain embodiments, one or more substrates are transformed to ferulic acid. In certain embodiments, the one or more substrates are transformed to coumaric acid. In certain embodiments, the one or more substrates comprise ferulic acid. In certain embodiments, the one or more substrates comprise feruloyl-CoA. In certain embodiments, the one or more substrates comprise feruloyl-diketide-CoA. In certain embodiments, the one or more substrates comprise feruloyl-diketide-CoA. In certain embodiments, the one or more substrates comprise curcumin. In certain embodiments, the one or more substrates comprise coumaric acid. In certain embodiments, the one or more substrates comprise demethoxycurcumin. In certain embodiments, the one or more substrates comprise coumaroyl-CoA. In certain embodiments, the one or more substrates comprise coumaroyl-diketide CoA. In certain embodiments, the one or more substrates comprise demethoxycurcumin.

[0062] In certain embodiments, the methods of the invention include that a feedstock is transformed to ferulic acid. In certain embodiments, a feedstock is transformed to ferulic acid in an engineered host cell. In certain embodiments, a feedstock is transformed to ferulic acid through enzymatic transformation. In certain embodiments, a feedstock is transformed to ferulic acid by a genetically engineered and / or optimized enzyme or enzymes. In certain embodiments, Attorney Docket No. DEBU-037 / 01WO 37396 / 185 the one or more enzymes responsible for transformation of a feedstock to ferulic acid belong to the shikimate pathway.

[0063] In certain embodiments, the invention provides that ferulic acid is transformed to feruloyl-CoA. In certain embodiments, ferulic acid is transformed to feruloyl-CoA in an engineered host cell. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through enzymatic transformation. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of ferulic acid to feruloyl-CoAis done by a ligase. In certain embodiments, the transformation of ferulic acid to feruloyl-CoAis done by a ligase. In certain embodiments, the ligase transforming ferulic acid to feruloyl-CoAis a feruloyl-CoA synthetase (FCS). In certain embodiments, the ligase is engineered to optimize transformation of ferulic acid to feruloyl-CoA. In certain embodiments, the ligase is engineered to optimize transformation of ferulic acid to feruloyl-CoA is a feruloyl- CoA synthetase (FCS). In certain preferred embodiments, feruloyl-CoA synthetase (FCS) enzymes used for microbial conversion of ferulic acid to feruloyl-CoA are provided in Table 2.

[0064] Table 2

[0065] Feruloyl-CoA synthetase (FCS) enzymes

[0066] In certain embodiments, feruloyl-CoA synthetase (FCS) is a genetically modified enzyme. In certain embodiments, FCS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from the enzymes provided in SEQ ID NOS: 12-17.

[0067] In certain embodiments, the invention provides that feruloyl-CoA is transformed to feruloyl-diketide-CoA. In certain embodiments, feruloyl-CoA is transformed to feruloyl- diketide-CoA in an engineered host cell. In certain embodiments, feruloyl-CoA is transformed to Attorney Docket No. DEBU-037 / 01WO 37396 / 185 feruloyl-diketide-CoA through enzymatic transformation. In certain embodiments, feruloyl-CoA is transformed to feruloyl-diketide-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of feruloyl-CoAto feruloyl-diketide-CoAis done by a diketide- CoA synthase (DCS). In certain embodiments, the diketide-CoA synthase (DCS) is engineered to optimize transformation of feruloyl-CoAto feruloyl-diketide-CoA. In certain preferred embodiments, diketide-CoA synthase (DCS) enzymes used for microbial conversion of feruloyl- CoAto feruloyl-diketide-CoA are provided in Table 3.

[0068] Table 3 Diketide-CoA synthase (DCS) enzymes

[0069] In certain embodiments, diketide-CoA synthase (DCS) is a genetically modified enzyme. In certain embodiments, DCS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes provided in SEQ ID NOS: 8-11.

[0070] In certain embodiments, the invention provides that feruloyl-diketide-CoA is transformed to curcumin. In certain embodiments, feruloyl-diketide-CoAis transformed to curcumin in an engineered host cell. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin through enzymatic transformation. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin through a genetically engineered enzyme. In certain embodiments, the transformation of feruloyl-diketide-CoAto curcumin is done by a curcumin synthase (CurS).In certain embodiments, the curcumin synthase (CurS) is engineered to optimize transformation of feruloyl-diketide-CoA to curcumin.

[0071] Table 4

[0072] Curcumin synthase (CurS) enzymes Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0073] In certain preferred embodiments, curcumin synthase (CurS) enzymes used for microbial conversion of feruloyl-diketide-CoA to curcumin are provided in Table 4. In certain embodiments, curcumin synthase (CurS) is a genetically modified enzyme. In certain embodiments, CurS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes provided in SEQ ID NOS: 2-7.

[0074] In certain embodiments, the methods of the invention include that a feedstock is transformed to coumaric acid. In certain embodiments, a feedstock is transformed to coumaric acid in an engineered host cell. In certain embodiments, a feedstock is transformed to coumaric acid through enzymatic transformation. In certain embodiments, a feedstock is transformed to coumaric acid by a genetically engineered and / or optimized enzyme or enzymes. In certain embodiments, the one or more enzymes responsible for transformation of a feedstock to coumaric acid belong to the shikimate pathway.

[0075] In certain embodiments, the invention provides that coumaric acid is transformed to coumaroyl-CoA. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA in an engineered host cell. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA through enzymatic transformation. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoA is done by a ligase. In certain embodiments, the ligase transforming coumaric acid to coumaroyl-CoA is a 4-coumarate-CoA ligase (4CL). In certain embodiments, the ligase is engineered to optimize transformation of coumaric acid to coumaroyl-CoA. In certain embodiments, the ligase engineered to optimize transformation of coumaric acid to coumaroyl-CoA is a 4-coumarate-CoA ligase (4CL). In certain preferred Attorney Docket No. DEBU-037 / 01WO 37396 / 185 embodiments, 4-coumarate-CoA ligase (4CL) enzymes used for microbial conversion of coumaric acid to coumaroyl-CoA are provided in Table 5.

[0076] Table 5 4-coumarate-CoA ligase (4CL) enzymes

[0077] In certain embodiments, 4-coumarate-CoA ligase (4CL) is a genetically modified enzyme. In certain embodiments, 4CL is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26. In certain embodiments, 4CL is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26. In certain embodiments, 4CL is selected from the enzymes provided in SEQ ID NOS: 18-26.

[0078] In certain embodiments, the invention provides that coumaroyl-CoA is transformed to coumaroyl-diketide-CoA. In certain embodiments, coumaroyl-CoAis transformed to coumaroyl- diketide-CoA in an engineered host cell. In certain embodiments, coumaroyl-CoA is transformed to coumaroyl-diketide-CoA through enzymatic transformation. In certain embodiments, coumaroyl-CoA is transformed to coumaroyl-diketide-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of coumaroyl-CoA to coumaroyl-diketide- CoA is done by a diketide-CoA synthase (DCS). In certain embodiments, the diketide-CoA synthase (DCS) is engineered to optimize transformation of coumaroyl-CoA to coumaroyl- diketide-CoA.

[0079] In certain preferred embodiments, diketide-CoA synthase (DCS) enzymes used for microbial conversion of coumaroyl-CoA to coumaroyl-diketide-CoA are provided in Table 3. In certain embodiments, diketide-CoA synthase (DCS) is a genetically modified enzyme. In certain embodiments, DCS is selected from enzymes having at least 80% amino acid sequence identity Attorney Docket No. DEBU-037 / 01WO 37396 / 185 from the enzymes provided in SEQ ID NOS: 8-11 . In certain embodiments, DCS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes provided in SEQ ID NOS: 8-11.

[0080] In certain embodiments, the invention provides that feruloyl-diketide-CoA and coumaroyl-diketide-CoA are transformed to demethoxy curcumin. In certain embodiments, feruloyl-diketide-CoA and coumaroyl-diketide-CoA are transformed to demethoxycurcumin in an engineered host cell. In certain embodiments, feruloyl-diketide-CoA and coumaroyl-diketide- CoA are transformed to demethoxycurcumin through enzymatic transformation. In certain embodiments, feruloyl-diketide-CoA and coumaroyl-diketide-CoA are transformed to demethoxycurcumin through a genetically engineered enzyme. In certain embodiments, the transformation of feruloyl-diketide-CoA and coumaroyl-diketide-CoA to demethoxycurcumin is done by a curcumin synthase (CurS). In certain embodiments, the curcumin synthase (CurS) is engineered to optimize transformation of feruloyl-diketide-CoA and coumaroyl-diketide-CoAto demethoxycurcumin.

[0081] In certain preferred embodiments, curcumin synthase (CurS) enzymes used for microbial conversion of feruloyl-diketide-CoA and coumaroyl-diketide-CoAto demethoxycurcumin are provided in Table 4. In certain embodiments, curcumin synthase (CurS) is a genetically modified enzyme. In certain embodiments, CurS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes provided in SEQ ID NOS: 2-7.

[0082] In certain embodiments, the invention provides a method for manufacturing tetrahydrocurcumin through one or more of the following steps:

[0083] (a) formation of ferulic acid from a feedstock;

[0084] (b) transformation of ferulic acid to feruloyl-CoA;

[0085] (c) transformation of feruloyl-CoA to feruloyl-diketide-CoA;

[0086] (d) transformation of feruloyl-diketide-CoAto curcumin; and

[0087] (e) transformation of curcumin to tetrahydrocurcumin. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0088] In certain embodiments, one or more steps of (a)-(e) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a)-(e) are engineered enzymes.

[0089] In certain embodiments, one or more steps (a)-(e) are conducted in an engineered host cell. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrocurcumin. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a)-(e).

[0090] In certain embodiments, steps (a)-(d) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a)-(d). In these embodiments, step (e) is optionally conducted in a cell-free medium. When step (e) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (e) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrocurcumin.

[0091] In certain embodiments, one or more steps (a)-(d) are conducted in an engineered host cell, and step (e) is conducted in a cell-free medium. In certain embodiments, step (e) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrocurcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a)-(e).

[0092] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a)-(e). In these embodiments, step (e) is optionally carried out in a cell-free medium.

[0093] In certain embodiments, the invention provides a method for manufacturing tetrahydrodem ethoxy curcumin through one or more of the following steps:

[0094] (al) formation of ferulic acid from a feedstock;

[0095] (bl) transformation of ferulic acid to feruloyl-CoA, and coumaric acid to coumaroyl- CoA;

[0096] (cl) transformation of feruloyl-CoA to feruloyl-diketide-CoA;

[0097] (dl) transformation of feruloyl-diketide-CoA and coumaroyl-CoA to demethoxycurcumin; and Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0098] (el) transformation of demeth oxy curcumin to tetrahydrodemethoxycurcumin.

[0099] In certain embodiments, one or more steps of (al)-(el) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (al)-(el) are engineered enzymes.

[0100] In certain embodiments, steps (al)-(dl) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (al)-(dl). In these embodiments, step (el) is optionally conducted in a cell-free medium. When step (el) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (el) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrodemethoxycurcumin.

[0101] In certain embodiments, one or more steps (al)-(el) are conducted in an engineered host cell. In certain embodiments, one or more steps (al)-(dl) are conducted in an engineered host cell, and step (el) is conducted in a cell-free medium. In certain embodiments, step (el) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrodemethoxycurcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (al)-(el).

[0102] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (al)-(el). In these embodiments, step (el) is optionally carried out in a cell-free medium.

[0103] In certain embodiments, the invention provides a method for manufacturing tetrahydrodemethoxycurcumin through one or more of the following steps:

[0104] (a2) formation of coumaric acid from a feedstock;

[0105] (b2) transformation of coumaric acid to coumaroyl-CoA, and ferulic acid to feruloyl- CoA;

[0106] (c2) transformation of coumaroyl-CoA to coumaroyl-diketide-CoA;

[0107] (d2) transformation of feruloyl -CoA and coumaroyl-diketide-CoA to demethoxycurcumin; and

[0108] (e2) transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0109] In certain embodiments, one or more steps of (a2)-(e2) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a2)-(e2) are engineered enzymes.

[0110] In certain embodiments, steps (a2)-(d2) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a2)-(d2). In these embodiments, step (e2) is optionally conducted in a cell-free medium. When step (e2) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (e2) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrodemethoxycurcumin.

[0111] In certain embodiments, one or more steps (a2)-(e2) are conducted in an engineered host cell. In certain embodiments, one or more steps (a2)-(d2) are conducted in an engineered host cell, and step (e2) is conducted in a cell-free medium. In certain embodiments, step (el) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrodemethoxycurcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a2)-(e2).

[0112] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a2)-(e2). In these embodiments, step (e2) is optionally carried out in a cell-free medium.

[0113] In certain embodiments, the engineered host cell is selected from a group consisting of bacteria, yeast, and / or fungal cells. In certain embodiments, one or more enzymes are introduced in the engineered host cell by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the engineered host cell. In certain embodiments, the engineered host cells are cultured in a medium. In certain embodiments, the medium in which engineered host cells are cultured may include one or more ingredients beneficial to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In various aspects, host cells may be engineered for enhanced production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve Attorney Docket No. DEBU-037 / 01WO 37396 / 185 cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin can increase yield, titer, purity, or rate of production.

[0114] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

[0115] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1.

[0116] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 1.

[0117] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1.

[0118] In some embodiments, the curcumin reductase comprises an amino acid sequence identical to SEQ ID NO: 1.

[0119] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0120] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0121] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0122] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0123] In some embodiments, the curcumin synthase comprises an amino acid sequence identical to or SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NO:7.

[0124] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0125] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0126] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0127] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0128] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence identical to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0129] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0130] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0131] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0132] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0133] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence identical to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0134] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0135] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0136] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0137] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0138] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0139] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:27.

[0140] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:27.

[0141] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:27.

[0142] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:27.

[0143] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence identical to SEQ ID NO:27.

[0144] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0145] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0146] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0147] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0148] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence identical to SEQ ID NO:28 or SEQ ID NO:29.

[0149] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:30. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0150] In some embodiments, the caffeic acid O-methyltransf erase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:30.

[0151] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:30.

[0152] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:30.

[0153] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence identical to SEQ ID NO: 30.

[0154] BRIEF DESCRIPTION OF THE DRAWINGS

[0155] FIG. 1 provides an overview of bioproduction of tetrahydrocurcumin (THCC) in cellbased and cell-free systems. Dashed lines box indicates steps that are operative when feeding ferulic acid.

[0156] FIG. 2 provides an HPLC chromatogram of tetrahydrocurcumin obtained by a cell-based process (THCC generation in strains). Experiments performed as described in Example 2. The curcuminoid products are 98% THCC.

[0157] FIG. 3 provides HPLC chromatograms of tetrahydrocurcumin obtained by a cell-free process from curcumin and turmeric extract as starting materials. Experiments performed as described in Example 1.

[0158] FIG. 4 provides an overview of bioproduction of tetrahydrodemethoxycurcumin (THDCC) in cell-based and cell-free systems. Dashed lines box indicates steps that are operative when feeding ferulic acid and coumaric acid.

[0159] FIG. 5 provides an HPLC chromatogram of tetrahydrodemethoxycurcumin obtained by a cell-based process. Experiments performed as described in Example 2.

[0160] FIG. 6 provides an HPLC chromatogram of tetrahydrodemethoxycurcumin obtained by a cell-free process (conversion of demethoxycurcumin to THDCC cell-free). Experiments performed as described in Example 1.

[0161] FIG. 7 provides an example of production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin obtained by a cell-based process. Experiments performed as described in Example 2. Production of THDCC with strain 1 using a ferulic acid (FA) and coumaric acid (CA) co-feed in a shake flask over 48 hours resulted in 450 pM THDCC. Strain 2 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 can produce 870 pM THCC as a sole product with a FA feed. HPLC chromatograms show production of specified products.

[0162] FIG. 8 provides a schematic representation of pathways for bioproduction of tetrahydrocurcumin and tetrahydrodemethoxycurcumin in a cell-based system. THCC and THDCC can be synthesized in E. coli through an engineered pathway utilizing ferulic acid (FA) alone or in combination with coumaric acid (CA), respectively. Both compounds use the same enzymes, a CoA ligase (4CL / FCS), diketide-CoA synthase (DCS), curcumin synthase (CurS) and curcumin reductase (CurA) to generate product.

[0163] FIG. 9 provides a schematic representation of bioproduction of tetrahydrocurcuminoids in a cell-free system. Bio-based mixtures of curcuminoids (curcumin, demethoxy curcumin, bisdem ethoxy curcumin) can be obtained from extracts from the turmeric plant (Curcuma longa) and enzymatically converted to their respective tetrahydrocurcuminoids.

[0164] FIG. 10 provides a schematic of bioproduction of tetrahydrocurcuminoids in a cell-free system. Experiments performed as described in Example 1. Curcumin reductase (CurA) rapidly converts all three curcuminoid species to their respective tetrahydrocurcuminoid analogs.

[0165] FIG. 11 provides examples of cell-based production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin, in engineered strains. Experiments performed as described in Example 3. De novo production of THCC and THDCC from glycerol in engineered strains. Product distribution can be altered by changing the levels of the different pathway enzymes.

[0166] DETAILED DESCRIPTION

[0167] The present application provides compositions and methods for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. One or more of the steps of the methods of the invention may be conducted in a cell-free medium, wherein one or more enzymes in a cell- free medium, wherein the one or more enzymes result in transformation of an organic material to tetrahydrocurcumin or tetrahydrodemethoxycurcumin. The one or more enzymes may be engineered. The engineered enzyme may be non-naturally occurring.

[0168] The term “non-naturally occurring”, when used in reference to an enzyme is intended to mean that nucleic acids or polypeptides include at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids, and polypeptides can be referred to as “wild-type” or “original”. A host cell, organism, or Attorney Docket No. DEBU-037 / 01WO 37396 / 185 microorganism that includes at least one genetic modification generated by human intervention can also be referred to as “non-naturally occurring”, “engineered”, “genetically engineered,” or “recombinant”.

[0169] As used herein, the singular forms “a,” “an,” and “the” are intended to include the plural forms as well, unless the context clearly indicates otherwise. Furthermore, to the extent that the terms “including,” “includes,” “having,” “has,” “with,” or variants thereof are used in either the detailed description and / or the claims, such terms are intended to be inclusive in a manner similar to the term “comprising.”

[0170] As used herein, “reaction solution” may refer to all components necessary for enzymebased chemical transformation. This is typically, but not limited to, buffering agent, salts, cofactor, and substrate (starting material).

[0171] As used herein, “reaction mixture” may refer to all components from the “reaction solution” plus the enzyme(s) and / or products from the reaction. In some embodiments, the “reaction mixture” may refer to just the reaction solution without any enzymes or reaction products. In some embodiments, “reaction solution” and “reaction mixture” may be used interchangeably.

[0172] As used herein, “buffering agents” may refer to chemicals added to water-based solutions that resist changes in pH by the action of acid-base conjugate components.

[0173] As used herein, “cofactors” may refer to a non-protein chemical compound that may bind to a protein and assist with a biological chemical reaction. Non-limiting examples of cofactors may include but are not limited to NADPH and NADH.

[0174] A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to Attorney Docket No. DEBU-037 / 01WO 37396 / 185 under-express or to have reduced expression of a gene, nucleic acid sequence, or to underexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Specifically included are gene disruptions, which include any insertions, deletions, or sequence mutations into or of the gene or a portion of the gene that affect its expression or the activity of the encoded polypeptide. Gene disruptions include “knockout” mutations that eliminate expression of the gene. Modifications to under-express a gene also include modifications to regulatory regions of the gene that can reduce its expression.

[0175] In the cell-free systems described herein, the critical components of the cell, namely cofactors and enzymes, are used in a chemical reaction without cellular components that can directly or indirectly inhibit the desired biochemical reaction. The same enzymes found in plants and other organisms may be created in vivo (typically through protein overexpression in hosts such as bacteria), isolated via chromatography and / or any other methods, and then added into a bioreactor with a substrate (starting material). The enzymes may also be used directly from plants without any isolation. The enzymes transform the substrate in the same way that occurs in the original organism without the organism’s complexity. Additionally, the biochemical reaction may be enhanced by the addition of co-solvents, detergents, or both, which would not be tolerated by, or simply would not work in a whole cell-based manufacturing method. In this way, natural products can be created without the plant, cell, or chemical synthesis.

[0176] The term “exogenous” or “heterologous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material that may be introduced on a vehicle such as a plasmid. Therefore, the term “endogenous” refers to a referenced molecule or activity that is naturally present in the host.

[0177] Genes or nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if Attorney Docket No. DEBU-037 / 01WO 37396 / 185 desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[0178] The percent identity (% identity) between two sequences is determined when sequences are aligned for maximum homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Omega, and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide or amino acid sequence similarity or identity and can be useful in identifying orthologs of genes of interest. Additional sequences added to a polypeptide sequence, such as but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity.

[0179] Tetrahydrocurcumin

[0180] Tetrahydrocurcumin has been isolated from Curcuma wenyujin. The chemical structure of tetrahydrocurcumin is provided below:

[0181] Tetrahydrocurcumin exists as keto-enol tautomeric isomers, which means that it rapidly converts between structural isomers. The keto-enol equilibrium is influenced by many factors, including: intra- and intermolecular interactions, solvent effects, physical state, hydrogen bond formation, and temperature.

[0182] The conventional methods of synthesis of tetrahydrocurcumin are resource intensive and lead to poor yields. Thus, these processes are economically inefficient.

[0183] Tetrahydrodemethoxycurcumin

[0184] Tetrahydrodemethoxy curcumin is a derivative of curcumin, structurally similar to tetrahydrocurcumin but lacking one methoxy group. The chemical structure of tetrahydrodemethoxycurcumin is provided below: Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0185] Tetrahydrodemethoxycurcumin also exists as keto-enol tautomeric isomers. The ketoenol equilibrium is influenced by many factors, including: intra- and intermolecular interactions, solvent effects, physical state, hydrogen bond formation, and temperature.

[0186] The invention provides methods and compositions for efficient production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin.

[0187] The invention provides novel methods for bioproduction of tetrahydrocurcumin and tetrahydrodemethoxycurcumin. The methods of the invention are economic and reliable as compared to existing methods of production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin. In addition, the methods provided in the invention provide higher titer and / or yield values of tetrahydrocurcumin and tetrahydrodemethoxycurcumin from these processes as compared to tetrahydrocurcumin and tetrahydrodemethoxycurcumin produced by existing methods.

[0188] Production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin

[0189] In certain aspects, the invention provides a method for production of tetrahydrocurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin through one or more intermediates.

[0190] In certain aspects, the invention provides a method for production of tetrahydrodemethoxycurcumin, in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrodemethoxycurcumin through one or more intermediates.

[0191] In one aspect, the invention provides a method for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin, wherein the method comprises providing an enzyme in a reaction medium, wherein the enzyme results in transformation of a substrate to tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the substrate to produce tetrahydrocurcumin is curcumin. In certain embodiments, the substrate to produce tetrahydrodemethoxycurcumin is demethoxycurcumin. In certain embodiments, the enzyme is a curcumin reductase enzyme.

[0192] Thus, the methods of the invention provide that tetrahydrocurcumin or tetrahydrodemethoxycurcumin may be produced in an engineered host cell or in a cell-free medium. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0193] In some embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxy curcumin has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0194] In other embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxy curcumin has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

[0195] Schematic representations of the cell-based and cell-free processes to form tetrahydrocurcumin and tetrahydrodemethoxycurcumin are provided in FIGS. 1, 4, 8, and 9.

[0196] Cell-free production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin

[0197] In certain aspects, the invention provides methods for cell-free production of tetrahydrocurcumin, wherein the method comprises providing an enzyme in a cell-free medium, wherein the enzyme results in transformation of one or more substrates to tetrahydrocurcumin. In other aspects, the invention provides methods for cell-free production of tetrahydrodemethoxycurcumin, wherein the method comprises providing an enzyme in a cell- free medium, wherein the enzyme results in transformation of one or more substrates to tetrahydrodemethoxycurcumin.

[0198] In certain embodiments, the enzyme is a curcumin reductase enzyme resulting in transformation of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxy curucmin. In certain embodiments, the curcumin reductase is engineered for efficient transformation of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxy curucmin.

[0199] In certain aspects, the invention provides methods for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin, wherein the method comprises providing a curcumin reductase enzyme in a cell-free medium, wherein the curcumin reductase enzyme results in transformation of one or more substrates to tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the substrate is curcumin. In certain embodiments, the substrate is demethoxycurcumin. In certain embodiments, the enzyme is a Attorney Docket No. DEBU-037 / 01WO 37396 / 185 curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is a curcumin reductase enzyme engineered for conversion of curcumin to tetrahydrocurcumin. In certain embodiments, the curcumin reductase enzyme is a curcumin reductase enzyme engineered for conversion of demethoxycurcumin to tetrahydrodemethoxycurcumin. In certain embodiments, the curcumin reductase enzyme is a curcumin reductase enzyme engineered for conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin.

[0200] In certain embodiments, the reaction medium comprises NADPH and / or NADH. In certain embodiments, the reaction medium further comprises components for regeneration of NADPH and / or NADH. In certain embodiments, the reaction medium further comprises glucose. In certain embodiments, the reaction medium further comprises glucose dehydrogenase (GDH). In certain embodiments, the glucose dehydrogenase (GDH) is purified. In certain embodiments, the glucose dehydrogenase (GDH) is in a lysate. In certain embodiments, the glucose dehydrogenase (GDH) is immobilized. In certain embodiments, the glucose dehydrogenase (GDH) is in a whole cell. In certain embodiments, the reaction medium is agitated to introduce oxygen in the reaction medium. In certain embodiments, the reaction medium further comprises oxygen. In certain embodiments, the curcumin reductase enzyme is produced in an engineered host cell. In certain embodiments, the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells. In certain embodiments, one or more enzymes are introduced in the host cell by integration into the genome of the host cell or on a plasmid. In certain embodiments, the engineered host cell expressing the one or more enzymes is cultured until a pre-determined biomass is achieved to produce the requisite quantity of the curcumin reductase enzyme.

[0201] In certain embodiments, the method further comprises lysing of the engineered host cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, one or more enzymes are purified from the cell lysate for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, one or more enzymes are immobilized on a solid support for cell-free production of tetrahydrocurcumin from curcumin or tetrahydrodemethoxycurcumin from demethoxycurcumin. In certain embodiments, one or more enzymes are in a solution for cell-free production of tetrahydrocurcumin from Attorney Docket No. DEBU-037 / 01WO 37396 / 185 curcumin or tetrahydrodemethoxy from demethoxycurcumin. In certain embodiments, the cell- free medium further comprises: buffer, curcumin or demethoxycurucumin, cell lysate, glucose, oxygen, GDH, and / or water. In certain preferred embodiments, the buffer is a phosphate buffer. In certain preferred embodiments, the buffer is a citrate buffer.

[0202] In certain embodiments, the methods of the invention provide that tetrahydrocurcumin or tetrahydrodemethoxycurcumin is produced in a cell-free medium. In certain embodiments, the invention provides methods for cell-free transformation of one or more substrates to tetrahydrocurcumin or tetrahydrodemethoxycurcumin in a cell-free medium. In certain embodiments, the invention provides cell-free enzymatic transformation of one or more substrates to tetrahydrocurcumin or tetrahydrodemethoxycurcumin through one or more intermediates. In certain embodiments, the invention provides cell-free transformation of curcumin to tetrahydrocurcumin. In certain embodiments, the invention provides cell-free transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin.

[0203] In certain beneficial aspects, the invention recognizes that the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin by methods of the invention provides higher titer values of tetrahydrocurcumin or tetrahydrodemethoxycurcumin as compared to existing methods. The methods of the invention have high efficiency in conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin in a cell-free medium. Surprisingly, the curcumin reductase enzyme provided in the methods of the invention provide for high efficiency cell-free and enzymatic conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurcumin. The pre-existing methods provide only trace amounts of generation of tetrahydrocurcumin or tetrahydrodemethoxycurcumin in cell-free conversion of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. Thus, beneficially, the curcumin reductase enzyme provided herein for tetrahydrocurcumin or tetrahydrodemethoxycurcumin production leads to highly efficient and high titer generation of tetrahydrocurcumin or tetrahydrodemethoxycurcumin.

[0204] In certain embodiments, the curcumin reductase enzyme used herein is listed in Table 1. In certain embodiments, the curcumin reductase enzyme is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase enzyme is selected from enzymes having at least 95% Attorney Docket No. DEBU-037 / 01WO 37396 / 185 amino acid sequence identity from the enzyme provided in SEQ ID NO: 1 . In certain embodiments, the curcumin reductase enzyme is the enzyme provided in SEQ ID NO: 1.

[0205] In certain embodiments, the curcumin reductase enzyme used in cell-free conversion of curcumin to tetrahydrocurcumin or demethoxy curcumin to tetrahydrodemethoxycurucmin is a purified enzyme. In certain embodiments, the purified curcumin reductase enzyme for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin is immobilized and used in batch or packed bed reactors.

[0206] In certain embodiments, the curcumin reductase enzyme for the cell free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin is generated from lysing a host cell overexpressing the curcumin reductase enzyme. In certain embodiments, the lysate generated from lysing said host cell overexpressing the curcumin reductase enzyme is utilized for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin. In certain embodiments, the curcumin reductase enzyme utilized for cell-free conversion of curcumin to tetrahydrocurcumin or demethoxycurcumin to tetrahydrodemethoxycurucmin is purified from the lysate of hosts expressing the curcumin reductase enzyme.

[0207] In certain aspects, the curcumin reductase enzyme required for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is expressed in a host organism. In certain embodiments, the host organism is selected from a group consisting of: bacteria, yeast, and / or fungal cells. In certain embodiments, the one or more enzymes are introduced in the host organism by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the host organism. In certain embodiments, host organisms expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. The predetermined biomass is calculated based on the quantity of the one or more enzymes required for the cell-free production of tetrahydrocurcumin or tetrahydrodemethoxycurucmin. In certain embodiments, the culture comprising host organisms expressing the one or more enzymes are lysed and used as the reaction medium for the methods provided in the invention. In certain other embodiments, once the culture comprising host organisms is lysed, the cell-debris is removed from the lysed matter to prepare the reaction medium for the methods of the invention. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0208] In some embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxy curcumin has a purity of about 10%, or about 20%, or about 30%, or about 40%, or about 50%, or about 60%, or about 70%, or about 80%, or about 90%, or about 95%, or about 99%, or about 100%.

[0209] In other embodiments, the isolated tetrahydrocurcumin or tetrahydrodemethoxy curcumin has a purity of from about 10% to 95%, or from about 10% to 90%, or from about 10% to 80% or from about 10% to 70%, or from about 10% to 60%, or from about 10% to 50%, or from about 10% to 40%, or from about 20% to 95%, or from about 20% to 90%, or from about 20% to 80% or from about 20% to 70%, or from about 20% to 60%, or from about 20% to 50%, or from about 20% to 40%, or from about 50% to 95%, or from about 50% to 90%, or from about 50% to 80% or from about 50% to 70%, or from about 50% to 60%.

[0210] Data illustrating the cell-free production of tetrahydrocurcumin and tetrahydrodemethoxy curcumin is provided in FIGS. 3 and 6.

[0211] Production of tetrahydrocurcumin and tetrahydrodemethoxy curcumin in an engineered host cell

[0212] In certain aspects, the invention provides a method for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for conversion of a substrate to tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the substrate is curcumin. In certain embodiments, the substrate is demethoxycurcumin. In certain embodiments, the one or more genetic modifications comprise expression and / or overexpression of a curcumin reductase enzyme. In certain embodiments, the engineered host cell is E. coli.

[0213] In certain embodiments, the engineered host cell is selected from the group consisting of bacteria, yeast, and / or fungal cells. In certain embodiments, the one or more enzymes are introduced in the engineered host cell by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the engineered host cell.

[0214] In certain embodiments, the engineered host cells are cultured in a medium. In various aspects, host cells may be engineered for enhanced production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct Attorney Docket No. DEBU-037 / 01WO 37396 / 185 formation, or improve cell fitness. Enhancing or improving production of tetrahydrocurcumin or tetrahydrodemethoxy curcumin can be increasing yield, titer, or rate of production.

[0215] In certain embodiments, the curcumin for production of tetrahydrocurcumin is produced in the engineered host cell. The pathways for generation of curcumin are also disclosed herein. In certain embodiments, the demethoxycurcumin for production of tetrahydrodemethoxycurcumin is produced in the engineered host cell. The pathways for generation of curcumin are also disclosed herein.

[0216] In certain aspects, the invention provides a method for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin or tetrahydrodemethoxycurcumin through one or more intermediates. In certain embodiments, the substrate is feedstock. In certain embodiments, the substrate is ferulic acid. In certain embodiments, the substrate is coumaric acid. In certain embodiments, the substrate is a combination of ferulic acid and coumaric acid.

[0217] In certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to precursors or cofactors for production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, the one or more intermediates are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, and curcumin. In certain embodiments, one more genetic modifications are selected from overexpression of a ligase, a diketide-CoA synthase (DCS), a curcumin synthase (CurS), and a curcumin reductase (CurA). In certain embodiments, the ligase is a feruloyl-CoA synthetase (FCS). In certain embodiments, the ligase is a 4-coumarate-CoA ligase (4CL).

[0218] In certain embodiments, feedstock is transformed to ferulic acid by enzymatic transformation through one or more enzymes via the shikimate pathway. In certain embodiments, ferulic acid is transformed to feruloyl-CoA. In certain embodiments, the transformation of ferulic to feruloyl-CoA is mediated by a ligase. In certain embodiments, the feruloyl-CoA is transformed to feruloyl-diketide-CoA. In certain embodiments, the transformation of feruloyl-CoA to feruloyl-diketide-CoA is mediated by a diketide-CoA synthase (DCS). In certain embodiments, feruloyl-CoA is transformed to curcumin. In certain embodiments, the transformation of feruloyl-CoA to curcumin is mediated by curcumin synthase (CurS). In certain embodiments, curcumin is transformed to tetrahydrocurcumin. In certain embodiments, the transformation of Attorney Docket No. DEBU-037 / 01WO 37396 / 185 curcumin to tetrahydrocurcumin is mediated by a curcumin reductase (CurA). In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts.

[0219] In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the medium further comprises NADPH and / or NADH.

[0220] In certain embodiments, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of tetrahydrocurcumin or tetrahydrodemethoxy curcumin from a carbon source through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin is through enzymatic transformation. In certain embodiments, the carbon source is feedstock. In certain embodiments, the engineered host cell is cultured in a medium comprising feedstock. In certain embodiments, the one or more genetic modifications leads to an increase in metabolic flux to precursors or cofactors to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more nonnative genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is E. coli.

[0221] In certain embodiments, one or more substrates are transformed to tetrahydrocurcumin. In certain embodiments, the one or more substrates that are transformed to tetrahydrocurcumin are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, and curcumin. In certain embodiments, the one or more substrates comprise curcumin. In certain embodiments, the one or more substrates comprise feruloyl-diketide-CoA. In certain embodiments, the one or more substrates comprise feruloyl-CoA. In certain embodiments, the one or more substrates comprise ferulic acid. In certain embodiments, the one or more substrates that are transformed to tetrahydrocurcumin is a carbon source. In certain embodiments, the carbon source is feedstock.

[0222] In certain embodiments, one or more substrates are transformed to tetrahydrodemethoxycurcumin. In certain embodiments, the one or more substrates that are Attorney Docket No. DEBU-037 / 01WO 37396 / 185 transformed to tetrahydrodemethoxy curcumin are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, coumaric acid, coumaroyl-CoA, coumaroyl-diketide- CoA, and tetrahydrocurcumin. In certain embodiments, the one or more substrates comprise curcumin. In certain embodiments, the one or more substrates comprise feruloyl-diketide-CoA. In certain embodiments, the one or more substrates comprise feruloyl-CoA. In certain embodiments, the one or more substrates comprise ferulic acid. In certain embodiments, the one or more substrates that are transformed to tetrahydrocurcumin is a carbon source. In certain embodiments, the carbon source is feedstock.

[0223] In various aspects, host cells may be engineered for enhanced production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, coumaric acid, coumaroyl-CoA, coumaroyl-diketide-CoA, curcumin, and tetrahydrocurcumin can increase yield, titer, or rate of production.

[0224] In certain embodiments, the methods of the invention provide that a carbon source, such as feedstock is transformed to ferulic acid. In certain embodiments, feedstock is transformed to feedstock in an engineered host cell. In certain embodiments, feedstock is transformed to ferulic acid by a genetically engineered and / or optimized enzyme via the shikimate pathway.

[0225] In certain embodiments, the invention provides that ferulic acid is transformed to feruloyl-CoA. In certain embodiments, the ferulic acid is transformed to feruloyl-CoA in an engineered host cell. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through enzymatic transformation. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation ferulic acid to feruloyl-CoA is by a ligase. In certain embodiments, the transformation ferulic acid to feruloyl-CoA is by a feruloyl-CoA synthetase (FCS). In certain embodiments, the feruloyl-CoA synthetase (FCS) is engineered to optimize transformation of ferulic acid to feruloyl-CoA.

[0226] In certain preferred embodiments, FCS enzymes used for microbial conversion of ferulic acid to feruloyl-CoA are provided Table 2. In certain embodiments, feruloyl-CoA synthetase (FCS) is selected from enzymes having at least 80% amino acid sequence identity from the Attorney Docket No. DEBU-037 / 01WO 37396 / 185 enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from the enzymes provided in SEQ ID NOS: 12-17.

[0227] In certain embodiments, the invention provides that feruloyl-CoAis transformed to feruloyl-diketide-CoA. In certain embodiments, feruloyl-CoAis transformed to feruloyl- diketide-CoA in an engineered host cell. In certain embodiments, the transformation of feruloyl- CoA to feruloyl-diketide-CoA is through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a diketide-CoA synthase (DCS).

[0228] In certain embodiments, the DCS is a genetically engineered enzyme. In certain embodiments, the DCS enzymes is selected from Table 3. In certain embodiments, diketide-CoA synthase (DCS) is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes provided in SEQ ID NOS: 8-11.

[0229] In certain embodiments, the invention provides that feruloyl-diketide-CoA is transformed to curcumin. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin in an engineered host cell. In certain embodiments, the transformation of feruloyl-diketide-CoA to curcumin is through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a curcumin synthase (CurS).

[0230] In certain embodiments, the CurS is a genetically engineered enzyme. In certain embodiments, the CurS enzymes are selected from Table 4. In certain embodiments, curcumin synthase (CurS) is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes provided in SEQ ID NOS: 2-7.

[0231] In certain embodiments, the invention provides that curcumin is transformed to tetrahydrocurcumin. In certain embodiments, the transformation of curcumin to tetrahydrocurcumin is in an engineered host cell. In certain embodiments, curcumin is Attorney Docket No. DEBU-037 / 01WO 37396 / 185 enzymatically transformed to tetrahydrocur cumin. In certain embodiments, the enzyme is genetically engineered to optimize transformation of curcumin to tetrahydrocurcumin. In certain embodiments, the enzyme is a curcumin reductase (CurA). In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes provided in SEQ ID NO: 1.

[0232] The method of the invention for production of tetrahydrocurcumin provide that the starting material for the process could be from any economically available carbon source. The invention beneficially provides that the starting material used for manufacture is feedstock. Because the methods of the invention provide manufacture of tetrahydrocurcumin with an economic carbon source, the methods of the invention are economically efficient.

[0233] In certain embodiments, one or more substrates are transformed to tetrahydrodemethoxy curcumin. In certain embodiments, the one or more substrates that are transformed to tetrahydrodemethoxy curcumin are selected from the group consisting of ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, coumaric aid, coumaroyl-CoA, coumaroyl-diketide- CoA, and demethoxycurcumin. In certain embodiments, the one or substrates comprise demethoxycurcumin. In certain embodiments, the one or substrates comprise feruloyl-diketide- CoA. In certain embodiments, the one or more substrates comprise feruloyl-CoA. In certain embodiments, the one or more substrates comprise ferulic acid. In certain embodiments, the one or substrates comprise coumaroyl-diketide-CoA. In certain embodiments, the one or more substrates comprise coumaroyl-CoA. In certain embodiments, the one or more substrates comprise coumaric acid. In certain embodiments, the one or more substrates that are transformed to tetrahydrodemethoxycurcumin is a carbon source. In certain embodiments, the carbon source is feedstock.

[0234] In certain embodiments, the invention provides that ferulic acid is transformed to feruloyl-CoA. In certain embodiments, the ferulic acid is transformed to feruloyl-CoA in an engineered host cell. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through enzymatic transformation. In certain embodiments, ferulic acid is transformed to feruloyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation ferulic Attorney Docket No. DEBU-037 / 01WO 37396 / 185 acid to feruloyl-CoA is by a ligase. Tn certain embodiments, the transformation ferulic acid to feruloyl-CoA is by a feruloyl-CoA synthetase (FCS). In certain embodiments, the feruloyl-CoA synthetase (FCS) is engineered to optimize transformation of ferulic acid to feruloyl-CoA.

[0235] In certain preferred embodiments, FCS enzymes used for microbial conversion of ferulic acid to feruloyl-CoA are provided Table 2. In certain embodiments, feruloyl-CoA synthetase (FCS) is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17. In certain embodiments, FCS is selected from the enzymes provided in SEQ ID NOS: 12-17.

[0236] In certain embodiments, the invention provides that coumaric acid is transformed to coumaroyl-CoA. In certain embodiments, the coumaric acid is transformed to coumaroyl-CoA in an engineered host cell. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA through enzymatic transformation. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoA is by a ligase. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoA is by a 4-coumarate-CoA ligase (4CL). In certain embodiments, the 4-coumarate-CoA ligase (4CL) is engineered to optimize transformation of coumaric acid to coumaroyl-CoA.

[0237] In certain preferred embodiments, 4CL enzymes used for microbial conversion of coumaric acid to coumaroyl-CoA are provided Table 5. In certain embodiments, 4-coumarate- CoA ligase (4CL) is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26. In certain embodiments, 4CL is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26. In certain embodiments, 4CL is selected from the enzymes provided in SEQ ID NOS: 18-26.

[0238] In certain embodiments, the invention provides that feruloyl-CoAis transformed to feruloyl-diketide-CoA. In certain embodiments, feruloyl-CoA is transformed to feruloyl- diketide-CoA in an engineered host cell. In certain embodiments, the transformation of feruloyl- CoAto feruloyl-diketide-CoAis through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a diketide-CoA synthase (DCS). In certain Attorney Docket No. DEBU-037 / 01WO 37396 / 185 embodiments, the invention provides that coumaroyl-CoA is transformed to count aroyl -di ketide- CoA. In certain embodiments, coumaroyl-CoAis transformed to coumaroyl-diketide-CoA in an engineered host cell. In certain embodiments, the transformation of coumaroyl-CoA to coumaroyl-diketide-CoAis through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a diketide-CoA synthase (DCS).

[0239] In certain embodiments, the DCS is a genetically engineered enzyme. In certain embodiments, the DCS enzymes is selected from Table 3. In certain embodiments, diketide-CoA synthase (DCS) is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes provided in SEQ ID NOS: 8-11.

[0240] In certain embodiments, the invention provides that feruloyl-diketide-CoAis transformed to curcumin. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin in an engineered host cell. In certain embodiments, feruloyl-diketide-CoA is transformed to curcumin in an engineered host cell. In certain embodiments, the transformation of feruloyl-diketide-CoA to curcumin is through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a curcumin synthase (CurS). In certain embodiments, the invention provides that coumaroyl-diketide-CoA is transformed to demethoxycurcumin. In certain embodiments, coumaroyl-diketide-CoA is transformed to demethoxycurcumin in an engineered host cell. In certain embodiments, coumaroyl-diketide-CoAis transformed to demethoxycurcumin in an engineered host cell. In certain embodiments, the transformation of coumaroyl-diketide-CoAto demethoxycurcumin is through one or more genetically engineered enzymes. In certain embodiments, the genetically engineered enzyme is a curcumin synthase (CurS).

[0241] In certain embodiments, the CurS is a genetically engineered enzyme. In certain embodiments, the CurS enzymes are selected from Table 4. In certain embodiments, curcumin synthase (CurS) is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0242] ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes provided in SEQ ID NOS: 2-7.

[0243] In certain embodiments, the invention provides that demethoxycurcumin is transformed to tetrahydrodemethoxycurcumin. In certain embodiments, the transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin is in an engineered host cell. In certain embodiments, demethoxycurcumin is enzymatically transformed to tetrahydrodemethoxycurcumin. In certain embodiments, the enzyme is genetically engineered to optimize transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin. In certain embodiments, the enzyme is a curcumin reductase (CurA). In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NO: 1. In certain embodiments, the curcumin reductase (CurA) enzyme is selected from the enzymes provided in SEQ ID NO: 1.

[0244] In certain embodiments, the methods of the invention include that feedstock is transformed to coumaric acid. In certain embodiments, feedstock is transformed to coumaric acid in an engineered host cell. In certain embodiments, feedstock is transformed to coumaric acid through enzymatic transformation. In certain embodiments, feedstock is transformed to coumaric acid by a genetically engineered and / or optimized enzyme or enzymes. In certain embodiments, the one or more enzymes responsible for transformation of feedstock to coumaric acid belong to the shikimate pathway.

[0245] In certain embodiments, the invention provides that coumaric acid is transformed to coumaroyl-CoA. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA in an engineered host cell. In certain embodiments, coumaric acid is transformed to feruloyl-CoA through enzymatic transformation. In certain embodiments, coumaric acid is transformed to coumaroyl-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoA is done by a ligase. In certain embodiments, the transformation of coumaric acid to coumaroyl-CoA is done by a ligase. In certain embodiments, the ligase transforming coumaric acid to coumaroyl-CoAis a 4-coumarate-CoA ligase (4CL). In certain embodiments, the ligase is engineered to optimize transformation of coumaric acid to coumaroyl-CoA. In certain embodiments, the ligase is engineered to optimize Attorney Docket No. DEBU-037 / 01WO 37396 / 185 transformation of coumaric acid to coumaroyl-CoA is a 4-coumarate-CoA ligase (4CL). Tn certain preferred embodiments, 4-coumarate-CoA ligase (4CL) enzymes used for microbial conversion of coumaric acid to feruloyl-CoA are provided in Table 5.

[0246] In certain embodiments, 4-coumarate-CoA ligase (4CL) is a genetically modified enzyme. In certain embodiments, 4CL is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26 In certain embodiments, 4CL is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26. In certain embodiments, 4CL is selected from the enzymes provided in SEQ ID NOS: 18-26.

[0247] In certain embodiments, the invention provides that coumaroyl-CoA is transformed to coumaroyl-diketide-CoA. In certain embodiments, coumaroyl-CoAis transformed to coumaroyl- diketide-CoA in an engineered host cell. In certain embodiments, coumaroyl-CoA is transformed to coumaroyl-diketide-CoA through enzymatic transformation. In certain embodiments, coumaroyl-CoA is transformed to coumaroyl-diketide-CoA through a genetically engineered enzyme. In certain embodiments, the transformation of coumaroyl-CoA to coumaroyl-diketide- CoA is done by a diketide-CoA synthase. In certain embodiments, the transformation of coumaroyl-CoA to coumaroyl-diketide-CoAis done by a diketide-CoA synthase (DCS). In certain embodiments, the diketide-CoA synthase (DCS) is engineered to optimize transformation of coumaroyl-CoA to coumaroyl-diketide-CoA.

[0248] In certain preferred embodiments, diketide-CoA synthase (DCS) enzymes used for microbial conversion of coumaroyl-CoAto coumaroyl-diketide-CoA are provided in Table 3. In certain embodiments, diketide-CoA synthase (DCS) is a genetically modified enzyme. In certain embodiments, DCS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11 In certain embodiments, DCS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11. In certain embodiments, DCS is selected from the enzymes provided in SEQ ID NOS: 8-11.

[0249] In certain embodiments, the invention provides that feruloyl-diketide-CoA or coumaroyl- diketide-CoA is transformed to demethoxycurcumin. In certain embodiments, feruloyl-diketide- CoA or coumaroyl-diketide-CoAis transformed to demethoxycurcumin in an engineered host cell. In certain embodiments, feruloyl-diketide-CoA or coumaroyl-diketide-CoAis transformed Attorney Docket No. DEBU-037 / 01WO 37396 / 185 to demethoxycurcumin through enzymatic transformation. In certain embodiments, feruloyl- diketide-CoA or coumaroyl-diketide-CoAis transformed to demethoxycurcumin through a genetically engineered enzyme. In certain embodiments, the transformation of feruloyl-diketide- CoA or coumaroyl-diketide-CoAto demethoxycurcumin is done by a curcumin synthase. In certain embodiments, the transformation of feruloyl-diketide-CoA or coumaroyl-diketide-CoAto demethoxycurcumin is done by a curcumin synthase (CurS). In certain embodiments, the curcumin synthase (CurS) is engineered to optimize transformation of coumaroyl-diketide-CoA to demethoxycurcumin.

[0250] In certain preferred embodiments, curcumin synthase (CurS) enzymes used for microbial conversion of coumaroyl-diketide-CoAto demethoxycurcumin are provided in Table 4. In certain embodiments, curcumin synthase (CurS) is a genetically modified enzyme. In certain embodiments, CurS is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7 In certain embodiments, CurS is selected from enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7. In certain embodiments, CurS is selected from the enzymes provided in SEQ ID NOS: 2-7.

[0251] In certain embodiments, tetrahydrodemethoxycurcumin is produced in a cell-based procees by co-feeding ferulic acid and coumaric acid.

[0252] In certain embodiments, the invention provides a method for manufacturing tetrahydrocurcumin through one or more of the following steps:

[0253] (a) formation of ferulic acid from feedstock;

[0254] (b) transformation of ferulic acid to feruloyl-CoA;

[0255] (c) transformation of feruloyl-CoA to feruloyl-diketide-CoA;

[0256] (d) transformation of feruloyl-diketide-CoAto curcumin; and

[0257] (e) transformation of curcumin to tetrahydrocurcumin.

[0258] In certain embodiments, one or more steps of (a)-(e) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a)-(e) are engineered enzymes.

[0259] In certain embodiments, one or more steps (a)-(e) are conducted in an engineered host cell. In certain embodiments, the engineered host cell is genetically modified for production of Attorney Docket No. DEBU-037 / 01WO 37396 / 185 tetrahydrocurcumin Tn certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a)-(e).

[0260] In certain embodiments, steps (a)-(d) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a)-(d). In these embodiments, step (e) is optionally conducted in a cell-free medium. When step (e) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (e) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrocurcumin.

[0261] In certain embodiments, one or more steps (a)-(d) are conducted in an engineered host cell, and step (e) is conducted in a cell-free medium. In certain embodiments, step (e) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrocurcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a)-(e).

[0262] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a)-(e). In these embodiments, step (e) is optionally carried out in a cell-free medium.

[0263] In certain embodiments, the invention provides a method for manufacturing tetrahydrodem ethoxy curcumin through one or more of the following steps:

[0264] (al) formation of ferulic acid from feedstock;

[0265] (bl) transformation of ferulic acid to feruloyl-CoA;

[0266] (cl) transformation of feruloyl-CoA to feruloyl-diketide-CoA;

[0267] (dl) transformation of feruloyl-diketide-CoAto demethoxycurcumin; and

[0268] (el) transformation of demethoxycurcumin to tetrahydrodemethoxy curcumin.

[0269] In certain embodiments, one or more steps of (al)-(el) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (al)-(el) are engineered enzymes.

[0270] In certain embodiments, steps (al)-(dl) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (al)-(dl). In these embodiments, step (el) is optionally conducted Attorney Docket No. DEBU-037 / 01WO 37396 / 185 in a cell-free medium. When step (el) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (el) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrodemethoxycurcumin.

[0271] In certain embodiments, one or more steps (al)-(el) are conducted in an engineered host cell. In certain embodiments, one or more steps (al)-(dl) are conducted in an engineered host cell, and step (el) is conducted in a cell-free medium. In certain embodiments, step (el) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrodemethoxycurcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (al)-(el).

[0272] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (al)-(el). In these embodiments, step (el) is optionally carried out in a cell-free medium.

[0273] In certain embodiments, the invention provides a method for manufacturing tetrahydrodemethoxycurcumin through one or more of the following steps:

[0274] (a2) formation of coumaric acid from feedstock;

[0275] (b2) transformation of coumaric acid to coumaroyl-CoA;

[0276] (c2) transformation of coumaroyl-CoA to coumaroyl-diketide-CoA;

[0277] (d2) transformation of coumaroyl-diketide-CoAto demethoxycurcumin; and

[0278] (e2) transformation of demethoxycurcumin to tetrahydrodemethoxycurcumin.

[0279] In certain embodiments, one or more steps of (a2)-(e2) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a2)-(e2) are engineered enzymes.

[0280] In certain embodiments, steps (a2)-(d2) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a2)-(d2). In these embodiments, step (e2) is optionally conducted in a cell-free medium. When step (e2) is conducted in a cell-free medium, the step may be optionally mediated by an engineered enzyme. In certain embodiments, step (e2) is mediated by a curcumin reductase enzyme. In certain embodiments, the curcumin reductase enzyme is an engineered enzyme optimized for conversion of curcumin to tetrahydrodemethoxycurcumin. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0281] In certain embodiments, one or more steps (a2)-(e2) are conducted in an engineered host cell. In certain embodiments, one or more steps (a2)-(d2) are conducted in an engineered host cell, and step (e2) is conducted in a cell-free medium. In certain embodiments, step (el) is conducted in a cell-free medium and curcumin is sourced from crude extracts. In certain embodiments, the engineered host cell is genetically modified for production of tetrahydrodemethoxy curcumin or a precursor thereof. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a2)-(e2).

[0282] In certain embodiments, the engineered host cell expresses one or more enzymes for the transformations listed in steps (a2)-(e2). In these embodiments, step (e2) is optionally carried out in a cell-free medium.

[0283] In certain embodiments, the invention provides a method for manufacturing tetrahydrodemethoxy curcumin wherein steps al-el and a2-e2 are all happening in the cell.

[0284] In certain embodiments, the engineered host cell is selected from a group consisting of bacteria, yeast, and / or fungal cells. In certain embodiments, one or more enzymes are introduced in the engineered host cell by integration into the genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be expressed by the engineered host cell. In certain embodiments, the engineered host cells are cultured in a medium. In certain embodiments, the medium in which engineered host cells are cultured may include one or more ingredients beneficial to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin. In various aspects, host cells may be engineered for enhanced production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin can increase yield, titer, purity, or rate of production.

[0285] Engineered Host Cells

[0286] An engineered host cell as provided herein can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanob acteri al cells. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0287] Host can be selected based on their ability to take up and utilize particular carbon sources, nitrogen sources, or precursor molecules or may be engineered to take up and utilize molecules that may be added to the culture medium.

[0288] Nonlimiting examples of suitable microbial hosts for the bio-production of tetrahydrocurcumin or tetrahydrodemethoxycurcumin include, but are not limited to: any gramnegative organisms, more particularly a member of the family Enterob acteriaceae, such as E. co / i, any gram-positive microorganism, for example Bacillus subtilis, Lactobacillus sp. or Lactococcus sp. a yeast, for example Saccharomyces cerevisiae, Pichia pastoris, Pichia stipites, Yarrowia lipolytica or Kluyveromyces marxianus; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobactei; Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Yarrowia, Kluyveromyces, and Saccharomyces.

[0289] Culture Medium

[0290] In yet another aspect, methods for producing tetrahydrocurcumin or tetrahydrodemethoxycurcumin that include incubating a culture of an engineered host cell as provided herein to produce said compounds. The methods can further include recovering the products-of-interest from the culture medium, whole culture, or cells.

[0291] The culture comprises of cells engineered to produce tetrahydrocurcumin or tetrahydrodemethoxycurcumin in a culture medium. In various embodiments the engineered cells can be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source that is also an energy source.

[0292] Culture conditions can include aerobic, microaerobic or any combination alternating aerobic / microaerobic growth conditions. Further, culture conditions can include microtiter well plates, shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving tetrahydrocurcumin or tetrahydrodemethoxycurcumin products include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing. Exemplary growth procedures include, for example, batch fermentation (such as in plates and shake flasks), fed-batch fermentation, and batch separation. In an exemplary batch fermentation protocol, the cells are grown in a bioreactor that Attorney Docket No. DEBU-037 / 01WO 37396 / 185 is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds. The desired temperature can be from, for example, 15-50 °C, depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH of the bioreactor can be controlled to range from 4.5-9 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for example, 1 to 48 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. To purify tetrahydrocurcumin or tetrahydrodemethoxy curcumin to homogeneity, further processing can be done, including by solvent extraction, ion exchange or silica-based chromatography, among many others.

[0293] EXAMPLES

[0294] Example 1: Cell-free bioproduction of tetrahydrocurcumin and tetrahydrodemethoxycurcumin

[0295] Cell-free production of tetrahydrocurcumin and tetrahydrodemethoxycurcumin is performed by producing a culture of E. coli expressing the CurA enzyme. The cells are lysed, cell debris is removed, and the resulting lysate can be used directly in cell-free reactions, or the enzyme can be further purified. In some embodiments, cell-free production conditions may be as shown in Table 6. Cell-free reactions contain CurA enzyme either in lysate or purified, either curcumin or demethoxy curcumin, NADP+or NADPH, and a buffer, and may also contain an organic solvent and / or a detergent. Optionally, reactions may also contain an NADPH recycling system, which may comprise glucose and glucose dehydrogenase. Reactions are incubated for up to about 48 hours and then quenched by addition of solvent. Analysis may be performed by HPLC.

[0296] Table 6 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0297] Example 2: Cell-Based Production of Tetrahydrocurcumin from Ferulic Acid, or Tetrahydrodemethoxycurcumin from Ferulic Acid and Coumaric Acid

[0298] For the production of THCC and THDCC in microbial hosts by feeding of ferulic acid and / or coumaric acid (see dotted box in the scheme in FIG. 1 and FIG. 4), E. coll strains derived from MG1655 were engineered to overexpress the following enzymes: FCS, DCS, CURS, and CurA from the sequences provided in Tables 7 to 11 . The strains were initially grown in rich medium (e.g. LB) as a seed culture. This was then used to inoculate a defined minimal medium containing a carbon source (e.g. glucose or glycerol), trace elements, vitamins, buffering components and ferulic acid and / or coumaric acid. Culturing was performed in shake-flasks or in microtiter plates (24-well, 48-well, or 96-well) with sufficient aeration and shaking at optimal temperatures for the strains and for the production of the desired products. To analyze production, the cell cultures were extracted 1 : 1 with methanol and centrifuged for 15 minutes. The supernatants were analyzed for tetrahydrocurcumin, tetrahydrodemethoxycurcumin, curcumin, demethoxycurcumin, and other byproducts by FfPLC. The production profile is illustrated in FIG. 2, FIG. 5, and FIG. 7.

[0299] Example 3: Cell-Based Production of Tetrahydrocurcumin and Tetrahydrodemethoxycurcumin from Glucose or Glycerol

[0300] THCC and THDCC were produced de novo in a microbial host growing in glucose or glycerol (See FIG. 1 and FIG. 4). An A. coli strain derived from MG1655 was engineered to overexpress the following enzymes: TAL, C3H, COMT, FCS, DCS, CURS and CurA from the sequences provided in Tables 7 to 14. The strains were initially grown in rich medium (e.g. LB) as a seed culture. This culture was then used to inoculate a defined minimal medium containing a carbon source (e g. glucose or glycerol), trace elements, vitamins and buffering components. Culturing was performed in shake-flasks or in microtiter plates (24-well, 48-well, or 96-well) with sufficient aeration and shaking at optimal temperatures for the strains and for the production Attorney Docket No. DEBU-037 / 01WO 37396 / 185 of the desired products. To analyze production, the cells cultures were extracted 1 : 1 with methanol and centrifuged for 15 minutes. The supernatants were analyzed for tetrahydrocurcumin, tetrahydrodemethoxycurcumin, curcumin, demethoxycurcumin, and other byproducts by HPLC. The production profiles of different strains are illustrated in FIG. 11. The varying profiles (ratio of THCC, THDCC, THBDCC) observed are an effect of different enzyme variants, as well as optimization on the levels of the enzymes to tune the product composition.

[0301] Table 7 provides an exemplary sequence for a curcumin reductase (CurA) enzyme in accordance with the methods of invention.

[0302] Table 7

[0303] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 90% identity to SEQ ID NO: 1.

[0304] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 1.

[0305] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 1.

[0306] In some embodiments, the curcumin reductase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 1 .

[0307] In some embodiments, the curcumin reductase comprises an amino acid sequence identical to SEQ ID NO: 1. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0308] Table 8 provides exemplary sequences for curcumin synthase (CurS) enzymes in accordance with the methods of invention.

[0309] Table 8 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0310] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0311] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0312] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7.

[0313] In some embodiments, the curcumin synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, or SEQ ID NO:7. In some embodiments, the curcumin synthase comprises an amino acid sequence identical to or SEQ ID NO:2, SEQ ID NO:3, SEQ ID NO:4, SEQ ID NO:5, SEQ ID NO:6, SEQ ID NOY.

[0314] Table 9 provides exemplary sequences for diketide-CoA synthase (DCS) enzymes in accordance with the methods of invention.

[0315] Table 9 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0316] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0317] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0318] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0319] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:8, SEQ ID NO:9, SEQ ID NO: 10, or SEQ ID NO: 11. Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0320] In some embodiments, the diketide-CoA synthase comprises an amino acid sequence identical to SEQ ID NO:8, SEQ ID N0:9, SEQ ID NO: 10, or SEQ ID NO: 11.

[0321] Table 10 provides exemplary sequences for feruloyl-CoA synthetase (FCS) enzymes in accordance with the methods of invention.

[0322] Table 10 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0323] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0324] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 95% identity to SEQ ID NO: 12, SEQ ID NO:13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0325] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17. In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0326] In some embodiments, the feruloyl-CoA synthetase comprises an amino acid sequence identical to SEQ ID NO: 12, SEQ ID NO: 13, SEQ ID NO: 14, SEQ ID NO: 15, SEQ ID NO: 16, or SEQ ID NO: 17.

[0327] Table 11 provides exemplary sequences for 4-coumarate-CoA ligase (4CL) enzymes in accordance with the methods of invention.

[0328] Table 11 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 Attorney Docket No. DEBU-037 / 01WO 37396 / 185 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0329] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0330] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0331] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 98% identity to SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0332] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence having at least 99% identity to SEQ ID NO: 18, SEQ ID NO:19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO:26.

[0333] In some embodiments, the 4-coumarate-CoA ligase comprises an amino acid sequence identical to SEQ ID NO: 18, SEQ ID NO: 19, SEQ ID NO:20, SEQ ID NO:21, SEQ ID NO:22, SEQ ID NO:23, SEQ ID NO:24, SEQ ID NO:25, or SEQ ID NO 26.

[0334] Table 12 provides an exemplary sequence for a tyrosine ammonia lyase (TAL) enzyme in accordance with the methods of invention.

[0335] Table 12 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0336] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:27.

[0337] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:27.

[0338] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:27.

[0339] In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:27. In some embodiments, the tyrosine ammonia lyase comprises an amino acid sequence identical to SEQ ID NO:27.

[0340] Table 13 provides an exemplary sequence for a p-coumarate 3-hydroxylase (C3H) enzyme in accordance with the methods of invention. Table 13 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0341] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0342] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0343] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:28 or SEQ ID NO:29.

[0344] In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:28 or SEQ ID NO:29. In some embodiments, the p-coumarate 3-hydroxylase comprises an amino acid sequence identical to SEQ ID NO:28 or SEQ ID NO:29.

[0345] Table 14 provides an exemplary sequence for a caffeic acid O-methyltransferase (COMT) enzyme in accordance with the methods of invention. Table 14 Attorney Docket No. DEBU-037 / 01WO 37396 / 185

[0346] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 90% identity to SEQ ID NO:30.

[0347] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 95% identity to SEQ ID NO:30.

[0348] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 98% identity to SEQ ID NO:30.

[0349] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence having at least 99% identity to SEQ ID NO:30.

[0350] In some embodiments, the caffeic acid O-methyltransferase comprises an amino acid sequence identical to SEQ ID NO: 30.

[0351] Incorporation by Reference

[0352] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0353] Equivalents

[0354] Various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including references to the scientific and patent literature cited herein. The subject matter herein contains important information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

Claims

Attorney Docket No. DEBU-037 / 01WO 37396 / 185CLAIMS1. A method for cell-free production of tetrahydrocurcumin, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to tetrahydrocurcumin.

2. The method of claim 1, wherein the substrate is curcumin.

3. The method of claim 2, wherein the enzyme is a curcumin reductase.

4. The method of claim 3, wherein the curcumin reductase is an engineered curcumin reductase enzyme for conversion of curcumin to tetrahydrocurcumin.

5. The method of claim 4, wherein the curcumin reductase is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; and the enzyme provided in SEQ ID NO: 1.

6. The method of claim 1, wherein the reaction medium comprises NADPH and / or NADH.

7. The method of claim 6, wherein the reaction medium further comprises components for regeneration of NADPH and / or NADH.

8. The method of claim 7, wherein the reaction medium further comprises glucose.

9. The method of claim 8, wherein the reaction medium further comprises glucose dehydrogenase (GDH).

10. The method of claim 1, wherein the reaction medium is agitated to introduce oxygen in the reaction medium.Attorney Docket No. DEBU-037 / 01WO 37396 / 18511. The method of claim 1, wherein the reaction medium further comprises oxygen.

12. The method of claim 1, wherein the curcumin reductase enzyme is produced in an engineered host cell.

13. The method of claim 12, wherein the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells.

14. The method of claim 12, wherein the one or more enzymes are introduced in the host cell by integration into the genome of the host cell or on a plasmid.

15. The method of claim 14, wherein the engineered host cell expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes.

16. The method of claim 15, further comprising lysing of the engineered host cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of tetrahydrocurcumin.

17. The method of claim 16, wherein the one or more enzymes is purified from the cell lysate for production of tetrahydrocurcumin.

18. The method of claim 17, wherein the one or more enzymes is immobilized on a solid support for cell-free production of tetrahydrocurcumin from curcumin.

19. The method of claim 17, wherein the one or more enzymes is in a solution for cell-free production of tetrahydrocurcumin from curcumin.

20. The method of any one of claims 1-19, wherein the cell-free medium further comprises a buffer, an organic solvent, a detergent, and water.Attorney Docket No. DEBU-037 / 01WO 37396 / 18521. The method of claim 20, wherein the buffer is a phosphate buffer.

22. The method of claim 20, wherein the buffer is a citrate buffer.

23. A method for cell-free production of tetrahydrodemethoxy curcumin, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of one or more substrates to tetrahydrodemethoxycurcumin.

24. The method of claim 23, wherein the substrate is curcumin.

25. The method of claim 24, wherein the enzyme is a curcumin reductase.

26. The method of claim 25, wherein the curcumin reductase is an engineered curcumin reductase enzyme for conversion of curcumin to tetrahydrodemethoxycurcumin.

27. The method of claim 26, wherein the curcumin reductase is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; and the enzyme provided in SEQ ID NO: 1.

28. The method of claim 23, wherein the reaction medium comprises NADPH and / or NADH.

29. The method of claim 28, wherein the reaction medium further comprises components for regeneration of NADPH and / or NADH.

30. The method of claim 29, wherein the reaction medium further comprises glucose.

31. The method of claim 30, wherein the reaction medium further comprises glucose dehydrogenase (GDH).Attorney Docket No. DEBU-037 / 01WO 37396 / 18532. The method of claim 23, wherein the reaction medium is agitated to introduce oxygen in the reaction medium.

33. The method of claim 23, wherein the reaction medium further comprises oxygen.

34. The method of claim 23, wherein the curcumin reductase enzyme is produced in an engineered host cell.

35. The method of claim 34, wherein the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells.

36. The method of claim 34, wherein the one or more enzymes are introduced in the host cell by integration into the genome of the host cell or on a plasmid.

37. The method of claim 36, wherein the engineered host cell expressing the one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes.

38. The method of claim 37, further comprising lysing of the engineered host cells followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of tetrahydrodemethoxycurcumin.

39. The method of claim 38, wherein the one or more enzymes is purified from the cell lysate for production of tetrahydrodemethoxycurcumin.

40. The method of claim 39, wherein the one or more enzymes is immobilized on a solid support for cell-free production of tetrahydrodemethoxycurcumin from curcumin.

41. The method of claim 39, wherein the one or more enzymes is in a solution for cell-free production of tetrahydrodemethoxycurcumin from curcumin.Attorney Docket No. DEBU-037 / 01WO 37396 / 18542. The method of any one of claims 22-41, wherein the cell-free medium further comprises a buffer, an organic solvent, a detergent, and water.

43. The method of claim 42, wherein the buffer is a phosphate buffer.

44. The method of claim 42, wherein the buffer is a citrate buffer.

45. A method for production of tetrahydrocurcumin in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrocurcumin, and wherein the engineered host cell is cultured in a medium comprising the one or more substrates.

46. The method of claim 45, wherein the one or more substrates are selected from the group consisting of glucose, glycerol, coumaric acid, caffeic acid, ferulic acid, feruloyl-CoA, feruloyl-diketide-CoA, and curcumin.

47. The method of claim 46, wherein the substrate ferulic acid is produced endogenously by the engineered host cell from glucose, glycerol, or tyrosine.

48. The method of claim 47, wherein the substrate ferulic acid is produced endogenously by the engineered host cell through overexpression of the TAL, C3H, and COMT enzymes.

49. The method of claim 45, wherein the one more genetic modifications are selected from overexpression of a ligase, a diketide-CoA synthase, a curcumin synthase, a curcumin reductase and any combinations thereof.

50. The method of any one of claims 45-49, wherein ferulic acid is transformed to feruloyl-CoA by a ligase.

51. The method of claim 50, wherein the ligase is a feruloyl-CoA synthetase.Attorney Docket No. DEBU-037 / 01WO 37396 / 18552. The method of claim 51, wherein the feruloyl-CoA synthetase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17; and enzymes provided in SEQ ID NOS: 12-17.

53. The method of any one of claims 45-49, wherein feruloyl-CoA is transformed to feruloyl- diketide-CoAby a diketide-CoA synthase.

54. The method of claim 53, wherein the diketide-CoA synthase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; and enzymes provided in SEQ ID NOS: 8-11.

55. The method of any one of claims 45-49, wherein feruloyl-diketide-CoAis transformed to curcumin by a curcumin synthase.

56. The method of claim 55, wherein the curcumin synthase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7; enzymes provided in SEQ ID NOS: 2-7.

57. The method of any one of claims 45-49, wherein curcumin is transformed to tetrahydrocurcumin by a curcumin reductase.

58. The method of claim 57, wherein the curcumin reductase is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; and the enzyme provided in SEQ ID NO: 1.Attorney Docket No. DEBU-037 / 01WO 37396 / 18559. A method for production of tetrahydrodemethoxycurcumin in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to tetrahydrodemethoxycurcumin, and wherein the engineered host cell is cultured in a medium comprising the one or more substrates.

60. The method of claim 59, wherein the one or more substrates are selected from the group consisting of glucose, glycerol, caffeic acid, ferulic acid, feruloyl-CoA, feruloyl-diketide- CoA, coumaric acid, coumaroyl-CoA, coumaroyl-diketide-CoA, and demethoxycurcumin.

61. The method of claim 59, wherein the substrates ferulic acid and coumaric acid are produced endogenously by the engineered host cell from glucose, glycerol or tyrosine.

62. The method of claim 59, wherein the substrates ferulic acid and coumaric acid are produced endogenously by the engineered host cell through overexpression of the TAL, C3H, and COMT enzymes.

63. The method of claim 59, wherein the one more genetic modifications are selected from overexpression of a ligase, a diketide-CoA synthase, a curcumin synthase, a curcumin reductase and any combinations thereof.

64. The method of any one of claims 59-63, wherein ferulic acid is transformed to feruloyl-CoA by a ligase.

65. The method of claim 64, wherein the ligase is a feruloyl-CoA synthetase.

66. The method of claim 65, wherein the feruloyl-CoA synthetase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 12-17; and enzymes provided in SEQ ID NOS: 12-17.Attorney Docket No. DEBU-037 / 01WO 37396 / 18567. The method of any one of claims 59-63, wherein feruloyl-CoA is transformed to feruloyl- diketide-CoA by a diketide-CoA synthase.

68. The method of claim 67, wherein the diketide-CoA synthase is selected from having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; and enzymes provided in SEQ ID NOS: 8-11.

69. The method of any one of claims 59-66, wherein coumaric acid is transformed to coumaroyl- CoAby a ligase.

70. The method of claim 69, wherein the ligase is a 4-coumarate-CoA ligase.

71. The method of claim 70, wherein the 4-coumarate-CoA ligase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 18-26; and the enzymes provided in SEQ ID NOS: 18-26.

72. The method of any one of claims 59-63, wherein coumaroyl-CoAis transformed to coumaroyl-diketide-CoA by a diketide-CoA synthase.

73. The method of claim 72, wherein the diketide-CoA synthase is selected from having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 8-11; and enzymes provided in SEQ ID NOS: 8-11.

74. The method of any one of claims 59-63, wherein feruloyl-diketide-CoA and coumaroyl- diketide-CoA are transformed to demethoxycurcumin by a curcumin synthase.

75. The method of claim 74, wherein the curcumin synthase is selected from enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7;Attorney Docket No. DEBU-037 / 01WO 37396 / 185 enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 2-7; enzymes provided in SEQ ID NOS: 2-7.

76. The method of any one of claims 59-63, wherein demethoxycurcumin is transformed to tetrahydrodem ethoxy curcumin by a curcumin reductase.

77. The method of claim 59, wherein the curcumin reductase is selected from enzymes having at least 80% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 85% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; enzymes having at least 95% amino acid sequence identity from the enzyme provided in SEQ ID NO: 1; and the enzyme provided in SEQ ID NO: 1.

78. The method of any one of claims 45-77, wherein the one or more genetic modifications cause reduction of formation of byproducts.

79. The method of any one of claims 45-77, wherein the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells.

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