Bioproduction of kermesic acid and carminic acid

Abstract

The invention is related to materials and methods for production of kermesic acid and / or carminic acid from substrates. The invention provides methods and materials for cell-free and host cell-based bioproduction of kermesic acid and / or carminic acid.

Description

[0001] Patent Application DEBU-033 / 01WO 37396 / 182 BIOPRODUCTION OF KERMESIC ACID AND CARMINIC ACID

[0002] I. FIELD OF THE INVENTION

[0003] The invention is related to materials and methods for bioproduction of kermesic acid and carminic acid. The invention provides methods and materials for cell-free and host cell-based 5 production of kermesic acid and carminic acid.

[0004] II. SEQUENCE LISTING

[0005] The present application is being filed along with a Sequence Listing in electronic format. The Sequence Listing is provided as a file named DEBU-033-01WO-Seq-Listing.xml, created on September 24, 2025, which is 65 KB in size. The information in the electronic format of the 10 Sequence Listing is incorporated herein by reference in its entirety.

[0006] III. BACKGROUND

[0007] Carminic acid is used as an additive in food. Carminic acid is one of the most frequently used dyes in food, medicine, cosmetics and textiles. Carminic acid is added to foods such as ketchup, strawberry milk, and candies. It is also added to cosmetics such as eye shadow, nail polish, 15 and lipstick.

[0008] Carminic acid is a colorant, which can be extracted from the female insect bodies of Dactylopius coccus costa (alternative name Coccus cacti L.). The insects live on Nopalea coccinellifera, Opuntia fidus indica and other plants of the family Cactaceae cultivated for instance in the desert areas of Mexico, Central and South America and Canary Islands. Carmine, 20 the most widely used form of carminic acid, is a highly stable pigment complex of carminic acid with certain metal salts. Depending on the pH the colorant may be a color in a spectrum from orange to red to purple and is generally known as cochineal or cochineal color. Carmine colorant is widely used in foods and beverages.

[0009] Kermesic acid is an anthraquinone derivative and the main component of the red dye 25 kermes (false carmine). Kermesic acid, like carminic acid and the laccaic acids, is an insect dye obtained from scale insects. Kermesic acid is found in insects of the genus Kermes.

[0010] 1 Patent Application DEBU-033 / 01WO 37396 / 182 In relation to current industrial relevant production, carminic acid is harvested by extraction from the insect's dried bodies with water or alcohol. The insects (Dactylopius coccus) are cultured on cacti.

[0011] IV. SUMMARY OF THE INVENTION

[0012] 5 The conventional methods of industrial production of kermesic acid and / or carminic acid involve extraction of kermesic acid and / or carminic acid from insect’s bodies. As a result, the supply of kermesic acid and / or carminic acid may be relatively expensive and subject to undesirable variations and price fluctuations.

[0013] The invention provides novel methods for bioproduction of kermesic acid and / or carminic 10 acid. The methods of the invention are economic and reliable as compared to other conventional methods of production of kermesic acid and / or carminic acid. In addition, the methods provided in the invention provide higher titer values of kermesic acid and / or carminic acid from these processes as compared to kermesic acid and / or carminic acid produced from other existing methods.

[0014] 15 Production of kermesic acid:

[0015] In one aspect, the invention provides a method for production of kermesic acid, wherein the method comprises: providing one or more enzymes in a reaction medium, wherein the one or more enzymes result in transformation of one or more substrates to kermesic acid. In certain embodiments, the substrate is flavokermesic acid. In certain embodiments, the enzyme is a 20 monooxygenase enzyme. In certain embodiments, the monooxygenase enzyme results in transformation of flavokermesic acid to kermesic acid.

[0016] In certain embodiments, the monooxygenase enzyme resulting in transformation of flavokermesic acid to kermesic acid is engineered for efficient transformation of flavokermesic acid to kermesic acid.

[0017] 25 Beneficially, the transformation of flavokermesic acid to kermesic acid may be conducted in an engineered host cell or in a cell-free medium. Thus, the methods of the invention provide that flavokermesic acid may be transformed to kermesic acid in an engineered host cell or in a cell- free medium. This is important because it provides the optimal modularity to develop efficient

[0018] 2 Patent Application DEBU-033 / 01WO 37396 / 182 methods for production of kermesic acid. Thus, manufacturing processes for kermesic acid can be engineered as either cell-free or cell-based transformation of flavokermesic acid to kermesic acid.

[0019] Cell-free production of kermesic acid from flavokermesic acid:

[0020] In certain aspects, the invention provides methods for cell-free production of kermesic acid.

[0021] 5 The invention beneficially recognizes that economically effective methods of cell-free conversion of flavokermesic acid to kermesic acid were not previously taught. Specifically, the use of economically viable and efficient monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid were not previously disclosed. The invention provides that the monooxygenase enzymes provided in the invention provide an increased efficiency for conversion of flavokermesic 10 acid to kermesic acid.

[0022] In certain embodiments, the methods for cell-free production of kermesic acid 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 kermesic acid. An overview of the cell-free transformation of flavokermesic acid to kermesic acid by monooxygenase enzymes is provided in 15 FIG. 1. FIG.2 provides the data for cell-free transformation of flavokermesic acid to kermesic acid by monooxygenase enzymes. In certain embodiments, the substrate is flavokermesic acid. In certain embodiments, the enzyme is a monooxygenase enzyme. In certain embodiments, the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid.

[0023] 20 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 reaction medium is agitated to introduce oxygen in the reaction medium. In 25 certain embodiments, the reaction medium further comprises oxygen. In certain embodiments, the monooxygenase 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

[0024] 3 Patent Application DEBU-033 / 01WO 37396 / 182 expressing one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes.

[0025] 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- 5 free production of kermesic acid. In certain embodiments, one or more enzymes are purified from the cell lysate for production of kermesic acid. In certain embodiments, one or more enzymes are immobilized on a solid support for cell-free production of kermesic acid from flavokermesic acid. In certain embodiments, one or more enzymes are in a solution for cell-free production of kermesic acid from flavokermesic acid. In certain embodiments, the cell-free medium further comprises: 10 buffer, flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, GDH, and / or water. In certain preferred embodiments, the buffer is a phosphate buffer.

[0026] In certain embodiments, the methods of the invention provide that kermesic acid is produced in a cell-free medium. In certain embodiments, the invention provides methods for cell- free transformation of one or more substrates to kermesic acid in a cell-free medium. In certain 15 embodiments, the invention provides cell-free enzymatic transformation of one or substrates to kermesic acid through one or more intermediates. In certain embodiments, the invention provides cell-free transformation of flavokermesic acid to kermesic acid.

[0027] In certain beneficial aspects, the invention recognizes that the cell-free production of kermesic acid by methods of the invention provides high titer values of kermesic acid as compared 20 to other conventional methods. The methods of the invention have high efficiency in conversion of flavokermesic acid to kermesic acid in cell-free medium. Surprisingly, the monooxygenase enzymes provided in the methods of the invention provide for high-efficiency cell-free and enzymatic conversion of flavokermesic acid to kermesic acid. The pre-existing methods provided only trace amounts of generation of kermesic acid in cell-free conversion of kermesic acid. Thus, 25 beneficially, the monooxygenase enzymes provided herein for kermesic acid production lead to highly efficient and high titer generation of kermesic acid.

[0028] In certain embodiments, the monooxygenase enzymes of the invention include P450s and flavin-dependent monooxygenases (FMOs). In certain preferred embodiments, the invention provides that FMOs provided a higher efficiency in conversion of flavokermesic acid to kermesic

[0029] 4 Patent Application DEBU-033 / 01WO 37396 / 182 acid. In certain embodiments, the monooxygenase enzymes used herein are listed below in the table below.

[0030] Monooxygenase enzymes for the production of kermesic acid from flavokermesic acid.

[0031] NCBI Accession # Organism

[0032] WP0141455421 Strtm ttl

[0033]

[0034] 5 In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in

[0035] 5 Patent Application DEBU-033 / 01WO 37396 / 182 SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0036] In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56. In 5 certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 48-56.

[0037] In certain embodiments, the monooxygenase enzymes may be either heme-dependent 10 (such as cytochrome P450s) or flavin-dependent. In the reactions, flavokermesic acid is combined with the monooxygenase enzyme, and the cofactor NADPH. In certain embodiments, the reaction mixture is agitated to introduce oxygen from the air. In certain embodiments, the reaction mixture may comprise a reaction system for generation of oxygen. In certain embodiments, the reaction medium further comprises a suitable recycling system for NADPH regeneration. As an example 15 of a NADPH regeneration system, glucose may be employed along with a glucose dehydrogenase (GDH).

[0038] In certain embodiments, the monooxygenase enzymes used in cell-free conversion of flavokermesic acid to kermesic acid are purified enzymes. In certain embodiments, the purified monooxygenase enzymes for cell-free conversion of flavokermesic acid to kermesic acid are 20 immobilized and used in batch or in packed-bed reactors.

[0039] In certain embodiments, the monooxygenase enzyme for the cell free conversion of flavokermesic acid to kermesic acid is generated from lysing a host cell overexpressing the monooxygenase enzyme. In certain embodiments, the lysate generated from lysing said host cell overexpressing the monooxygenase enzyme is utilized for cell-free conversion of flavokermesic 25 acid to kermesic acid. In certain embodiments, the monooxygenase enzyme utilized for cell-free conversion of flavokermesic acid to kermesic acid is purified from the lysate of hosts expressing the monooxygenase enzyme.

[0040] In certain aspects, the one or more enzymes required for the cell-free production of kermesic acid are expressed in a host organism. In certain embodiments, the host organism is

[0041] 6 Patent Application DEBU-033 / 01WO 37396 / 182 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 5 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 kermesic acid. 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 10 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.

[0042] Production of kermesic acid from flavokermesic acid in an engineered host cell:

[0043] In certain aspects, the invention provides a method for production of kermesic acid in an 15 engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for conversion of a substrate to kermesic acid. An overview of the production of kermesic acid from flavokermesic acid produced in an engineered host cell is provided in FIG.3. The data demonstrating the production of kermesic acid from flavokermesic acid is provided in FIG.4. In certain embodiments, the substrate is flavokermesic acid. In certain embodiments, the host cell 20 produces flavokermesic acid. In certain embodiments, the one or more genetic modifications comprise expression and / or overexpression of monooxygenase enzyme. In certain embodiments, the engineered host cell further comprises one or more genetic modifications for reduction of use of precursors for production of kermesic acid. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is cultured in a medium comprising 25 flavokermesic acid. In certain embodiments, the engineered host cell comprises further genetic modifications for production of flavokermesic acid. In certain embodiments, one or more genetic modifications comprise overexpression of monooxygenase enzyme. In certain embodiments, one or more genetic modifications comprise overexpression of glucose dehydrogenase. In certain embodiments, the medium further comprises NADPH and / or NADH.

[0044] 7 Patent Application DEBU-033 / 01WO 37396 / 182 In certain aspects, the invention provides methods of production of kermesic acid from flavokermesic acid in an engineered host cell. In certain embodiments, flavokermesic acid is transformed to kermesic acid by an enzyme. In certain embodiments, the enzyme is a monooxygenase. In certain embodiments, the monooxygenase enzyme is selected from the 5 enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 85% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39- 56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In 10 certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0045] In certain embodiments, in order to improve cofactor availability for the monooxygenase, the engineered host cell comprises some additional modifications. In certain embodiments, the genetic modifications in engineered host cells include deleting or downregulating genes encoding 15 for enzymes that consume NADPH. In E. coli, there are about 80 NADPH-consuming genes. In certain embodiments, one or more genes consuming NADPH are deleted or downregulated. In certain embodiments, this list includes yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD. In certain embodiments, deletion or downregulation of one or more genes responsible consuming NADPH improves availability of the cofactor NADPH for conversion of 20 flavokermesic acid to kermesic acid by a monooxygenase.

[0046] In certain embodiments, the engineered host cell 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 engineered host cell by integration into genome of the host organism or on a plasmid. In certain embodiments, the plasmid comprises extrachromosomal DNA, which can be 25 expressed by the engineered host cell.

[0047] 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 for the production of kermesic acid. In certain embodiments, the medium may comprise flavokermesic acid. In various aspects, host cells may be engineered for enhanced 30 production of kermesic acid by introducing additional exogenous pathways and / or modifying

[0048] 8 Patent Application DEBU-033 / 01WO 37396 / 182 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 kermesic acid can be increasing yield, titer, or rate of production.

[0049] 5 In certain embodiments, the flavokermesic acid for production of kermesic acid is produced in the engineered host cell. The pathways for generation of flavokermesic acid are also disclosed herein.

[0050] Production of flavokermesic acid (FK) and / or kermesic acid (KA) in engineered host cells: In certain aspects, the invention provides a method for production of flavokermesic acid 10 and / or kermesic acid, in an engineered host cell.

[0051] In certain embodiments, the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to flavokermesic acid, and / or kermesic acid through one or more intermediates. In certain embodiments, the one or more substrates is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino 15 acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more substrate is glucose, glycerol, or any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

[0052] 20 In certain embodiments, the one or more genetic modifications lead to increase in metabolic flux to precursors or cofactors for production of flavokermesic acid and / or kermesic acid. In certain embodiments, the one or more intermediates are selected from the group consisting of acetyl-CoA, malonyl-CoA, an octaketide, and flavokermesic acid anthrone (FKA). In certain embodiments, one more genetic modifications are selected from overexpression of polyketide 25 synthase (PKS), aromatase, cyclases, and / or monooxygenase.

[0053] In certain embodiments, glucose or glycerol are transformed to acetyl-CoA and malonyl- CoA by enzymatic transformation through one or more enzymes. In certain embodiments, acetyl- CoA and malonyl-CoA are transformed to an octaketide. In certain embodiments, the transformation is mediated by polyketide synthase (PKS). In certain embodiments, the octaketide

[0054] 9 Patent Application DEBU-033 / 01WO 37396 / 182 is transformed to flavokermesic acid anthrone (FKA). In certain embodiments, the transformation is mediated by one or more cyclases or aromatases.

[0055] In certain embodiments, flavokermesic acid anthrone (FKA) is transformed to flavokermesic acid. In certain embodiments, said transformation is mediated by an oxygenase 5 enzyme. In certain embodiments, said transformation is mediated by a monooxygenase enzyme.

[0056] In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications comprise downregulation and / or deletion of one or more genes selected from the group consisting of yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD.

[0057] 10 In certain embodiments, one or more genetic modifications are selected from the group consisting of overexpression of ACC, ACS, MCS, matC, mdcA, panK, hemA, and / or ALAS. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the medium further comprises NADPH and / or NADH.

[0058] In certain aspects, the invention provides a method for production of flavokermesic acid 15 (FK), and / or kermesic acid (KA) in an engineered host cell. In certain embodiments, the engineered host cells comprise one or more genetic modifications, wherein the one or more genetic modifications lead to production of flavokermesic acid and / or kermesic acid from one or more substrates through one or more intermediates. An overview of the pathway for production of flavokermesic acid and kermesic acid is provided in FIG.3.

[0059] 20 In certain embodiments, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of flavokermesic acid and / or kermesic acid 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 flavokermesic acid, and / or kermesic acid is through enzymatic transformation. In certain embodiments, the carbon source is 25 selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the one or more 30 genetic modifications lead to an increase in metabolic flux to precursors or cofactors for production

[0060] 10 Patent Application DEBU-033 / 01WO 37396 / 182 of flavokermesic acid and / or kermesic acid. 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 5 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 non- native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, 10 biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid. In certain embodiments, the engineered host cell is E. coli.

[0061] In certain embodiments, one or more substrates are transformed to flavokermesic acid and / or kermesic acid through the PKS pathway. The schematic version of the pathway is provided in FIG.3.

[0062] 15 In certain embodiments, one or more substrates are transformed to kermesic acid. In certain embodiments, the one or more substrates that are transformed to kermesic acid are selected from the group consisting of glucose, glycerol, acetyl-CoA, malonyl-CoA, an octaketide, flavokermesic acid anthrone, and flavokermesic acid. In certain embodiments, the one or substrates is flavokermesic acid. In certain embodiments, the one or more substrate is flavokermesic acid 20 anthrone. In certain embodiments, the one or more substrates is an octaketide. In certain embodiments, the one or more substrates is acetyl-CoA and / or malonyl-CoA. In certain embodiments, the one or more substrates that are transformed to kermesic acid are a carbon source. In certain embodiments, the carbon source is a sugar. In certain embodiments, the carbon source is glucose or glycerol. In certain embodiments, the pathway for conversion of glycerol to kermesic 25 acid is provided in FIG.3.

[0063] In certain embodiments, the methods of the invention provide that a carbon source, such as glucose or glycerol is transformed to acetyl-CoA and / or malonyl-CoA. In certain embodiments, glycerol is transformed to acetyl-CoA and / or malonyl-CoA in an engineered host cell. In certain embodiments, glycerol is transformed to acetyl-CoA and / or malonyl-CoA through enzymatic

[0064] 11 Patent Application DEBU-033 / 01WO 37396 / 182 transformation. In certain embodiments, glucose is transformed to acetyl-CoA and / or malonyl- CoA by a genetically engineered and / or optimized enzyme.

[0065] In certain embodiments, the one or more enzymes responsible for transformation of glucose and / or glycerol to acetyl-CoA and / or malonyl-CoA are selected from the group consisting of 5 acetyl-CoA carboxylase (ACC), acetyl-CoA synthetase (ACS), malonyl-CoA synthetase (MCS), malonate transporter (matC), malonate-CoA transferase (mdcA), pantothenate kinase (PanK), glutamyl-tRNA reductase (hemA), and 5-aminolevulinic acid synthase (ALAS) enzymes. In certain embodiments, one or more of these enzymes are genetically engineered and optimized for preparation of malonyl-CoA and acetyl-CoA. In certain embodiments, the pathways for generation 10 of malonyl-CoA and acetyl-CoA are provided in PCT / US2022 / 024591, which is incorporated by reference in its entirety.

[0066] In certain embodiments, acetyl-CoA carboxylase (ACC) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 85-88. In certain embodiments, ACC is selected from the 15 enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 85-88. In certain embodiments, ACC is selected from the enzymes provided in SEQ ID NOS: 85-88.

[0067] In certain embodiments, acetyl-CoA synthase (ACS) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the 20 enzyme provided in SEQ ID NO: 89. In certain embodiments, ACS is selected from the enzymes having at least 95% amino acid sequence identity with the enzyme provided in SEQ ID NO: 89. In certain embodiments, ACS is selected from the enzyme provided in SEQ ID NO: 89.

[0068] In certain embodiments, malonyl-CoA synthetase (MCS) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from 25 the enzymes provided in SEQ ID NOS: 90-93. In certain embodiments, MCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 90-93. In certain embodiments, MCS is selected from the enzymes provided in SEQ ID NOS: 90-93.

[0069] 12 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, malonate transporter (matC) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 94-98. In certain embodiments, matC is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID 5 NOS: 94-98. In certain embodiments, matC is selected from the enzymes provided in SEQ ID NOS: 94-98.

[0070] In certain embodiments, malonate-CoA transferase (mdcA) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 99-103. In certain embodiments, mdcA is selected from 10 the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 99-103. In certain embodiments, mdcA is selected from the enzymes provided in SEQ ID NOS: 99-103.

[0071] In certain embodiments, pantothenate kinase (panK) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the 15 enzymes provided in SEQ ID NOS: 104-107. In certain embodiments, panK is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 104-107. In certain embodiments, panK is selected from the enzymes provided in SEQ ID NOS: 104-107.

[0072] In certain embodiments, glutamyl-tRNA reductase (hemA) is selected from the enzymes 20 having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 108-111. In certain embodiments, hemA is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 108-111. In certain embodiments, hemA is selected from the enzymes provided in SEQ ID NOS: 108-111.

[0073] 25 In certain embodiments, ALA synthase (ALAS) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 112-116. In certain embodiments, ALAS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID

[0074] 13 Patent Application DEBU-033 / 01WO 37396 / 182 NOS: 112-116. In certain embodiments, ALAS is selected from the enzymes provided in SEQ ID NOS: 112-116.

[0075] In certain embodiments, the invention provides that acetyl-CoA and / or malonyl-CoA are transformed to an octaketide. In certain embodiments, the acetyl-CoA and / or malonyl-CoA are 5 transformed to an octaketide in an engineered host cell. In certain embodiments, acetyl-CoA and / or malonyl-CoA are transformed to an octaketide through enzymatic transformation. In certain embodiments, acetyl-CoA and / or malonyl-CoA are transformed to an octaketide through a genetically engineered enzyme. In certain embodiments, the transformation of acetyl-CoA and / or malonyl-CoA to an octaketide by polyketide synthase (PKS). In certain embodiments, polyketide 10 synthase (PKS) is engineered to optimize transformation of acetyl-CoA and / or malonyl-CoA to an octaketide. In certain embodiments, PKS is a PKSIII or PKSII. In certain preferred embodiments, PKSIII enzymes used for microbial conversion of acetyl-CoA and / or malonyl-CoA are provided in the Table below.

[0076] PKSIIIs used for microbial production of flavokermesic acid (FK)

[0077] NCBI Accession Number Organism

[0078] 15

[0079]

[0080] In certain preferred embodiments, PKSII enzymes and / or accessory enzymes used for microbial conversion of acetyl-CoA and / or malonyl-CoA are provided in the Table below.

[0081] 14 Patent Application DEBU-033 / 01WO 37396 / 182 PKSIIs and accessory enzymes used for microbial production of flavokermesic acid (FK) NCBI Accession Number Organism WP0111482951 Photorhabdus laumondii subsp Laumondii

[0082]

[0083] In certain embodiments, polyketide synthase (PKS) is a genetically modified enzyme. In certain embodiments, PKS is selected from the enzymes having at least 80%, 85%, 90%, 95%, 5 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 57-74. In certain embodiments, PKS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 57-74. In certain embodiments, PKS is selected from the enzymes provided in SEQ ID NOS: 57-74.

[0084] In certain embodiments, the invention provides that an octaketide is transformed to 10 flavokermesic acid anthrone. In certain embodiments, an octaketide is transformed to flavokermesic acid anthrone. In certain embodiments, an octaketide is transformed to flavokermesic acid anthrone in an engineered host cell. In certain embodiments, an octaketide is transformed to flavokermesic acid anthrone in an engineered host cell. In certain embodiments, the transformation of an octaketide to flavokermesic acid anthrone is through one or more 15 genetically engineered enzymes. In certain embodiments, the one or more genetically engineered enzymes are one or more aromatases and / or cyclases. In certain embodiments, the genetically engineered enzyme is a cyclase. In certain embodiments, the genetically engineered enzyme is an aromatase. In certain embodiments, the cyclase and / or aromatase enzymes are optimized by a genetically engineered enzyme. In certain embodiments, the aromatase and / or cyclase enzymes 20 are selected from the table below.

[0085] 15 Patent Application DEBU-033 / 01WO 37396 / 182 Cyclases and aromatases used for microbial production of flavokermesic acid or kermesic acid NCBI Accession Number Organism

[0086] AAG301961 Strtm R1128

[0087]

[0088] In certain embodiments, aromatase and / or cyclase enzymes are selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from 5 the enzymes provided in SEQ ID NOS: 75-84. In certain embodiments, aromatase and / or cyclase enzymes are selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 75-84. In certain embodiments, aromatase and / or cyclase enzymes are selected from the enzymes provided in SEQ ID NOS: 75-84.

[0089] In certain embodiments, the invention provides that flavokermesic acid anthrone is 10 transformed to flavokermesic acid. In certain embodiments, the transformation of flavokermesic acid anthrone to flavokermesic acid is in an engineered host cell. In certain embodiments, the transformation of flavokermesic acid anthrone to flavokermesic acid is mediated through one or more enzymes. In certain embodiments, the one or more enzymes for transformation of flavokermesic acid anthrone to flavokermesic acid are genetically engineered or modified.

[0090] 15 In certain embodiments, for generation of flavokermesic acid in an engineered host cell, the genes for PKS pathway (described in FIG.3), such as PKSII, PKSIII, or both, are combined with the genes for cyclases and aromatases to produce flavokermesic acid anthrone. The produced

[0091] 16 Patent Application DEBU-033 / 01WO 37396 / 182 flavokermesic acid anthrone spontaneously oxidizes to flavokermesic acid. The genes for said enzymes may be in plasmids or integrated into the chromosome of the engineered host cell. Additionally, other modifications to the host may be required to improve the flux towards the products-of-interest.

[0092] 5 In certain embodiments, the invention provides a method for manufacturing kermesic acid through one or more of the following steps:

[0093] (a) transformation of glycerol or another carbon source to acetyl-CoA and / or malonyl- CoA;

[0094] (b) transformation of acetyl-CoA and / or malonyl-CoA to an octaketide;

[0095] 10 (c) transformation of an octaketide to flavokermesic acid anthrone;

[0096] (d) transformation of flavokermesic acid anthrone to flavokermesic acid; and (e) transformation of flavokermesic acid to kermesic acid.

[0097] 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 15 steps (a)-(e) are engineered enzymes.

[0098] 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 kermesic acid. In certain embodiments, the engineered host cells are modified to express one or more enzymes for transformation in steps (a)-(e).

[0099] 20 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 monooxygenase enzyme. In 25 certain embodiments, the monooxygenase enzyme is an engineered enzyme optimized for conversion of flavokermesic acid to kermesic acid.

[0100] 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

[0101] 17 Patent Application DEBU-033 / 01WO 37396 / 182 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 for the production of flavokermesic acid and / or 5 kermesic acid. In various aspects, host cells may be engineered for enhanced production of flavokermesic acid and / or kermesic acid 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 flavokermesic acid 10 and / or kermesic acid can increase yield, titer, or rate of production.

[0102] Cell-free production of carminic acid from flavokermesic acid:

[0103] In certain aspects, the invention provides a method of cell free production of carminic acid. Flavokermesic acid (FK) is converted to carminic acid (CA) through a hydroxylation and glycosylation step. The enzymes that catalyze these reactions in Dactylopius coccus are either not 15 known or not suitable for efficient bioproduction. The enzymes provided herein are selected and / or further optimized for cell-free conversion of FK to CA in a single step. The methods of the invention beneficially overcome the instability of kermesic acid (KA) as an intermediate. Advantageously, the methods of the invention may be scaled up from lab scale to commercial scale while maintaining the efficiency of the enzymes for conversion of FK to CA.

[0104] 20 In certain embodiments, the method of cell-free production of carminic acid comprises:

[0105] providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of flavokermesic acid to carminic acid through one or more intermediates. In certain embodiments, one or more intermediates are selected from the group consisting of kermesic acid and C-glucosylated flavokermesic acid. In certain embodiments, one or more enzymes are 25 selected from the group consisting of monooxygenase, C-glucosyltransferase, glucose dehydrogenase (GDH), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK).

[0106] 18 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, one or more enzymes are selected from the group consisting of monooxygenase and / or C-glucosyltransferase. In certain embodiments, the cell-free medium further comprises glucose dehydrogenase (GDH) enzyme. In certain embodiments, the cell-free medium comprises flavokermesic acid and monooxygenase enzyme. In certain embodiments, 5 UDP-glucose and CGT are added to the cell-free medium. In certain embodiments, the cell-free medium comprises UDP-glucose, monooxygenase and / or C-glucosyltransferase. In certain embodiments, UDP-glucose is synthesized in the cell-free medium by the one or more enzymes. In certain embodiments, UDP-glucose is synthesized from one or ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6-phosophate, glucose-1-10 phosphate, and / or polyphosphate. In certain embodiments, one or more enzymes is selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK).

[0107] In certain embodiments, the cell-free medium is agitated to include oxygen from the 15 atmosphere. In certain embodiments, the cell-free medium further comprises NADPH / NADH. In certain embodiments, the cell-free medium further comprises 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 20 consisting of: bacteria, yeast, and / or fungal cells. In certain embodiments, one or more enzymes are 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 one or more enzymes are cultured until a pre- determined biomass is achieved to produce the requisite quantity of the one or more enzymes. In certain embodiments, the engineered host cells followed by removal of cell debris to generate a 25 cell lysate for use in the cell-free medium for cell-free production of carminic acid.

[0108] In certain embodiments, one or more enzymes are purified from the cell lysate for production of carminic acid. In certain embodiments, one or more enzymes are immobilized on a solid support for cell-free production of carminic acid from flavokermesic acid. In certain embodiments, one or more enzyme are in a solution for cell-free production of carminic acid from 30 flavokermesic acid. In certain embodiments, the cell-free medium further comprises: buffer,

[0109] 19 Patent Application DEBU-033 / 01WO 37396 / 182 flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, 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 one or more enzymes are in a solution. In certain embodiments, the reaction is conducted in a packed bed reactor, wherein the one or more enzymes 5 are immobilized. In certain embodiments, the method results in titer values of produced carminic acid from about 10 times to about 5000 times higher than methods for cell-based production of carminic acid.

[0110] In one aspect, the invention provides a method for cell-free production of carminic acid, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the 10 one or more enzymes result in transformation of flavokermesic acid to carminic acid through one or more intermediates. In certain embodiments, the one or more enzymes that transform flavokermesic acid to carminic acid are C-glucosyltransferase (CGT), monooxygenase (MOX), or a combination thereof. In certain embodiments, one or more intermediates are selected from the group consisting of kermesic acid (KA) and C-glucosylated flavokermesic acid (DCII). An 15 overview of the synthetic pathway for cell-free conversion of flavokermesic acid to carminic acid is provided in FIG.5. FIG.6 provides the data demonstrating the conversion of flavokermesic acid to carminic acid.

[0111] In certain embodiments, the method of the invention provides that monooxygenase and C- glucosyltransferase transform flavokermesic acid to carminic acid. In certain embodiments, 20 monooxygenase and C-glucosyltransferase are added to the cell-free reaction simultaneously. In these embodiments, UDP-glucose and other reaction components are added to the reaction medium simultaneously. In these embodiments, kermesic acid or C-glucosyltransferase are intermediates, but they are not isolated or purified. In certain embodiments, the cell-free reaction may proceed more favorably via the generation of C-glucosylated flavokermesic acid because the C-25 glucosyltransferase enzyme acts first to convert flavokermesic acid to C-glucosylated flavokermesic acid, and monooxygenase acts second to convert C-glucosylated flavokermesic acid to carminic acid.

[0112] In certain embodiments, a first step is carried out by addition of monooxygenase to the reaction system, and the reaction is carried to completion. Subsequently, C-glucosyltransferase is 30 added to the reaction medium. In these embodiments, monooxygenase enzyme transforms

[0113] 20 Patent Application DEBU-033 / 01WO 37396 / 182 flavokermesic acid to kermesic acid. In certain embodiments, the details of the cell-free reaction for transformation of flavokermesic acid to kermesic acid are provided herein. In certain embodiments, kermesic acid is not purified or isolated. In certain embodiments, kermesic acid is further transformed to carminic acid through one or more enzymes. In certain embodiments, C- 5 glucosyltransferase (CGT) transforms kermesic acid to carminic acid in a cell-free reaction medium. In certain embodiments, the reaction medium for C-glucosyltransferase (CGT) mediated enzymatic transformation of kermesic acid to carminic acid further comprises an activated sugar. In certain embodiments, the activated sugar is uridine diphosphate glucose (UDP-glucose). In certain embodiments, UDP-glucose is an essential co-factor for the transformation of kermesic 10 acid to carminic acid. An overview of the conversion of kermesic acid to carminic acid is provided in FIG.7.

[0114] In certain embodiments, the monooxygenase enzymes used herein are listed below in the table below.

[0115] NCBI Accession # Organism

[0116]

[0117] 21 Patent Application DEBU-033 / 01WO 37396 / 182 WP_028998915 Azohydromonas australica TNF55200 Burkholderiales bacterium

[0118]

[0119] In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 5 85% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0120] 10 In certain embodiments, the one or more enzymes involved in transformation of kermesic acid to carminic acid are glucosyltransferase (GT), C-glucosyltransferase (CGT), and / or sucrose synthase (SuSy). An overview of the process where kermesic acid is converted to carminic acid and UDP-glucose is generated from sucrose is provided in FIG. 7. In certain embodiments, GT / CGT is selected from the enzymes having at least 80% amino acid sequence identity from the 15 enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, GT / CGT is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, GT / CGT is selected from the enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, SuSy is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 16-25. In certain 20 embodiments, SuSy is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 16-25. In certain embodiments, SuSy is selected from the enzymes provided in SEQ ID NOS: 16-25.

[0121] In certain embodiments, the invention provides methods for production of carminic acid. In certain embodiments, the methods of the invention provide that carminic acid is produced by 25 enzymatic transformation of flavokermesic acid to carminic acid through one or more

[0122] 22 Patent Application DEBU-033 / 01WO 37396 / 182 intermediates. In certain embodiments, the one or more enzymes responsible for transformation of flavokermesic acid are selected from the group consisting of monooxygenase and CGT. In certain embodiments, flavokermesic acid is enzymatically transformed to kermesic acid through monooxygenase. In certain embodiments, kermesic acid is enzymatically transformed to carminic 5 acid through CGT.

[0123] In certain embodiments, the process for transformation of kermesic acid to carminic acid is provided in PCT / US2024 / 035784, which is incorporated by reference in its entirety. In certain embodiments, the one more enzymes expressed in an engineered host cell for transformation of kermesic acid to carminic acid include C-glucosyltransferase (CGT), sucrose synthase (SuSy), 10 glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, C-glucosyltransferase (CGT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase 15 (NDK). An outline for the enzymatic pathway for conversion of kermesic acid to carminic acid is provided in FIG.1, FIG.2, and FIG.3 of PCT / US2024 / 035784, which is incorporated by reference in its entirety. In certain embodiments, glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, 20 GLK, PGM, PPK, UGP, and / or NDK are enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 26-38. In certain embodiments, GLK, PGM, PPK, UGP, and / or NDK are enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 26-38. In certain embodiments, GLK, PGM, PPK, UGP, and / or NDK are enzymes are selected from the enzymes provided in SEQ ID NOS: 26-38.

[0124] 25 In certain embodiments, the C-glucosyltransferase (CGT) enzymes used herein are provided in the Table below.

[0125] NCBI Accession # Organism

[0126]

[0127] 23 Patent Application DEBU-033 / 01WO 37396 / 182 KAG8368435 Buddleja alternifolia AFJ52991 Linum usitatissimum

[0128]

[0129] In certain embodiments, the invention provides engineered enzymes for the cell-free production of kermesic acid and / or carminic acid. In certain embodiments, the engineered enzymes are optimized for cell-free production of kermesic acid and / or carminic acid. In certain 5 embodiments, engineered enzymes may include genetic modifications. In certain embodiments, the genetic modifications may be selected from a group consisting of: point mutations, insertions, deletions, and / or any other modifications such that those enzymes result in efficient and optimal cell-free production of kermesic acid and / or carminic acid. In certain embodiments, the enzymes used in the cell-free production of kermesic acid and / or carminic acid are any enzymes disclosed 10 herein.

[0130] In certain aspects of the invention, where the one or more steps for production of kermesic acid and / or carminic acid are conducted in a cell-free medium, the method of the cell-free production does not require the purification of the one or more enzymes from the lysed host organisms. Accordingly, in certain embodiments, the methods of the invention do not require the 15 purification of one or more enzymes from the lysed biomass comprising host cell expressing the one or more enzymes for the cell-free production of kermesic acid and / or carminic acid.

[0131] 24 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the monooxygenase and C-glucosyltransferase enzymes used in cell-free conversion of flavokermesic acid to carminic acid are purified enzymes. In certain embodiments, the purified monooxygenase and C-glucosyltransferase enzymes for cell-free conversion of flavokermesic acid to carminic acid are used in batch or immobilized packed bed 5 reactors.

[0132] In certain embodiments, the monooxygenase and C-glucosyltransferase enzymes for the cell free conversion of flavokermesic acid to carminic acid are generated from lysing a host cell overexpressing the monooxygenase and C-glucosyltransferase enzymes. In certain embodiments, the lysate generated from lysing said host cell overexpressing the monooxygenase and C-10 glucosyltransferase enzymes is utilized for cell-free conversion of flavokermesic acid to carminic acid. In certain embodiments, the monooxygenase and C-glucosyltransferase enzymes utilized for cell-free conversion of flavokermesic acid to carminic acid are purified from the lysate of hosts expressing the enzymes.

[0133] In certain aspects, the one or more enzymes required for the cell-free production of 15 carminic acid are 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 20 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 kermesic acid. 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 25 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.

[0134] In certain embodiments, the cell-free medium for production of carminic acid from flavokermesic acid may further comprise any other additional ingredients required for the cell-free

[0135] 25 Patent Application DEBU-033 / 01WO 37396 / 182 production of carminic acid. In certain embodiments, the cell-free reaction medium further comprises UDP-glucose.

[0136] In certain embodiments, the UDP-glucose for the reaction medium is generated from sucrose. In certain embodiments, the UDP-glucose is generated in the reaction medium by one or 5 more enzymes. In certain embodiments, the generation of UDP-sugar is mediated by sucrose synthase (SuSy). In certain embodiments, sucrose synthase (SuSy) mediates the conversion of sucrose to UDP-glucose. Thus, in certain embodiments, the reaction medium comprises sucrose, which is subsequently converted to UDP-glucose in course of the reaction. An overview of the process where kermesic acid is converted to carminic acid and UDP-glucose is generated from 10 sucrose is provided in FIG.7.

[0137] In certain beneficial aspects, the invention provides that the UDP-glucose used in the reaction is generated from other substrates in course of the reaction. In certain embodiments, the UDP moiety in UDP-glucose is recycled in course of the process. The recycling of UDP provides economic efficiency of the processes of the invention. Accordingly, in certain aspects, the 15 production of carminic acid from kermesic acid catalyzed by CGT is conducted in conjunction with other methods for UDP-glucose production or recycling of UDP. In certain embodiments, the overview of the synthetic scheme involving the UDP-glucose production and / or recycling is provided in FIG.8. In certain embodiments, UDP-glucose is generated by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of 20 UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridylyltransferase (UGP). In certain preferred embodiments, the UGP may be galU.

[0138] In certain aspects, glucose-1-phosphate is the source for formation of UDP-glucose. Accordingly, in certain embodiments, glucose-1-phosphate may be added to the reaction for generation of carminic acid. In certain embodiments, the glucose-1-phosphate may be supplied to 25 the reaction for preparation of carminic acid.

[0139] In certain embodiments, glucose-1-phosphate is generated during the course of the reaction. In certain embodiments, glucose-1-phosphate is generated enzymatically during the course of the reaction. Accordingly, in certain embodiments, glucose is converted to glucose-6- phosphate by a reaction of ATP (adenosine triphosphate) and glucose. In certain embodiments,

[0140] 26 Patent Application DEBU-033 / 01WO 37396 / 182 reaction between glucose and ATP results in generation of glucose-6-phosphate and adenosine diphosphate (ADP). In certain embodiments, conversion of glucose to glucose-6-phosphate is catalyzed by glucokinase (GLK). In certain embodiments, conversion of glucose to glucose-6- phosphate is catalyzed by hexokinase (HK). In certain embodiments, glucose-6-phosphate is 5 converted to glucose-1-phosphate. In certain embodiments, the conversion of glucose-6-phosphate to glucose-1-phsophate is catalyzed by phosphoglucomutase (PGM).

[0141] In certain aspects, the invention further provides that the nucleoside triphosphate species used in the reaction are recycled. In certain embodiments, the recycled nucleoside trisphosphates are ATP and UTP. In certain embodiments, UTP is generated by reaction of UDP and ATP. In 10 certain embodiments, the generation of UTP from UDP and ATP is catalyzed by nucleoside diphosphate kinase (NDK). In certain embodiments, ATP may be generated from ADP and phosphate or polyphosphate. In certain embodiments, the conversion of ADP to ATP is catalyzed by polyphosphate kinase (PPK).

[0142] In certain embodiments, the cell-free medium comprises buffer, an activated sugar, 15 magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water. In certain embodiments, the cell-free medium comprises buffer, flavokermesic acid, an activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for enzymatic conversion of kermesic acid to carminic acid. In certain embodiments, the 20 buffer maintains the pH of about 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 present at a concentration of about 1 mM to about 200 mM. In certain embodiments, the buffer is present at a concentration of 25 about 5 mM to about 100 mM.

[0143] In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.001 mM to about 50 mM. In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.01 mM to about 10 mM. In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 30 0.01 mM to about 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0144] 27 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the cell-free reaction medium comprises magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium comprises magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0145] In certain embodiments, the cell-free reaction medium further comprises sucrose. In certain 5 embodiments, the sucrose is converted to UDP-glucose. Thus, the concentration of sucrose in the reaction mixture is determined by the quantity of UDP-glucose needed for the reaction for cell- free production of carminic acid. In certain embodiments, sucrose is present at a concentration of about 10 mM to about 1000 mM in the cell-free reaction medium. In certain embodiments, sucrose is present at a concentration of about 20 mM to about 800 mM in the cell-free reaction medium.

[0146] 10 In certain embodiments, sucrose is present at a concentration of about 50 mM to about 600 mM in the cell-free reaction medium.

[0147] In certain embodiments, the quantity of the one or more enzymes for the cell-free production of carminic acid is dependent on the target quantity of carminic acid to be produced and / or the concentration of other ingredients present in the reaction mixture. In certain 15 embodiments, the one or more enzymes are present in a concentration of from about 1% to about 50% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 2.5% to about 45% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 5% to about 40% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 7.5% to about 30% (v / v).

[0148] 20 In certain embodiments, the reaction for production of carminic acid is conducted for a duration until the desired quantity of carminic acid is obtained. In certain embodiments, the reaction for cell-free production of carminic acid is carried out from about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out from about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free 25 production of kermesic and / or carminic acid is carried out from about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out from about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of kermesic and / or carminic acid is carried out from about 1 hour to about 15 hours.

[0149] 28 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is varied to obtain optimal results for the production of carminic acid. In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is

[0150] 5 for cell- embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is -free production of carminic acid may influence the rate of production of carminic acid. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to 10 obtain optimal yield of carminic acid in cell-free production of carminic acid.

[0151] 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 carminic acid. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column reactor / bioreactor, the one or 15 more enzymes involved in cell-free production of carminic acid are in a solution. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a bubble column reactor / bioreactor comprises the lysate from the host organism. In certain embodiments, it is advantageous to use the bubble column reactor / bioreactor for cell-free production of carminic acid when the reaction mixture involves the lysate (or lysate with cellular debris removed) from 20 the host cell organisms in which the one or more enzymes responsible for cell-free production of carminic acid were utilized. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a packed bed reactor / bioreactor. In certain embodiments, the one or more enzymes are immobilized in the packed bed reactor / bioreactor. The packed bed reactors / bioreactors are preferred for the purified enzymes playing a role in cell-free production of 25 carminic acid. In certain embodiments, the one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, the one or more enzymes may be immobilized in different reactors / bioreactors, wherein these reactors / bioreactors are linked sequentially.

[0152] In certain beneficial aspects, the methods of the invention provided herein are novel methods for cell-free production of carminic acid from flavokermesic acid. The methods of the 30 invention beneficially provide high titer values of carminic acid. The titer values of carminic acid

[0153] 29 Patent Application DEBU-033 / 01WO 37396 / 182 achieved using the methods of the invention are significantly higher than comparable cell-based methods of production of carminic acid. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 250 µM or higher. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a 5 concentration of 500 µM or higher. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 750 µM or higher. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 1 mM or higher.

[0154] In certain aspects, the invention provides compositions for cell-free production of kermesic 10 acid and / or carminic acid. The compositions of the invention are utilized for cell-free production of kermesic acid and / or carminic acid in accordance with the methods described above.

[0155] Advantageously, the methods of invention for production of kermesic acid and / or carminic acid provided herein provides significantly higher titer values for kermesic acid and / or carminic acid as compared to the existing methods. The higher titer values of kermesic acid and / or carminic 15 acid provide additional cost advantages for production of kermesic acid and / or carminic acid because the higher titers of kermesic acid and / or carminic acid provide efficiency in purifying and / or concentrating carminic acid from the reaction mixture. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 5-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic 20 acid and / or carminic acid titer values at least 10-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 50-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 100-fold higher than the conventional methods. In certain embodiments, the methods of the invention 25 provide kermesic acid and / or carminic acid titer values at least 500-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 1000-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 5000-fold higher than the conventional methods. The methods of the invention 30 provide a fast and efficient method for production of carminic acid. As demonstrated by the data

[0156] 30 Patent Application DEBU-033 / 01WO 37396 / 182 provided herein, the methods of the invention may be used for production of carminic acid up to 2.4 g / L within 30 hours. FIG.9 provides data related to production of carminic acid produced by the methods of the invention.

[0157] In certain embodiments, carminic acid produced from the methods of the invention may be 5 further converted to carmine by adding carminic acid to alum.

[0158] In certain embodiments, the invention provides a method for manufacturing carminic acid through one or more of the following steps:

[0159] (a) transformation of glycerol or another carbon source to acetyl-CoA and / or malonyl- CoA;

[0160] 10 (b) transformation of acetyl-CoA and / or malonyl-CoA to an octaketide;

[0161] (c) transformation of an octaketide to flavokermesic acid anthrone;

[0162] (d) transformation of flavokermesic acid anthrone to flavokermesic acid;

[0163] (e) transformation of flavokermesic acid to kermesic acid; and

[0164] (f) transformation of kermesic acid to carminic acid.

[0165] 15 In certain embodiments, one or more steps of (a)-(f) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a)-(f) are engineered enzymes.

[0166] In certain embodiments, one or more steps (a)-(e) are conducted in an engineered host cell, and step (f) is conducted in a cell-free medium. In certain embodiments, the engineered host cell 20 is genetically modified for production of kermesic acid 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).

[0167] In certain embodiments, steps (a)-(d) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one more enzymes for the transformations listed 25 in steps (a)-(d). In these embodiments, steps (e) and (f) are optionally carried out in a cell-free medium. In certain embodiments, steps (a)-(e) are carried out in an engineered host cell and step (f) is carried out in a cell-free medium.

[0168] V. BRIEF DESCRIPTION OF THE FIGURES

[0169] 31 Patent Application DEBU-033 / 01WO 37396 / 182 FIG. 1 provides the overview of conversion of flavokermesic acid (FK) to kermesic acid (KA).

[0170] FIG. 2 provides HPLC data demonstrating the conversion of flavokermesic acid (FK) to kermesic acid (KA) catalyzed by a monooxygenase enzyme.

[0171] FIG. 3 provides the pathway for conversion of carbon feedstocks to kermesic acid (KA) through 5 one or more intermediates.

[0172] FIG. 4 provides HPLC data demonstrating the conversion of carbon feedstocks to kermesic acid through one or more enzymes.

[0173] FIG. 5 provides the pathway for conversion of flavokermesic acid (FK) to carminic acid (CA) through one or more intermediates.

[0174] 10 FIG. 6 provides HPLC data demonstrating the conversion of flavokermesic acid (FK) to carminic acid (CA) through one or more intermediates.

[0175] FIG. 7 provides a schematic showing the conversion of kermesic acid to carminic acid, and the conversion of sucrose to UDP-glucose.

[0176] FIG. 8 provides a schematic showing the conversion of kermesic acid to carminic acid, wherein 15 the UDP is recycled.

[0177] FIG. 9 provides the data related to cell-free production of carminic acid from flavokermesic acid..

[0178] FIG. 10 provides data related to generation of kermesic acid from flavokermesic acid in cell-free reactions employing MOX enzyme lysates.

[0179] 20 VI. DETAILED DESCRIPTION

[0180] The present application provides compositions and methods for production of kermesic acid and / or carminic acid. 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 kermesic acid and / or 25 carminic acid. The one or more enzymes may be engineered. The engineered enzyme may be non- naturally occurring.

[0181] 32 Patent Application DEBU-033 / 01WO 37396 / 182 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 5 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”.

[0182] 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 10 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.”

[0183] As used herein, “reaction solution” may refer to all components necessary for enzyme- based chemical transformation. This is typically, but not limited to, buffering agent, salts, cofactor, 15 and substrate (starting material).

[0184] 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 20 interchangeably.

[0185] 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.

[0186] 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 25 include but are not limited to NADPH and NADH.

[0187] 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 30 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

[0188] 33 Patent Application DEBU-033 / 01WO 37396 / 182 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 under-express or to 5 have reduced expression of a gene, nucleic acid sequence, or to under-express 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 10 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.

[0189] 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 15 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 20 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.

[0190] The term “exogenous” or “heterologous” is intended to mean that the referenced molecule 25 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.

[0191] 30 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,

[0192] 34 Patent Application DEBU-033 / 01WO 37396 / 182 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 desired.

[0193] 5 Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.

[0194] 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 10 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 15 (presence or absence), etc., do not affect the % identity.

[0195] Kermesic acid

[0196] Kermesic acid is a natural dye. It can be extracted from female kermes insect, Kermes vermilio, which lives on the kermes oak tree (Quercus coccifera L.). It gives a blood-red color and is used as a colorant in the pharmaceutical and textile industries.

[0197] 20 Kermesic acid is the penultimate intermediate in the biosynthesis of carminic acid, and a required intermediate in most routes to carminic acid. Kermesic acid (KA) is derived via hydroxylation of its polyketide-derived precursor flavokermesic acid (FK), however the native oxygenase performing this function in the D. coccus insect is not known, frustrating attempts to reconstitute the pathway in industrially-tractable microbial hosts. Previous efforts to produce 25 kermesic acid in heterologous hosts have relied on oxygenases giving extremely low conversion levels, or have proceeded via unidentified host enzymes, preventing further enzyme and process optimization. Yang, D.; Jang, W. D.; Lee, S. Y. Production of Carminic Acid by Metabolically Engineered Escherichia coli. J. Am. Chem. Soc.2021, 143, 5364-5377. Zhang, Q.; Wang, X.; Zeng, W.; Xu, S.; Li, D.; Yu, S.; Zhou, J. De novo biosynthesis of carminic acid in Saccharomyces 30 cerevisiae. Metabolic Engineering 2023, 76, 50-62. Frandsen, R. J. N.; Khorsand-Jamal, P.;

[0198] 35 Patent ApplicationDEBU-033 / 01WO 37396 / 182Kongstad, K. T.; Nafisi, M.; Kannangara, R. M.; Staerk, D.; Okkels, F. T. Binderup, K.; Madsen, B.; Møller, B. L.; Thrane, U.; Mortensen, U. H. Heterologous production of the widely used naturalfood colorant carminic acid in Aspergillus nidulans. Sci. Reports 2018, 8, 12853.

[0199] The chemical structure for kermesic acid is provided below:

[0200] 5

[0201]

[0202] The conventional methods of synthesis of kermesic acid are resource intensive and lead to poor yields. Thus, these processes are economically inefficient.

[0203] Carminic Acid:

[0204] Carminic acid is an anthraquinone compound containing a glucose molecule attached via10 a glycosidic bond. The anthraquinone portion strongly absorbs light, giving the compound itsintense color. Depending on the pH the colorant may be a color in a spectrum from orange to red to purple and is generally known as cochineal or cochineal color. Carminic acid and derivativesare utilized in a large range of foods such as ice cream, confectionery, culinary, desserts, beverages,dairy and others; carminic acid is also widely used in cosmetics. Carmine, the most widely used15 form of carminic acid, is a highly stable pigment that can withstand heat processing and adversestorage conditions. Carmine is obtained by adding aluminum salts to the cochineal extract underbasic conditions. Complexation of carminic acid to the metal causes a bathochromic shift of thechromophore and gives a red lake formulation of the colorant.

[0205] The widespread use of carminic acid derivatives in the food and cosmetic industries may 20 be explained in part by the fact that they are one of a few rare examples of naturally derived colors exhibiting comparable stability to artificial dyes. Indeed, processing conditions such as heat or storage under light and oxygen have a minor effect on the dye’s color and purity.

[0206] The chemical structure of carminic acid is provided below.

[0207] 36 Patent ApplicationDEBU-033 / 01WO 37396 / 182

[0208] Carminic acid is har

[0209]

[0210] us insect's dried bodies withwater or alcohol. During the extraction of carminic acid from the insect, an amount of insect protein is also released from the insect and will be contained in the color extract. Hereinafter this 5 extract is referred to as cochineal extract solution.

[0211] Very specific climatic conditions are required for cultivating carminic acid-producinginsects, and thus carmine production is limited to relatively few geographies, including Peru,Mexico, and the Canary Islands. Harvesting and processing of the insects is not an easy task: inorder to obtain 1 kg of extract for colorants, about 100,000 insects must be processed.

[0212] 10 Due to these poor yields and the fact that until a few years ago artificial colors were widelyaccepted by the global food industry, local economies in carmine-producing regions favored moreprofitable businesses such as vegetable farming (peppers, asparagus, etc.). Nevertheless, due to therecent trend for natural colors in the global food industry, the consumption of carmine increasedgreatly from 2004 to 2010 and the resulting shortage raised the price to levels never before seen.

[0213] 15 Since it is derived from animal sources, Halal and Kosher preferences both prohibit the use of carmine. Thus, alternative methods of synthesis of carminic acid are required.

[0214] Carminic acid is a natural, long-lasting, brilliant red dye used widely as a colorant in foods and cosmetics. The dye is produced by the scale insects Dactylopius coccus, which must be cultivated on Opuntia cacti under specific climate conditions, limiting D. coccus production to20 certain regions and leading to fluctuations in the availability and price of the dye. Production ofcarminic acid by synthetic biology would enable reliable manufacturing of an insect-free, high purity version of this natural red dye.

[0215] The invention provides methods and compositions for efficient production of kermesic acid and / or carminic acid.

[0216] 37 Patent Application DEBU-033 / 01WO 37396 / 182 The invention provides novel methods for bioproduction of kermesic acid and / or carminic acid. The methods of the invention are economic and reliable as compared to existing methods of production of kermesic acid and / or carminic acid. In addition, the methods provided in the invention provide higher titer and / or yield values of kermesic acid and / or carminic acid from these 5 processes as compared to kermesic acid and / or carminic acid produced from existing methods.

[0217] Production of kermesic acid:

[0218] In one aspect, the invention provides a method for production of kermesic acid, wherein the method comprises: providing one or more enzymes in a reaction medium, wherein the one or more enzymes result in transformation of one or more substrates to kermesic acid. In certain 10 embodiments, the substrate is flavokermesic acid. In certain embodiments, the enzyme is a monooxygenase enzyme. In certain embodiments, the monooxygenase enzyme results in transformation of flavokermesic acid to kermesic acid. An overview of the transformation of flavokermesic acid to kermesic acid by monooxygenase enzymes is provided in FIG.1.

[0219] In certain embodiments, the monooxygenase enzyme resulting in transformation of 15 flavokermesic acid to kermesic acid is engineered for efficient transformation of flavokermesic acid to kermesic acid.

[0220] Beneficially, the transformation of flavokermesic acid to kermesic acid may be conducted in an engineered host cell or in a cell-free medium. Thus, the methods of the invention provide that flavokermesic acid may be transformed to kermesic acid in an engineered host cell or in a cell-20 free medium. This is important because it provides the optimal modularity to develop efficient methods for production of kermesic acid. Thus, according to the invention, manufacturing processes for kermesic acid can be engineered as either cell-free or cell-based transformation of flavokermesic acid to kermesic acid.

[0221] Cell-free production of kermesic acid:

[0222] 25 In certain aspects, the invention provides methods for cell-free production of kermesic acid, 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 kermesic acid. In certain embodiments, the substrate is flavokermesic acid. In certain embodiments, the enzyme is a

[0223] 38 Patent Application DEBU-033 / 01WO 37396 / 182 monooxygenase enzyme. In certain embodiments, the monooxygenase enzyme is a monooxygenase enzyme engineered for conversion of flavokermesic acid to kermesic acid.

[0224] In certain embodiments, the reaction medium comprises NADPH and / or NADH. In certain embodiments, the reaction medium further comprises components for regeneration of NADPH 5 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 reaction medium is agitated to introduce oxygen in the reaction medium. In certain embodiments, the reaction medium further comprises oxygen. In certain embodiments, the monooxygenase enzyme is produced in an engineered host cell. In certain embodiments, the 10 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 one or more enzymes.

[0225] 15 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 kermesic acid. In certain embodiments, one or more enzymes are purified from the cell lysate for production of kermesic acid. In certain embodiments, one or more enzymes are immobilized on a solid support for cell-free production of kermesic acid from flavokermesic acid.

[0226] 20 In certain embodiments, one or more enzymes are in a solution for cell-free production of kermesic acid from flavokermesic acid. In certain embodiments, the cell-free medium further comprises: buffer, flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, GDH, and / or water. In certain preferred embodiments, the buffer is a phosphate buffer.

[0227] In certain embodiments, the methods of the invention provide that kermesic acid is 25 produced in a cell-free medium. In certain embodiments, the invention provides methods for cell- free transformation of one or more substrates to kermesic acid in a cell-free medium. In certain embodiments, the invention provides cell-free enzymatic transformation of one or more substrates to kermesic acid through one or more intermediates. In certain embodiments, the invention provides cell-free transformation of flavokermesic acid to kermesic acid.

[0228] 39 Patent Application DEBU-033 / 01WO 37396 / 182 In certain beneficial aspects, the invention recognizes that the cell-free production of kermesic acid by methods of the invention provides higher titer values of kermesic acid as compared to existing methods. The methods of the invention have high efficiency in conversion of flavokermesic acid to kermesic acid in cell-free medium. Surprisingly, the monooxygenase 5 enzymes provided in the methods of the invention provide for high efficiency cell-free and enzymatic conversion of flavokermesic acid to kermesic acid. The pre-existing methods provided only trace amounts of generation of kermesic acid in cell-free conversion of kermesic acid. Thus, beneficially, the monooxygenase enzymes provided herein for kermesic acid production lead to highly efficient and high titer generation of kermesic acid.

[0229] 10 In certain embodiments, the monooxygenase enzymes used herein are listed below in the table below.

[0230] Monooxygenases discovered for production of kermesic acid from flavokermesic acid.

[0231] NCBI Accession # Organism

[0232]

[0233] 40 Patent Application DEBU-033 / 01WO 37396 / 182 TNF55200 Burkholderiales bacterium WP013233037 Herbaspirillum seropedicae

[0234]

[0235] In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 5 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0236] In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56. In 10 certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 48-56.

[0237] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 15 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 48. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 48.

[0238] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID 20 NO: 49. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 49.

[0239] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 50. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID 25 NO: 50.

[0240] 41 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 51. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 51.

[0241] 5 In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 52. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 52.

[0242] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 10 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 53. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 53.

[0243] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID 15 NO: 54. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 54.

[0244] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 55. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID 20 NO: 55.

[0245] In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 55. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 55.

[0246] 25 In certain embodiments, the monooxygenase enzyme is an enzyme having at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity with the enzyme provided in SEQ ID NO: 56. In certain embodiments, the monooxygenase enzyme is an enzyme provided in SEQ ID NO: 56.

[0247] 42 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the monooxygenase enzymes may be either heme-dependent (such as cytochrome P450s) or flavin-dependent. In the reactions, flavokermesic acid is combined with the monooxygenase enzyme and the cofactor NADPH. In certain embodiments, the reaction mixture is agitated to introduce oxygen from the air. In certain embodiments, the reaction mixture 5 may comprise a medium for generation of oxygen. In certain embodiments, the reaction medium further comprises a suitable recycling system for NADPH regeneration. As an example of a NADPH regeneration system, glucose may be employed along with a glucose dehydrogenase (GDH).

[0248] In certain embodiments, the monooxygenase enzymes used in cell-free conversion of 10 flavokermesic acid to kermesic acid are purified enzymes. In certain embodiments, the purified monooxygenase enzymes for cell-free conversion of flavokermesic acid to kermesic acid are immobilized and used in batch or packed bed reactors.

[0249] In certain embodiments, the monooxygenase enzyme for the cell free conversion of flavokermesic acid to kermesic acid is generated from lysing a host cell overexpressing the 15 monooxygenase enzyme. In certain embodiments, the lysate generated from lysing said host cell overexpressing the monooxygenase enzyme is utilized for cell-free conversion of flavokermesic acid to kermesic acid. In certain embodiments, the monooxygenase enzyme utilized for cell-free conversion of flavokermesic acid to kermesic acid is purified from the lysate of hosts expressing the monooxygenase enzyme.

[0250] 20 In certain aspects, the one or more enzymes required for the cell-free production of kermesic acid are 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 25 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 kermesic acid. In certain embodiments, the culture comprising host organisms expressing the 30 one or more enzymes are lysed and used as the reaction medium for the methods provided in the

[0251] 43 Patent Application DEBU-033 / 01WO 37396 / 182 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.

[0252] Production of kermesic acid from flavokermesic acid in an engineered host cell:

[0253] 5 In certain aspects, the invention provides a method for production of kermesic acid in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for conversion of a substrate to kermesic acid. In certain embodiments, the substrate is flavokermesic acid. In certain embodiments, the one or more genetic modifications comprise expression and / or overexpression of a monooxygenase enzyme. In certain embodiments, the 10 engineered host cell further comprises one or more genetic modifications for reduction of use of precursors for production of kermesic acid. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the engineered host cell is cultured in a medium comprising flavokermesic acid. In certain embodiments, the engineered host cell comprises further genetic modifications for production of flavokermesic acid. In certain embodiments, one or more genetic 15 modifications comprise overexpression of a monooxygenase enzyme. In certain embodiments, one or more genetic modifications comprise overexpression of glucose dehydrogenase. In certain embodiments, the medium further comprises NADPH and / or NADH.

[0254] In certain aspects, the invention provides methods of production of kermesic acid from flavokermesic acid in an engineered host cell. In certain embodiments, flavokermesic acid is 20 transformed to kermesic acid by an enzyme. In certain embodiments, the enzyme is monooxygenase. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 85% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-25 56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0255] 44 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, in order to improve cofactor availability for the monooxygenase, the engineered host cell comprises some additional modifications. In certain embodiments, the genetic modifications in engineered host cells include deleting or downregulating genes encoding for enzymes that consume NADPH. In E. coli, there are about 80 NADPH-consuming genes. In 5 certain embodiments, one more genes consuming NADPH are deleted or downregulated. In certain embodiments, this list includes yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD. In certain embodiments, deletion or downregulation of one or more genes responsible for consuming NADPH improves availability of the cofactor NADPH for conversion of flavokermesic acid to kermesic acid by a monooxygenase.

[0256] 10 In certain embodiments, the engineered host cell is selected from a group consisting of:

[0257] 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.

[0258] 15 In certain embodiments, the engineered host cells are cultured in a medium. In certain embodiments, the medium in which the engineered host cells are grown may include one or more ingredients beneficial for the production of kermesic acid. In certain embodiments, the medium may comprise flavokermesic acid. In various aspects, host cells may be engineered for enhanced production of kermesic acid by introducing additional exogenous pathways and / or modifying 20 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 kermesic acid can be increasing yield, titer, or rate of production.

[0259] In certain embodiments, the flavokermesic acid for production of kermesic acid is produced 25 in the engineered host cell. The pathways for generation of flavokermesic acid are also disclosed herein.

[0260] In certain aspects, the invention provides a method for production of flavokermesic acid and / or kermesic acid in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of one or more substrates to flavokermesic acid

[0261] 45 Patent Application DEBU-033 / 01WO 37396 / 182 and / or kermesic acid through one or more intermediates. In certain embodiments, the one or more substrates is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more substrate is glucose, glycerol, or any combination thereof. In certain 5 embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

[0262] In certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to precursors or cofactors for production of flavokermesic acid and / or kermesic 10 acid. In certain embodiments, the one or more intermediates are selected from the group consisting of acetyl-CoA, malonyl-CoA, an octaketide, and flavokermesic acid anthrone (FKA). In certain embodiments, one more genetic modifications are selected from overexpression of polyketide synthase (PKS), aromatase, cyclases, and / or monooxygenase.

[0263] In certain embodiments, glucose or glycerol are transformed to acetyl-CoA and malonyl-15 CoA by enzymatic transformation through one or more enzymes. In certain embodiments, acetyl- CoA and malonyl-CoA are transformed to an octaketide. In certain embodiments, the transformation is mediated by a polyketide synthase (PKS). In certain embodiments, the octaketide is transformed to flavokermesic acid anthrone (FKA). In certain embodiments, the transformation is mediated by one or more cyclases or aromatases. In certain embodiments, flavokermesic acid 20 anthrone (FKA) is transformed to flavokermesic acid. In certain embodiments, said transformation is mediated by an oxygenase enzyme. In certain embodiments, said transformation is mediated by a monooxygenase enzyme. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications comprise downregulation and / or deletion of one or more genes selected from the group consisting 25 of yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD.

[0264] In certain embodiments, one or more genetic modifications are selected from the group consisting of overexpression of ACC, ACS, MCS, matC, mdcA, panK, hemA, and / or ALAS. In certain embodiments, the engineered host cell is E. coli. In certain embodiments, the medium further comprises NADPH and / or NADH.

[0265] 46 Patent Application DEBU-033 / 01WO 37396 / 182 In certain aspects, the invention provides a method for production of flavokermesic acid (FK) and / or kermesic acid (KA) in an engineered host cell. In certain embodiments, the engineered host cells comprise one or more genetic modifications, wherein the one or more genetic modifications lead to production of flavokermesic acid and / or kermesic acid from one or more 5 substrates through one or more intermediates. An overview of pathway for production of flavokermesic acid and kermesic acid is provided in FIG.3.

[0266] In certain embodiments, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of flavokermesic acid and / or kermesic acid from a carbon source that can also be an energy source, through multiple chemical intermediates, 10 by the engineered host cell. In certain embodiments, the production of flavokermesic acid and / or kermesic acid is through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group 15 consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications leads to an increase in metabolic flux to precursors or cofactors for production of flavokermesic acid and / or kermesic acid. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or 20 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 non-native genes in the engineered host cells; and (iv) a combination thereof. In certain 25 embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid. In certain embodiments, the engineered host cell is E. coli.

[0267] 47 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, one or more substrates is transformed to flavokermesic acid and / or kermesic acid through the PKS pathway. The schematic version of the pathway is provided in FIG.

[0268] 3.

[0269] In certain embodiments, one or more substrates are transformed to kermesic acid. In certain 5 embodiments, the one or more substrates that are transformed to kermesic acid are selected from the group consisting of glucose, glycerol, acetyl-CoA, malonyl-CoA, an octaketide, flavokermesic acid anthrone, and flavokermesic acid. In certain embodiments, the one or substrates is flavokermesic acid. In certain embodiments, the one or more substrates is flavokermesic acid anthrone. In certain embodiments, the one or more substrates is an octaketide. In certain 10 embodiments, the one or more substrates is acetyl-CoA and / or malonyl-CoA. In certain embodiments, the one or more substrates that are transformed to kermesic acid is a carbon source. In certain embodiments, the carbon source is a sugar. In certain embodiments, the carbon source is glucose or glycerol. In certain embodiments, the pathway for conversion of glycerol to kermesic acid is provided in FIG.3.

[0270] 15 In various aspects, host cells may be engineered for enhanced production of flavokermesic acid and / or kermesic acid 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 flavokermesic acid, kermesic acid, 20 and / or carminic acid can increase yield, titer, or rate of production.

[0271] In certain embodiments, the methods of the invention provide that a carbon source, such as glucose or glycerol is transformed to acetyl-CoA and / or malonyl-CoA. In certain embodiments, glycerol is transformed to acetyl-CoA and / or malonyl-CoA in an engineered host cell. In certain embodiments, glycerol is transformed to acetyl-CoA and / or malonyl-CoA through enzymatic 25 transformation. In certain embodiments, glycerol is transformed to acetyl-CoA and / or malonyl- CoA by a genetically engineered and / or optimized enzyme.

[0272] In certain embodiments, the one or more enzymes responsible for transformation of glucose and / or glycerol to acetyl-CoA and / or malonyl-CoA are selected from the group consisting of acetyl-CoA carboxylase (ACC), acetyl-CoA synthetase (ACS), malonyl-CoA synthetase (MCS),

[0273] 48 Patent Application DEBU-033 / 01WO 37396 / 182 malonate transporter (matC), malonate-CoA transferase (mdcA), pantothenate kinase (PanK), glutamyl-tRNA reductase (hemA), and 5-aminolevulinic acid synthase (ALAS) enzymes. In certain embodiments, one or more of these enzymes are genetically engineered and optimized for preparation of malonyl-CoA and acetyl-CoA. In certain embodiments, the pathways for generation 5 of malonyl-CoA and acetyl-CoA are provided in PCT / US2022 / 024591, which is incorporated by reference in its entirety.

[0274] Thus, a host cell engineered for the production of flavokermesic acid and / or kermesic acid can be engineered to include any or any combination of: overexpression of an acetyl-CoA carboxylase (ACC) or an ACC variant; expression or overexpression of at least one enzyme for 10 increasing the cell’s malonyl-CoA supply that does not rely on the ACC step; expression or overexpression of at least one enzyme to increase CoA availability for synthesizing precursors malonyl-CoA; deletion or downregulation of at least one fatty acid synthesis enzyme; lactate dehydrogenase, pyruvate oxidase, phosphate acetyl transferase, or acetate kinase; at least one enzyme of a fatty acid degradation pathway, at least one thioesterase, or at least one TCA gene.

[0275] 15 The foregoing list of modifications is nonlimiting.

[0276] Malonyl-CoA is the direct precursor for making the anthraquinone core of the molecule. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation of acetyl-CoA in a multistep reaction. First, the biotin carboxylase domain catalyzes the ATP-dependent 20 carboxylation of biotin using bicarbonate as a CO2 donor. In the second reaction, the carboxyl- group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes, including fungi, both reactions are catalyzed by a large single chain protein, but in E. coli and other bacteria, the activity is catalyzed by a multi-subunit enzyme. Host cells can be engineered for example to express an exogenous acetyl-CoA carboxylase or a variant ACC to increase malonyl-CoA 25 synthesis from acetyl-CoA. For example, Mucor circinelloides acetyl-CoA carboxylase can be introduced into the host cells. Additional examples of ACC genes that may be used in the engineered cells provided herein include, without limitation, genes with SEQ ID NOS: 85-88, naturally occurring orthologs of these ACCs, or variants having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to referenced 30 genes. Further, naturally occurring acetyl-CoA carboxylase genes can be further engineered to

[0277] 49 Patent Application DEBU-033 / 01WO 37396 / 182 introduce single or multiple amino acid mutations to increase catalytic activity and / or remove feedback inhibition. In certain embodiments, ACC is selected from the group consisting of:

[0278] NCBI Accession Number Organism

[0279] AT603211 Li tki

[0280]

[0281] In certain embodiments, acetyl-CoA carboxylase (ACC) is selected from the enzymes 5 having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 85- 88. In certain embodiments, ACC is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 85-88. In certain embodiments, ACC is selected from the enzymes provided in SEQ ID NOS: 85-88.

[0282] Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is 10 converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). Acetyl-CoA can be synthesized from acetate by an acyl-CoA ligase in an ATP-dependent reaction. Acetyl-CoA synthetase (ACS) or acetate-CoA ligase (EC 6.2.1.1.) catalyzes the formation of a new chemical bond between acetate and CoA coenzyme A (CoA). ACSs with native activity on acetate will provide the function of increasing acetyl-CoA supply when cells are either supplied with acetate as a co-feed, or where

[0283] 50 Patent Application DEBU-033 / 01WO 37396 / 182 acetate is produced as a by-product. Other acyl-CoA ligases, having their main activity on other acid substrates, may also have substantial activity on acetate, and are viable candidates for providing acetate-CoA ligase activity in the engineered cells provided herein. The ACSs expressed in the host cells can be prokaryotic or eukaryotic. Cultures of engineered host cells that overexpress 5 a nucleic acid sequence encoding ACS can optionally include acetate in the culture medium.

[0284] Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce FK and / or KA include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium, and orthologs of these ACSs in other species having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these 10 ACSs.

[0285] In certain embodiments, acetyl-CoA synthase (ACS) is selected from the enzymes having at least 80% amino acid sequence identity with the enzyme provided in SEQ ID NO: 89. In certain embodiments, ACS is selected from the enzymes having at least 95% amino acid sequence identity with the enzyme provided in SEQ ID NO: 89. In certain embodiments, ACS is selected from the 15 enzyme provided in SEQ ID NO: 89.

[0286] Alternatively, or in addition, an engineered host cell can overexpress a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA, to increase acetyl-CoA supply. PDH catalyzes an irreversible metabolic step, and the control of its activity is complex and involves control by its substrates and products. Nicotinamide adenine dinucleotide hydrogen 20 (NADH), a product of the PDH reaction, is a competitive inhibitor of the PDH complex. The NADH sensitivity of the PDH complex has been demonstrated to reside in LPD, the enzyme that interacts with NAD+ as a substrate. Thus, a variant of the Lpd subunit of PDH can be expressed that includes one or more mutations that reduces inhibition of PDH by NADH. Such an example is a LPD variant in E. coli that contains E354K mutation, and the mutated enzyme was less 25 sensitive to NADH inhibition than the native LPD.

[0287] Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. For example, a cell engineered to produce flavokermesic acid, kermesic acid, and / or carminic acid, as provided herein can include an exogenous nucleic 30 acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from

[0288] 51 Patent Application DEBU-033 / 01WO 37396 / 182 malonate. Acyl-CoA synthetase catalyzes the conversion of a carboxylic acid to its acyl-CoA thioester through an ATP-dependent two-step reaction. In the first step, the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate. In the second step, the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA 5 product. Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor, matB of Rhodopseudomonas palustris, matB of Rhizobium sp, BUS003, matB of Ochrobacrum sp.,or other homologs having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced sequences. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered 10 to express a malonyl-CoA synthetase. In Rhizobium trifolii, the matB gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, 15 for example of Streptomyces coelicolor, of Rhizobiales bacterium, of Rhizobium leguminosarum, of Agrobacterium vitis, of Neorhizobium sp., or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a naturally- occurring malonate transporter. Cell cultures of a host cell engineered to express a malonyl-CoA 20 synthetase and a malonate transporter can include a culture medium that includes malonate.

[0289] In certain embodiments, malonyl-CoA synthetase (MCS) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 90-93. In certain embodiments, MCS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID 25 NOS: 90-93. In certain embodiments, MCS is selected from the enzymes provided in SEQ ID NOS: 90-93.

[0290] In certain embodiments, malonate transporter (matC) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 94-98. In certain embodiments, matC is selected from the 30 enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID

[0291] 52 Patent Application DEBU-033 / 01WO 37396 / 182 NOS: 94-98. In certain embodiments, matC is selected from the enzymes provided in SEQ ID NOS: 94-98.

[0292] In additional embodiments, a cell engineered to produce flavokermesic acid and / or kermesic acid is further engineered to include an exogenous nucleic acid sequence encoding 5 malonate CoA-transferase (EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase) that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity. The mdcA gene product, the subunit, is malonate CoA-transferase, and mdcD gene product, the subunit, is a malonyl-10 CoA decarboxylase. The mdcE gene product, the subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the subunit, was an When the subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an subunit to exchange the acetyl group for a malonyl group. As the 15 thioester transfer should be thermodynamically favorable, the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply. Examples of mdcAs that can be expressed in an engineered cell as provided herein include, without limitation, mdcA of Acinetobacter calcoaceticus, mdcAs with SEQ ID NOS: 99-103, or a transferase having at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% 20 identity to any of these or other naturally occurring malonate CoA-transferases.

[0293] In certain embodiments, the mdcA are selected from the group consisting of:

[0294] NCBI Accession Number Organism

[0295]

[0296] 53 Patent Application DEBU-033 / 01WO 37396 / 182 WP_064617969.1 Moraxella catarrhalis MBL02837421 Zooloea s

[0297]

[0298] In certain embodiments, malonate-CoA transferase (mdcA) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 99-103. In certain embodiments, mdcA is selected from 5 the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 99-103. In certain embodiments, mdcA is selected from the enzymes provided in SEQ ID NOS: 99-103.

[0299] In some embodiments, a cell engineered to produce flavokermesic acid and / or kermesic acid is further engineered to increase the supply of coenzyme A (CoA) to increase its availability 10 for producing acetyl-CoA, malonyl-CoA, and / or p-coumaroyl-CoA. Strategies for increasing CoA supply include expressing or overexpressing at least one enzyme of a CoA biosynthesis pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4'- phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-15 limiting step in the biosynthesis of CoA. Three distinct types of PanK have been identified - PanK- I (found in bacteria), PanK-II (mainly found in eukaryotes, but also in the Staphylococci) and PanK-III, also known as CoaX (found in bacteria). In E. coli, pantothenate kinase is competitively inhibited by CoA itself, as well as by some CoA esters. The type III enzymes CoaX are not subject to feedback inhibition by CoA. In some embodiments, a host cell can be engineered to include a 20 nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, without limitation, CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33), PanK of Pseudomonas putida. WP_274132471, Bacillus sp., or Thermotoga sp. Cultures of cells engineered for the production of flavokermesic acid and / or kermesic acid can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and 25 can optionally also include cysteine, used in the CoA biosynthesis.

[0300] 54 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, pantothenate kinase (panK) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 104-107. In certain embodiments, panK is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID 5 NOS: 104-107. In certain embodiments, panK is selected from the enzymes provided in SEQ ID NOS: 104-107.

[0301] Additional strategies to increase malonyl-CoA flux to the flavokermesic acid and / or kermesic acid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavokermesic acid and / or 10 kermesic acid biosynthesis for the precursor malonyl-CoA and thus limits flavokermesic acid and / or kermesic acid formation. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavokermesic acid and / or kermesic acid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) can be disrupted, attenuated or deleted to reduce fatty acid biosynthesis.

[0302] 15 Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl- ACP synthase I enzyme (E. coli fabB) and / or acyl carrier protein (E. coli acpP).

[0303] Additional genetic modifications that may be present in a host cell engineered to produce 20 flavokermesic acid and / or kermesic acid include downregulation, disruption, or deletion of the gene targets that divert carbon flux to form byproducts such as ethanol, acetate, and lactate. They include genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In an E. coli host cell, genes that are downregulated, disrupted, or deleted can include adhE, ldhA, poxB, and ackA-pta.

[0304] 25 Further, a cell engineered for the production of flavokermesic acid and / or kermesic acid can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, acetyl-CoA, and / or p-coumaryol-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme

[0305] 55 Patent Application DEBU-033 / 01WO 37396 / 182 A. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and / or ybgC can be downregulated, disrupted, or deleted.

[0306] In further embodiments, a cell engineered for the production of flavokermesic acid and / or kermesic acid can have one or more of fatty acid degradation genes downregulated, disrupted, or 5 deleted to improve precursor supply to the flavokermesic acid and / or kermesic acid pathway. In E.

[0307] coli, for example, the acyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and / or gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted, or deleted. Alternatively, or in addition, other TCA enzymes that can be downregulated including citrate 10 synthase that converts acetyl-CoA to citrate.

[0308] In certain embodiments, the invention provides an engineered host cell for the production of flavokermesic acid and / or kermesic acid further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an 15 effective strategy to improve the production of kermesic acid, and / or carminic acid from flavokermesic acid. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There exist two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA synthase (ALAS). The C4 pathway is 20 restricted to mammals, fungi and purple nonsulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions, including assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chains, and incorporation of reduced iron into the molecule, are required 25 to convert ALA to heme. In an E. coli host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (hemA), and glutamate-1- semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and / or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be 30 overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium

[0309] 56 Patent Application DEBU-033 / 01WO 37396 / 182 (EC:1.1.1.70) and hemA with SEQ ID NOS: 108-111. Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway. Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Rhodobacter capsulatus, ALAS with SEQ ID NOS: 112-116, or an ALAS having at least 85%, at least 90%, at least 95%,

[0310]

[0311] 5 at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally- occurring ALAS. Further, one or more of the downstream genes (E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavokermesic acid and / or kermesic acid can in some embodiments include a 10 medium that includes succinate and / or glycine, precursors of heme biosynthesis via the C4 pathway.

[0312] In certain embodiments, glutamyl-tRNA reductase (hemA) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 108-111. In certain embodiments, hemA is selected from 15 the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 108-111. In certain embodiments, hemA is selected from the enzymes provided in SEQ ID NOS: 108-111.

[0313] In certain embodiments, ALA synthase (ALAS) is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the 20 enzymes provided in SEQ ID NOS: 112-116. In certain embodiments, ALAS is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 112-116. In certain embodiments, ALAS is selected from the enzymes provided in SEQ ID NOS: 112-116.

[0314] In certain embodiments, the invention provides that acetyl-CoA and / or malonyl-CoA are 25 transformed to an octaketide. In certain embodiments, the acetyl-CoA and / or malonyl-CoA are transformed to an octaketide in an engineered host cell. In certain embodiments, acetyl-CoA and / or malonyl-CoA are transformed to an octaketide through enzymatic transformation. In certain embodiments, acetyl-CoA and / or malonyl-CoA are transformed to an octaketide through a genetically engineered enzyme. In certain embodiments, the transformation of acetyl-CoA and / or 30 malonyl-CoA to an octaketide by polyketide synthase (PKS). In certain embodiments, polyketide

[0315] 57 Patent Application DEBU-033 / 01WO 37396 / 182 synthase (PKS) is engineered to optimize transformation of acetyl-CoA and / or malonyl-CoA to an octaketide. In certain embodiments, PKS is a PKSIII or PKSII. In certain preferred embodiments, PKSIII enzymes used for microbial conversion of acetyl-CoA and / or malonyl-CoA are provided in the Table below.

[0316] 5 PKSIIIs used for microbial production of flavokermesic acid (FK)

[0317] NCBI Accession Number Organism

[0318] A772 Pl i

[0319]

[0320] In certain preferred embodiments, PKSII enzymes used for microbial conversion of acetyl- CoA and / or malonyl-CoA are provided in the Table below.

[0321] PKSII used for microbial production of flavokermesic acid (FK)

[0322] NCBI Accession Number Organism

[0323]

[0324] 58 Patent Application DEBU-033 / 01WO 37396 / 182 WP_011148297.1 Photorhabdus laumondii subsp. Laumondii CAA448581 Bacillus subtilis subs subtilis str168

[0325]

[0326] In certain embodiments, PKS is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 57-64. In certain embodiments, PKS is selected from the enzymes having at least 95% 5 amino acid sequence identity from the enzymes provided in SEQ ID NOS: 57-74. In certain embodiments, PKS is selected from the enzymes provided in SEQ ID NOS: 57-74.

[0327] In certain embodiments, the invention provides that octaketide is transformed to flavokermesic acid anthrone. In certain embodiments, octaketide is transformed to flavokermesic acid anthrone. In certain embodiments, octaketide is transformed to flavokermesic acid anthrone 10 is in an engineered host cell. In certain embodiments, octaketide is transformed to flavokermesic acid anthrone in an engineered host cell. In certain embodiments, the transformation of octaketide to flavokermesic acid anthrone is through one or more genetically engineered enzymes. In certain embodiments, the one or more genetically engineered enzymes are one or more aromatases and / or cyclases. In certain embodiments, the genetically engineered enzyme is a cyclase. In certain 15 embodiments, the genetically engineered enzyme is an aromatase. In certain embodiments, the cyclase and / or aromatase enzymes are optimized by a genetically engineered enzyme. In certain embodiments, the aromatase and / or cyclase enzymes are selected from the table below.

[0328] Cyclases and aromatases used for microbial production of flavokermesic acid or kermesic acid NCBI Accession Number Organism

[0329]

[0330] 59 Patent Application DEBU-033 / 01WO 37396 / 182 WP_189057072.1 Longimycelium tulufanense WP2231035871 Dactlosoranium vinaceum

[0331]

[0332] In certain embodiments, aromatase and / or cyclase enzymes are selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 75-84. In certain embodiments, aromatase and / or cyclase 5 enzymes are selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 75-84. In certain embodiments, aromatase and / or cyclase enzymes are selected from the enzymes provided in SEQ ID NOS: 75-84.

[0333] In certain embodiments, the invention provides that flavokermesic acid anthrone is transformed to flavokermesic acid. In certain embodiments, the transformation of flavokermesic 10 acid anthrone to flavokermesic acid is in an engineered host cell. In certain embodiments, the transformation of flavokermesic acid anthrone to flavokermesic acid is mediated through one or more enzymes. In certain embodiments, the one or more enzymes for transformation of flavokermesic acid anthrone to flavokermesic acid are genetically engineered or modified.

[0334] In certain embodiments, for generation of flavokermesic acid in an engineered host cell, 15 the genes for PKS pathway (described in FIG.3), such as PKSII, PKSIII, or both, are combined with the genes for cyclases and aromatases to produce flavokermesic acid anthrone. The produced flavokermesic acid anthrone spontaneously oxidizes to flavokermesic acid. The genes for said enzymes may be in in plasmids or integrated into the chromosome of the engineered host cell. Additionally, other modifications to the host may be required to improve the flux towards the 20 products-of-interest.

[0335] In certain embodiments, the invention provides that flavokermesic acid is transformed to kermesic acid. In certain embodiments, the transformation of flavokermesic acid to kermesic acid is in an engineered host cell. In certain embodiments, flavokermesic acid is enzymatically transformed to kermesic acid. In certain embodiments, the enzyme is genetically engineered to 25 optimize transformation of flavokermesic acid to kermesic acid. In certain embodiments, the enzyme is monooxygenase (MOX). In certain embodiments, the monooxygenase enzyme is

[0336] 60 Patent Application DEBU-033 / 01WO 37396 / 182 selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain 5 embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80%, 85%, 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the 10 enzymes provided in SEQ ID NOS: 48-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 48-56.

[0337] In certain embodiments, in order to improve cofactor availability for the monooxygenase, the engineered host cell comprises some additional modifications. In certain embodiments, the genetic modifications in engineered host cells include deleting or downregulating genes encoding 15 for enzymes that consume NADPH. In E. coli, there are about 80 NADPH-consuming genes. IN certain embodiments, one more genes consuming NADPH are deleted or downregulated. In certain embodiments, this list includes yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD. In certain embodiments, deletion or downregulation of one or more genes responsible consuming NADPH improves availability of the cofactor NADPH for conversion of flavokermesic 20 acid to kermesic acid by a monooxygenase.

[0338] The method of the invention for production of kermesic acid 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 glycerol. Because the methods of the invention provide manufacture of kermesic acid with an economic carbon source, 25 the methods of the invention are economically efficient.

[0339] In certain embodiments, the invention provides a method for manufacturing kermesic acid through one or more of the following steps:

[0340] (a) transformation of glycerol or a carbon source to acetyl-CoA and / or malonyl-CoA; (b) transformation of acetyl-CoA and / or malonyl-CoA to an octaketide;

[0341] 61 Patent Application DEBU-033 / 01WO 37396 / 182 (c) transformation of an octaketide to flavokermesic acid anthrone;

[0342] (d) transformation of flavokermesic acid anthrone to flavokermesic acid; and (e) transformation of flavokermesic acid to kermesic acid.

[0343] In certain embodiments, one or more steps of (a)-(e) are mediated by enzymatic 5 transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a)-(e) are engineered enzymes.

[0344] 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 kermesic acid. In certain embodiments, the engineered host cells are modified to express one or more 10 enzymes for transformation in steps (a)-(e).

[0345] In certain embodiments, all the steps (a)-(e) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one more enzymes for the transformations listed in steps (a)-(e).

[0346] In certain embodiments, steps (a)-(d) are conducted in an engineered host cell. In certain 15 embodiments, the engineered host cell expresses one more enzymes for the transformations listed in steps (a)-(d). In these embodiments, step (e) is optionally carried out in a cell-free medium.

[0347] 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 20 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 grown may include one or more ingredients beneficial for the production of flavokermesic acid and / or kermesic acid. In various aspects, host cells may be engineered for enhanced production of 25 flavokermesic acid and / or kermesic acid 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 flavokermesic acid and / or kermesic acid can increase yield, titer, or rate of production.

[0348] 62 Patent Application DEBU-033 / 01WO 37396 / 182 ENGINEERED HOST CELLS

[0349] 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 5 cyanobacterial cells.

[0350] 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.

[0351] Nonlimiting examples of suitable microbial hosts for the bio-production of flavokermesic 10 acid and / or kermesic acid include, but are not limited to: any gram-negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coli; 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 15 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, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, Yarrowia, Kluyveromyces, and Saccharomyces.

[0352] 20 CULTURE MEDIUM

[0353] In yet another aspect, methods for producing flavokermesic acid and / or kermesic acid 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.

[0354] 25 The culture comprises cells engineered for the production of flavokermesic acid and / or kermesic acid 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. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. Such carbon sources may be purified or crude, including a biomass comprising glycerol, for example,

[0355] 63 Patent Application DEBU-033 / 01WO 37396 / 182 crude glycerol produced as a byproduct of biodiesel production from corn waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply such as, without limitation, acetate, malonate, succinate, glycine, bicarbonate, biotin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and 5 ascorbate.

[0356] 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 flavokermesic acid and / or kermesic acid products include aerobic or 10 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 is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds. The desired 15 temperature can be from, for example, 20-37 ºC, 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 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for example, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to 20 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 FK and / or KA, to homogeneity, further processing can be done, including by solvent extraction, ion exchange or silica-based chromatography, among many others.

[0357] Cell-free production of carminic acid from flavokermesic acid:

[0358] 25 In certain aspects, the invention provides a method of cell free production of carminic acid, wherein the method comprises providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of flavokermesic acid to carminic acid through one or more intermediates. In certain embodiments, one or more intermediates are selected from the group consisting of kermesic acid and C-glucosylated flavokermesic acid. In certain embodiments, 30 one or more enzymes are selected from the group consisting of monooxygenase, C- 64 Patent Application DEBU-033 / 01WO 37396 / 182 glucosyltransferase (CGT), glucose dehydrogenase (GDH), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP— glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK).

[0359] In certain embodiments, one or more enzymes are selected from the group consisting of 5 monooxygenase and / or C-glucosyltransferase. In certain embodiments, the cell-free medium further comprises glucose dehydrogenase (GDH) enzyme. In certain embodiments, the cell-free medium comprises flavokermesic acid and monooxygenase enzyme. In certain embodiments, UDP-glucose and CGT are added to the cell-free medium. In certain embodiments, the cell-free medium comprises UDP-glucose, monooxygenase and / or C-glucosyltransferase. In certain 10 embodiments, UDP-glucose is UDP-glucose is synthesized in the cell-free medium by the one or more enzymes. In certain embodiments, UDP-glucose is synthesized from one or ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6-phosophate, glucose-1-phosphate, and / or polyphosphate. In certain embodiments, one or more enzymes is selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase 15 (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK).

[0360] In certain embodiments, the cell-free medium is agitated to include oxygen from the atmosphere. In certain embodiments, the cell-free medium further comprises NADPH / NADH. In certain embodiments, the cell-free medium further comprises an NADPH regeneration system. In 20 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, one or more enzymes are introduced in the host cell by integration into the genome of the host cell or on a plasmid. In 25 certain embodiments, the engineered host cell expressing one or more enzymes are cultured until a pre-determined biomass is achieved to produce the requisite quantity of the one or more enzymes. In certain embodiments, the engineered host cells are grown followed by removal of cell debris to generate a cell lysate for use in the cell-free medium for cell-free production of carminic acid.

[0361] In certain embodiments, one or more enzymes are purified from the cell lysate for 30 production of carminic acid. In certain embodiments, one or more enzymes are immobilized on a

[0362] 65 Patent Application DEBU-033 / 01WO 37396 / 182 solid support for cell-free production of carminic acid from flavokermesic acid. In certain embodiments, one or more enzymes are in a solution for cell-free production of carminic acid from flavokermesic acid. In certain embodiments, the cell-free medium further comprises: buffer, flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, GDH, and / or water.

[0363] 5 In certain embodiments, the buffer is a phosphate buffer. In certain embodiments, the reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution. In certain embodiments, the reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized. In certain embodiments, the method results in titer values of produced carminic acid from about 10 times to about 5000 times higher than methods for cell-based production of 10 carminic acid.

[0364] In one aspect, the invention provides a method for cell-free production of carminic acid, wherein the method comprises: providing one or more enzymes in a cell-free medium, wherein the one or more enzymes result in transformation of flavokermesic acid to carminic acid through one or more intermediates. In certain embodiments, the one or more enzymes that transforms 15 flavokermesic acid to carminic acid are C-glucosyltransferase (CGT), monooxygenase (MOX), or a combination thereof. In certain embodiments, one or more intermediates are selected from the group consisting of kermesic acid (KA) and C-glucosylated flavokermesic acid (DCII). An overview of the synthetic pathway for cell-free conversion of flavokermesic acid to carminic acid is provided in FIG.5.

[0365] 20 In certain embodiments, the method of the invention provides that monooxygenase and C- glucosyltransferase transform flavokermesic acid to carminic acid. In certain embodiments, monooxygenase and C-glucosyltransferase are added to the cell-free reaction simultaneously. In these embodiments, UDP-glucose and other reaction components are added to the reaction medium simultaneously. In these embodiments, kermesic acid or C-glucosyltransferase are intermediates, 25 but they are not isolated or purified. In certain embodiments, the cell-free reaction may proceed more favorably via the generation of C-glucosylated flavokermesic acid because the C- glucosyltransferase enzyme acts first to convert flavokermesic acid to C-glucosylated flavokermesic acid, and monooxygenase acts second to convert C-glucosylated flavokermesic acid to carminic acid.

[0366] 66 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, a first step is carried out by addition of monooxygenase to the reaction system, and the reaction is carried to completion. Subsequently, C-glucosyltransferase is added to the reaction medium. In these embodiments, monooxygenase enzyme transforms flavokermesic acid to kermesic acid. In certain embodiments, the details of the cell-free reaction 5 for transformation of flavokermesic acid to kermesic acid are provided herein. In certain embodiments, kermesic acid is not purified or isolated. In certain embodiments, kermesic acid is further transformed to carminic acid through one or more enzymes. In certain embodiments, C- glucosyltransferase (CGT) transforms kermesic acid to carminic acid in a cell-free reaction medium. In certain embodiments, the reaction medium for C-glucosyltransferase (CGT) mediated 10 enzymatic transformation of kermesic acid to carminic acid further comprises an activated sugar.

[0367] In certain embodiments, the activated sugar is uridine diphosphate glucose (UDP-glucose). In certain embodiments, UDP-glucose is an essential co-factor for the transformation of kermesic acid to carminic acid. An overview of the conversion of kermesic acid to carminic acid is provided in FIG.7.

[0368] 15 In certain embodiments, the monooxygenase enzymes used herein are listed below in the table below.

[0369] NCBI Accession # Organism

[0370]

[0371] 67 Patent Application DEBU-033 / 01WO 37396 / 182 KPF58291 beta proteobacterium AAP51 WP284615091 Rubrivivax pictus

[0372]

[0373] In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 5 85% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56. In certain embodiments, the monooxygenase enzyme is selected from the enzymes provided in SEQ ID NOS: 39-56.

[0374] 10 In certain embodiments, the C-glucosylated flavokermesic acid (DCII) is produced by enzymatic conversion of flavokermesic acid to C-glucosylated flavokermesic acid (DCII). In certain embodiments, the enzyme for conversion of flavokermesic acid to C-glucosylated flavokermesic acid (DCII) is C-glucosyltransferase (CGT). In certain embodiments, the CGT enzymes for production of C-glucosylated flavokermesic acid (DCII) are provided in the Table 15 below.

[0375] In certain embodiments, the CGT enzymes used herein are listed in the Table below:

[0376] NCBI Accession # Organism

[0377]

[0378] 68 Patent Application DEBU-033 / 01WO 37396 / 182 KAD6795309 Mikania micrantha XP006282251 Capsella rubella

[0379]

[0380] In certain aspects, the invention provides methods for production of carminic acid. In certain embodiments, carminic acid is produced by enzymatic transformation of one or more substrates to carminic acid. In certain embodiments, the substrate is kermesic acid. Thus, in certain 5 embodiments, kermesic acid is enzymatically transformed to carminic acid. In certain embodiments, the enzyme responsible for enzymatic transformation of kermesic acid to carminic acid is glucosyltransferase. In certain embodiments, the enzymatic transformation of kermesic acid to carminic acid is conducted in a cell-free medium.

[0381] In certain embodiments, the one or more enzymes involved in transformation of kermesic 10 acid to carminic acid are glucosyltransferase (GT), C-glucosyltransferase (CGT), and / or sucrose synthase (SuSy). In certain embodiments, kermesic acid is transformed to carminic acid in a cell free medium comprising C-glucosyltransferase (CGT) and / or sucrose synthase (SuSy) enzymes. An overview of the process where kermesic acid is converted to carminic acid and UDP-glucose is generated from sucrose is provided in FIG.7. In certain embodiments, GT / CGT is selected from 15 the enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, GT / CGT is selected from the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, GT / CGT is selected from the enzymes provided in SEQ ID NOS: 1-15. In certain embodiments, SuSy is selected from the enzymes having at least 80% amino acid sequence identity 20 from the enzymes provided in SEQ ID NOS: 16-25. In certain embodiments, SuSy is selected from

[0382] 69 Patent Application DEBU-033 / 01WO 37396 / 182 the enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 16-25. In certain embodiments, SuSy is selected from the enzymes provided in SEQ ID NOS: 16-25.

[0383] In certain embodiments, the invention provides methods for production of carminic acid.

[0384] 5 In certain embodiments, the methods of the invention provide that carminic acid is produced by enzymatic transformation of flavokermesic acid to carminic acid through one or more intermediates. In certain embodiments, the enzymatic transformation of flavokermesic acid to carminic acid is conducted in a cell-free medium. In certain embodiments, the one or more enzymes responsible for transformation of flavokermesic acid are selected from the group 10 consisting of monooxygenase and CGT. In certain embodiments, flavokermesic acid is enzymatically transformed to kermesic acid through a monooxygenase. In certain embodiments, kermesic acid is enzymatically transformed to carminic acid through CGT.

[0385] In certain beneficial aspects, the invention provides that the UDP-glucose used in the reaction is generated from other substrates in course of the reaction. In certain embodiments, the 15 UDP moiety in UDP-glucose is recycled in course of the process. The recycling of UDP provides economic efficiency of the processes of the invention. Accordingly, in certain aspects, the production of carminic acid from kermesic acid catalyzed by CGT is conducted in conjunction with other methods for UDP-glucose production or recycling of UDP. In certain embodiments, the overview of the synthetic scheme involving the UDP-glucose production and / or recycling is 20 provided in FIG.8. In certain embodiments, UDP-glucose is generated by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridylyltransferase (UGP). In certain preferred embodiments, the UGP may be galU.

[0386] In certain embodiments, the process for transformation of kermesic acid to carminic acid 25 is provided in PCT / US2024 / 035784, which is incorporated by reference in its entirety. In certain embodiments, the one more enzymes expressed in an engineered host cell for transformation of kermesic acid to carminic acid include C-glucosyltransferase (CGT), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase 30 (NDK). In certain embodiments, C-glucosyltransferase (CGT), sucrose synthase (SuSy),

[0387] 70 Patent Application DEBU-033 / 01WO 37396 / 182 glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). An outline for the enzymatic pathway for conversion of kermesic acid to carminic acid is provided in FIG.1, FIG.2, and FIG.3 of PCT / US2024 / 035784, which is incorporated by reference 5 in its entirety. In certain embodiments, glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK). In certain embodiments, GLK, PGM, PPK, UGP, and / or NDK are enzymes having at least 80% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 26-38. In certain embodiments, GLK, PGM, 10 PPK, UGP, and / or NDK are enzymes having at least 95% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 26-38. In certain embodiments, GLK, PGM, PPK, UGP, and / or NDK are enzymes are selected from the enzymes provided in SEQ ID NOS: 26-38.

[0388] In certain embodiments, the invention provides engineered enzymes for the cell-free production of kermesic acid and / or carminic acid. In certain embodiments, the engineered enzymes 15 are optimized for cell-free production of kermesic acid and / or carminic acid. In certain embodiments, engineered enzymes may include genetic modifications. In certain embodiments, the genetic modifications may be selected from a group consisting of: point mutations, insertions, deletions, and / or any other modifications such that those enzymes result in efficient and optimal cell-free production of kermesic acid and / or carminic acid. In certain embodiments, the enzymes 20 used in the cell-free production of kermesic acid and / or carminic acid are any enzymes disclosed herein.

[0389] In certain aspects of the invention, where the one or more steps for production of kermesic acid and / or carminic acid are conducted in a cell-free medium, the method of the cell-free production does not require the purification of the one or more enzymes from the lysed host 25 organisms. Accordingly, in certain embodiments, the methods of the invention do not require the purification of one or more enzymes from the lysed biomass comprising host cell expressing the one or more enzymes for the cell-free production of kermesic acid and / or carminic acid.

[0390] In certain embodiments, the cell-free medium may further comprise any other additional ingredients required for the cell-free production of carminic acid. In certain embodiments, the cell-30 free medium comprises buffer, kermesic acid, an activated sugar, magnesium chloride, cell lysate,

[0391] 71 Patent Application DEBU-033 / 01WO 37396 / 182 sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water. In certain embodiments, the cell-free medium comprises buffer, flavokermesic acid, kermesic acid, an activated sugar, magnesium chloride, cell lysate, sucrose, and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for enzymatic 5 conversion of kermesic acid to carminic acid. In certain embodiments, the buffer maintains the pH of about 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 present at a concentration of about 1 mM to about 200 10 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 100 mM.

[0392] In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.001 mM to about 50 mM. In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.01 mM to about 10 mM. In certain 15 embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.01 mM to about 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0393] In certain embodiments, the cell-free reaction medium comprises magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction medium comprises magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0394] 20 In certain embodiments, the cell-free reaction medium further comprises sucrose. In certain embodiments, the sucrose is converted to UDP-glucose. Thus, the concentration of sucrose in the reaction mixture is determined by the quantity of UDP-glucose needed for the reaction for cell- free production of carminic acid. In certain embodiments, sucrose is present at a concentration of about 10 mM to about 1000 mM in the cell-free reaction medium. In certain embodiments, sucrose 25 is present at a concentration of about 20 mM to about 800 mM in the cell-free reaction medium.

[0395] In certain embodiments, sucrose is present at a concentration of about 50 mM to about 600 mM in the cell-free reaction medium.

[0396] In certain embodiments, the quantity of the one or more enzymes for the cell-free production of kermesic acid and / or carminic acid is dependent on the target quantity of kermesic

[0397] 72 Patent Application DEBU-033 / 01WO 37396 / 182 acid and / or carminic acid to be produced and / or the concentration of other ingredients present in the reaction mixture. In certain embodiments, the one or more enzymes are present in a concentration of from about 1% to about 50% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 2.5% to about 45% (v / v). In certain 5 embodiments, the one or more enzymes are present in a concentration of from about 5% to about 40% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 7.5% to about 30% (v / v).

[0398] In certain embodiments, the reaction for production of kermesic and / or carminic acid is conducted for a duration until the desired quantity of kermesic acid and / or carminic acid is 10 obtained. In certain embodiments, the reaction for cell-free production of kermesic and / or carminic acid is carried out from about 10 minutes to about 48 hours. In certain embodiments, the reaction for cell-free production of kermesic acid and / or carminic acid is carried out from about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of kermesic and / or carminic acid is carried out from about 20 minutes to about 24 hours. In certain embodiments, the 15 reaction for cell-free production of kermesic acid and / or carminic acid is carried out from about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of kermesic acid and / or carminic acid is carried out from about 1 hour to about 15 hours.

[0399] In certain embodiments, the temperature of the reaction mixture for cell-free production of kermesic acid and / or carminic acid is varied to obtain optimal results for the production of 20 kermesic acid and / or carminic acid. In certain embodiments, the temperature of the reaction mixture for cell-free production of kermesic acid and / or

[0400] -free production of kermesic acid and / or

[0401] the temperature of the reaction mixture for cell-free production of kermesic acid and / or carminic 25 -free production of kermesic acid and / or carminic acid may influence the rate of production of kermesic acid and / or carminic acid. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to obtain optimal yield of kermesic acid and / or carminic acid in cell-free production of kermesic acid and / or carminic acid.

[0402] 73 Patent Application DEBU-033 / 01WO 37396 / 182 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 kermesic acid and / or carminic acid. In certain embodiments, the reaction for cell-free production of kermesic acid and / or carminic acid is conducted in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column 5 reactor / bioreactor, the one or more enzymes involved in cell-free production of kermesic acid and / or carminic acid are in a solution. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a bubble column reactor / bioreactor comprises the lysate from the host organism. In certain embodiments, it is advantageous to use the bubble column reactor / bioreactor for cell-free production of kermesic acid and / or carminic acid when the reaction 10 mixture involves the lysate (or lysate with cellular debris removed) from the host cell organisms in which the one or more enzymes responsible for cell-free production of kermesic acid and / or carminic acid were utilized. In certain embodiments, the reaction for cell-free production of kermesic acid and / or carminic acid is conducted in a packed bed reactor / bioreactor. In certain embodiments, the one or more enzymes are immobilized in the packed bed reactor / bioreactor. The 15 packed bed reactors / bioreactors are preferred for the purified enzymes playing a role in cell-free production of carminic acid. In certain embodiments, the one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, the one or more enzymes may be immobilized in different reactors / bioreactors, wherein these reactors / bioreactors are linked sequentially. In certain embodiments, the bioreactor system provided in PCT / US2021 / 064049, 20 incorporated by reference in its entirety.

[0403] The methods provided in the invention are advantageous over other conventional methods of production of kermesic acid and / or carminic acid. In certain embodiments, the methods of the invention provide cell-free production of kermesic acid and / or carminic acid. Because the methods of the invention are conducted in cell-free medium, they provide significant economic efficiency 25 by reducing the cost of production of carminic acid in other conventional methods. In certain embodiments, because the reaction for production of kermesic acid and / or carminic acid is conducted from the lysates from the host organisms expressing the one or more enzymes involved in the reaction, the methods of the invention are cost-efficient. In particular, in certain embodiments, the methods of the invention do not involve the purification of the one or more 30 enzymes. Because purification of individual enzymes is not required in the methods of the

[0404] 74 Patent Application DEBU-033 / 01WO 37396 / 182 invention, it provides further economic efficiency by reducing the cost that would have been otherwise required in purifying individual enzymes.

[0405] Advantageously, the cell-free production of kermesic acid and / or carminic acid provided herein provides significantly higher titer values for kermesic acid and / or carminic acid as 5 compared to the conventional methods. The higher titer values provide additional cost advantages for production of kermesic acid and / or carminic acid because the higher titers provide efficiency in purifying and / or concentrating kermesic acid and / or carminic acid from the reaction mixture. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 5-fold higher than the conventional methods. In certain embodiments, the 10 methods of the invention provide kermesic acid and / or carminic acid titer values at least 10-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 50-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 100-fold higher than the conventional methods. In certain 15 embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 500-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 1000-fold higher than the conventional methods. In certain embodiments, the methods of the invention provide kermesic acid and / or carminic acid titer values at least 5000-fold higher than the conventional methods. 20 In some embodiments, the isolated kermesic acid and / or carminic acid 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%.

[0406] In other embodiments, the isolated kermesic acid and / or carminic acid 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 25 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%.

[0407] 75 Patent Application DEBU-033 / 01WO 37396 / 182 In certain aspects, the invention provides compositions for cell-free production of kermesic acid and / or carminic acid. The compositions of the invention are utilized for cell-free production of kermesic acid and / or carminic acid in accordance with the methods described above.

[0408] In certain embodiments, the monooxygenase enzyme and C-glucosyltransferase enzymes 5 used in cell-free conversion of flavokermesic acid to carminic acid are purified enzymes. In certain embodiments, the purified monooxygenase enzyme and C-glucosyltransferase enzymes for cell- free conversion of flavokermesic acid to carminic acid are used in batch or immobilized packed bed reactors.

[0409] In certain embodiments, the monooxygenase enzyme and C-glucosyltransferase enzymes 10 for the cell free conversion of flavokermesic acid to carminic acid are generated from lysing a host cell overexpressing the monooxygenase enzyme and C-glucosyltransferase enzymes. In certain embodiments, the lysate generated from lysing said host cell overexpressing the monooxygenase enzyme and C-glucosyltransferase enzymes is utilized for cell-free conversion of flavokermesic acid to carminic acid. In certain embodiments, the monooxygenase enzyme and C-15 glucosyltransferase enzymes utilized for cell-free conversion of flavokermesic acid to carminic acid is purified from the lysate of hosts expressing the monooxygenase enzyme.

[0410] In certain aspects, the one or more enzymes required for the cell-free production of carminic acid are 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, 20 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 25 calculated based on the quantity of the one or more enzymes required for the cell-free production of kermesic acid. 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 30 the invention.

[0411] 76 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, the cell-free medium for production of carminic acid from flavokermesic acid may further comprise any other additional ingredients required for the cell-free production of carminic acid. In certain embodiments, the cell-free reaction medium further comprises UDP-glucose.

[0412] 5 In certain embodiments, the UDP-glucose for the reaction medium is generated from sucrose. In certain embodiments, the UDP-glucose is generated in the reaction medium by one or more enzymes. In certain embodiments, the generation of UDP-sugar is mediated by sucrose synthase (SuSy). In certain embodiments, sucrose synthase (SuSy) mediates the conversion of sucrose to UDP-glucose. Thus, in certain embodiments, the reaction medium comprises sucrose, 10 which is subsequently converted to UDP-glucose in the course of the reaction. An overview of the process where kermesic acid is converted to carminic acid and UDP-glucose is generated from sucrose is provided in FIG.7.

[0413] In certain beneficial aspects, the invention provides that the UDP-glucose used in the reaction is generated from other substrates in course of the reaction. In certain embodiments, the 15 UDP moiety in UDP-glucose is recycled in course of the process. The recycling of UDP provides economic efficiency of the processes of the invention. Accordingly, in certain aspects, the production of carminic acid from kermesic acid catalyzed by CGT is conducted in conjunction with other methods for UDP-glucose production or recycling of UDP. In certain embodiments, the overview of the synthetic scheme involving the UDP-glucose production and / or recycling is 20 provided in FIG.8. In certain embodiments, UDP-glucose is generated by the reaction of uridine triphosphate (UTP) with glucose-1-phosphate. In certain preferred embodiments, the reaction of UTP and glucose-1-phosphate is catalyzed by UTP-glucose-1-phosphate uridylyltransferase (UGP). In certain preferred embodiments, the UGP may be galU.

[0414] In certain aspects, glucose-1-phosphate is the source for formation of UDP-glucose.

[0415] 25 Accordingly, in certain embodiments, glucose-1-phosphate may be added to the reaction for generation of carminic acid. In certain embodiments, the glucose-1-phosphate may be supplied to the reaction for preparation of carminic acid.

[0416] In certain embodiments, glucose-1-phosphate is generated during the course of the reaction. In certain embodiments, glucose-1-phosphate is generated enzymatically during the

[0417] 77 Patent Application DEBU-033 / 01WO 37396 / 182 course of the reaction. Accordingly, in certain embodiments, glucose is converted to glucose-6- phosphate by a reaction of ATP (adenosine triphosphate) and glucose. In certain embodiments, reaction between glucose and ATP results in generation of glucose-6-phosphate and adenosine diphosphate (ADP). In certain embodiments, conversion of glucose to glucose-6-phosphate is 5 catalyzed by glucokinase (GLK). In certain embodiments, conversion of glucose to glucose-6- phosphate is catalyzed by hexokinase (HK). In certain embodiments, glucose-6-phosphate is converted to glucose-1-phosphate. In certain embodiments, the conversion of glucose-6-phosphate to glucose-1-phsophate is catalyzed by phosphoglucomutase (PGM).

[0418] In certain aspects, the invention further provides that the nucleoside triphosphate species 10 used in the reaction are recycled. In certain embodiments, the recycled nucleoside trisphosphates are ATP and UTP. In certain embodiments, UTP is generated by reaction of UDP and ATP. In certain embodiments, the generation of UTP from UDP and ATP is catalyzed by nucleoside diphosphate kinase (NDK). In certain embodiments, ATP may be generated from ADP and phosphate or polyphosphate. In certain embodiments, the conversion of ADP to ATP is catalyzed 15 by polyphosphate kinase (PPK).

[0419] In certain embodiments, the cell-free medium comprises buffer, an activated sugar, magnesium chloride, cell lysate, sucrose, glucose, glucose-1-phosphate, glucose-6-phosphate, UDP, UTP, ATP, polyphosphate, and / or water. In certain embodiments, the cell-free medium comprises buffer, flavokermesic acid, an activated sugar, magnesium chloride, cell lysate, sucrose, 20 and / or water. In certain embodiments, the buffer used in the cell-free reaction medium is any buffer suitable for enzymatic conversion of kermesic acid to carminic acid. In certain embodiments, the buffer maintains the pH of about 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 25 buffer is a phosphate buffer. In certain embodiments, the buffer is present at a concentration of about 1 mM to about 200 mM. In certain embodiments, the buffer is present at a concentration of about 5 mM to about 100 mM.

[0420] In certain embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.001 mM to about 50 mM. In certain embodiments, the cell-free reaction 30 medium comprises activated sugar at a concentration of about 0.01 mM to about 10 mM. In certain

[0421] 78 Patent Application DEBU-033 / 01WO 37396 / 182 embodiments, the cell-free reaction medium comprises activated sugar at a concentration of about 0.01 mM to about 5 mM. In certain embodiments, the activated sugar is UDP-glucose.

[0422] In certain embodiments, the cell-free reaction medium comprises magnesium chloride at a concentration of about 0.5 mM to about 40 mM. In certain embodiments, the cell-free reaction 5 medium comprises magnesium chloride at a concentration of about 1 mM to about 20 mM.

[0423] In certain embodiments, the cell-free reaction medium further comprises sucrose. In certain embodiments, the sucrose is converted to UDP-glucose. Thus, the concentration of sucrose in the reaction mixture is determined by the quantity of UDP-glucose needed for the reaction for cell- free production of carminic acid. In certain embodiments, sucrose is present at a concentration of 10 about 10 mM to about 1000 mM in the cell-free reaction medium. 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.

[0424] In certain embodiments, the quantity of the one or more enzymes for the cell-free 15 production of carminic acid is dependent on the target quantity of carminic acid to be produced and / or the concentration of other ingredients present in the reaction mixture. In certain embodiments, the one or more enzymes are present in a concentration of from about 1% to about 50% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 2.5% to about 45% (v / v). In certain embodiments, the one or more enzymes are present 20 in a concentration of from about 5% to about 40% (v / v). In certain embodiments, the one or more enzymes are present in a concentration of from about 7.5% to about 30% (v / v).

[0425] In certain embodiments, the reaction for production of carminic acid is conducted for a duration until the desired quantity of carminic acid is obtained. In certain embodiments, the reaction for cell-free production of carminic acid is carried out from about 10 minutes to about 48 25 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out from about 10 minutes to about 36 hours. In certain embodiments, the reaction for cell-free production of kermesic and / or carminic acid is carried out from about 20 minutes to about 24 hours. In certain embodiments, the reaction for cell-free production of carminic acid is carried out

[0426] 79 Patent Application DEBU-033 / 01WO 37396 / 182 from about 30 minutes to about 20 hours. In certain embodiments, the reaction for cell-free production of kermesic and / or carminic acid is carried out from about 1 hour to about 15 hours.

[0427] In certain embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is varied to obtain optimal results for the production of carminic acid. In certain 5 embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is

[0428] for cell- embodiments, the temperature of the reaction mixture for cell-free production of carminic acid is -free 10 production of carminic acid may influence the rate of production of carminic acid. Consequently, the duration of reaction may be adjusted according to the temperature of the reaction mixture to obtain optimal yield of carminic acid in cell-free production of carminic acid.

[0429] 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 carminic acid. In certain embodiments, the 15 reaction for cell-free production of carminic acid is conducted in a bubble column reactor / bioreactor. In certain embodiments, in the bubble column reactor / bioreactor, the one or more enzymes involved in cell-free production of carminic acid are in a solution. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a bubble column reactor / bioreactor comprises the lysate from the host organism. In certain embodiments, it 20 is advantageous to use the bubble column reactor / bioreactor for cell-free production of carminic acid when the reaction mixture involves the lysate (or lysate with cellular debris removed) from the host cell organisms in which the one or more enzymes responsible for cell-free production of carminic acid were utilized. In certain embodiments, the reaction for cell-free production of carminic acid is conducted in a packed bed reactor / bioreactor. In certain embodiments, the one or 25 more enzymes are immobilized in the packed bed reactor / bioreactor. The packed bed reactors / bioreactors are preferred for the purified enzymes playing a role in cell-free production of carminic acid. In certain embodiments, the one or more enzymes may be immobilized in a single reactor / bioreactor. In certain other embodiments, the one or more enzymes may be immobilized in different reactors / bioreactors, wherein these reactors / bioreactors are linked sequentially.

[0430] 80 Patent Application DEBU-033 / 01WO 37396 / 182 In certain beneficial aspects, the methods of the invention provided herein are novel methos for cell-free production of carminic acid from flavokermesic acid. The methods of the invention beneficially provide high titer values of carminic acid. The titer values of carminic acid achieved using the methods of the invention are significantly higher than comparable cell-based methods of 5 production of carminic acid. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 250 µM or higher. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 500 µM or higher. In certain preferred embodiments, the methods of the invention lead to production of carminic acid with a concentration of 750 µM or higher. In certain preferred 10 embodiments, the methods of the invention lead to production of carminic acid with a concentration of 1 mM or higher.

[0431] In certain embodiments, the invention provides a method for manufacturing carminic acid through one or more of the following steps:

[0432] (a) transformation of glycerol or another carbon source to acetyl-CoA and / or malonyl-15 CoA;

[0433] (b) transformation of acetyl-CoA and / or malonyl-CoA to an octaketide;

[0434] (c) transformation of an octaketide to flavokermesic acid anthrone;

[0435] (d) transformation of flavokermesic acid anthrone to flavokermesic acid;

[0436] (e) transformation of flavokermesic acid to kermesic acid; and

[0437] 20 (f) transformation of kermesic acid to carminic acid.

[0438] In certain embodiments, one or more steps of (a)-(f) are mediated by enzymatic transformation. In certain embodiments, the one or more enzymes for transformations provided in steps (a)-(f) are engineered enzymes.

[0439] In certain embodiments, one or more steps (a)-(e) are conducted in an engineered host cell, 25 and step (f) is conducted in a cell-free medium. In certain embodiments, the engineered host cell is genetically modified for production of kermesic acid 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).

[0440] 81 Patent Application DEBU-033 / 01WO 37396 / 182 In certain embodiments, steps (a)-(d) are conducted in an engineered host cell. In certain embodiments, the engineered host cell expresses one more enzymes for the transformations listed in steps (a)-(d). In these embodiments, steps (e) and (f) are optionally carried out in a cell-free medium.

[0441] 5 Examples

[0442] Example 1: Cell-free conversion of flavokermesic acid to kermesic acid:

[0443] Exemplary reaction parameters for cell-free conversion of flavokermesic acid to kermesic acid are provided in the Table below.

[0444] Component Concentration

[0445]

[0446] 10 An exemplary reaction protocol for cell-free conversion of flavokermesic acid to kermesic acid is also provided herein. Seed cultures with 500 µL Luria Broth (LB), 100 µg / mL carbenicillin in 2 mL deep-well plates are inoculated from single colonies or with 5 µL of a glycerol stock (15% glycerol). These cultures are grown for 16-20 hours at 30 °C, 750 RPM (70% humidity).5 µL of the seed cultures are used to inoculate an expression culture (500 µL TB, 100 ug / mL carbenicillin, 15 0-2.0 mM 5-ALA, 0-1 µg / mL riboflavin in 2 mL deep-well plates). Expression cultures are grown overnight for 24 hours at 30 °C, 750 RPM (70% humidity). The following day, culture plates are centrifuged at 4000 xg (4 °C) for 15 minutes. The media supernatant is decanted and the cell pellets are frozen at –80 °C overnight. After thawing cell pellets for at least 15 minutes, they are resuspended with 200 µL lysis buffer (50 mM KPi pH 8.0, 2.5 mg (20k units) / mL lysozyme, 0.1

[0447] 82 Patent Application DEBU-033 / 01WO 37396 / 182 mg / mL DNaseI, 10 mM MgSO4, 1 mM DTT, 0.1% (w / v) Triton X-100. The plates are incubated at 30 °C, 400 RPM (70% humidity) for 45 minutes and then centrifuged at 4000 xg (4 °C) for 20 minutes; the supernatant is then employed in lysate-based reactions.

[0448] Activity assays are conducted in 150 mM KPi pH 7.0 using 25 mM glucose, 1 mM NADP+, 5 0.5 mg / mL glucose dehydrogenase (GDH), a given concentration of FK, and 1-40 vol% lysate.

[0449] Reactions are performed in flat-bottom 96-well assay plates at 30 °C, 750 RPM (70% humidity). After 20h, reactions are quenched with 100% MeOH (10x dilution), incubated at –20 °C for 1 hour, filtered through a 0.45 um filter, and analyzed by HPLC.

[0450] FIG. 2 provides the HPLC data related to the cell-free conversion of flavokermesic acid to 10 kermesic acid catalyzed by a monooxygenase enzyme. The top panel and middle panel in FIG.2 provide the analytical standards for flavokermesic acid and kermesic acid. The bottom panel provides HPLC data demonstrating the conversion of flavokermesic acid to kermesic acid catalyzed by a monooxygenase enzyme.

[0451] FIG. 10 provides data for production of kermesic acid by various enzymes provided herein.

[0452] 15 Example 2: Production of flavokermesic acid and kermesic acid in microbial strains An E. coli cell derived from MG1655 was engineered to integrate the key pathway genes in different loci in the chromosome to produce flavokermesic acid. Integrated genes include PKS and accessory enzymes (SEQ IDs #57-74) and a cyclase and an aromatase (SEQ IDs #75-84). Other genome edits were also present to increase the flux towards the product. For kermesic acid, 20 a monooxygenase (SEQ IDs #39-56) was expressed in the strain producing flavokermesic acid either in the chromosome or in a plasmid.

[0453] The strains were initially grown in rich medium (e.g. LB) as a seed culture. This was then inoculated to a defined minimal medium that contains a carbon source (e.g. glucose or glycerol), trace elements, vitamins, and other necessary components for buffering. To screen strains, initial 25 culturing was performed in shake-flasks or in microtiter plates (24-well, 48-well, or 96-well) with enough aeration and shaking at optimal temperatures for the strains and for the production of the desired products. To analyze production, the cells cultures were extracted with methanol at 1:1 ratio, and centrifuged for 15 minutes. The supernatant was analyzed for flavokermesic acid and / or kermesic acid by HPLC or LCMS.

[0454] 83 Patent Application DEBU-033 / 01WO 37396 / 182 Select strains were run scaled up in larger bioreactors. The bioreactor cultivation process was initiated with an initial volume of approximately 850 mL. The pH was continuously controlled at 7.0, and the temperature was maintained at 30°C throughout the cultivation. Dissolved oxygen (DO) levels were regulated above 40%, achieved through an airflow rate of 1.2 VVM and variable 5 stirring speeds ranging from 350 to 1500 RPM. pH adjustments were made using an NH4OH solution, and an antifoam agent was periodically added to mitigate foaming. The fed-batch process employed a DO-stat mode, with a feed solution containing glycerol and MgSO4 initiated when the DO level dropped to 45%. The cultivation was sustained for a total duration of 70 hours.

[0455] The example provides data for production of kermesic acid in an engineered host cell, 10 wherein the engineered host cells express monooxygenase, PKS, cyclase, and aromatase enzymes.

[0456] An overview of the pathway is provided in FIG.3. The HPLC data demonstrating the production of flavokermesic acid, which is subsequently converted to kermesic acid is provided in FIG.4.

[0457] Panel (i) provides HPLC chromatograms demonstrating production of flavokermesic acid in microbial strains containing PKS / cyclase / aromatase enzymes. Panel (ii) provides LCMS 15 chromatograms showing increased kermesic acid production in microbial strains expressing monooxygenase enzymes.

[0458] Example 3: Cell-free production of carminic acid from flavokermesic acid

[0459] Exemplary reaction parameters for cell-free conversion of flavokermesic acid to carminic acid are provided in the Table below.

[0460] Component Working Range

[0461]

[0462] 84 Patent Application DEBU-033 / 01WO 37396 / 182 Temperature 20 °C-40 °C

[0463]

[0464] Microbes expressing MOX and CGT enzymes are grown according to the exemplary protocol in Example 1, and then used to generate either lysates as described in Example 1, or purified protein following standard protocols. Activity assays are conducted in 150 mM KPi pH 5 7.0 using 25 mM glucose, 1 mM NADP+, 0.5 mg / mL glucose dehydrogenase (GDH), 1-20 mM magnesium chloride, a given concentration of FK, a given concentration of UDP-glucose with or without UDP recycling components, 1-40 vol% lysate containing MOX or 0.05-50 M purified MOX, and 1-40 vol% lysate containing CGT or 0.1-100 M purified CGT. Reactions are performed in flat-bottom 96-well assay plates or in Erlenmeyer flasks at 30 °C. After a given time, 10 reactions or reaction samples are quenched with 100% MeOH (10x dilution), incubated at –20 °C for 1 hour, filtered through a 0.45 um filter, and analyzed by HPLC.

[0465] FIG. 6 provides the HPLC chromatograms demonstrating the production of carminic acid from flavokermesic acid. The top panel shows the standard for carminic acid and the bottom panel provides an HPLC chromatogram demonstrating the cell-free production of carminic acid from 15 flavokermesic acid.

[0466] FIG. 9 provides data related to production of carminic acid produced by the methods of the protocol provided herein.

[0467] The table below provides exemplary sequences for glycosyltransferase enzymes in accordance with the methods of invention.

[0468] Glycosyltransferase Enzymes

[0469]

[0470] D L V S

[0471]

[0472] 85 Patent Application DEBU-033 / 01WO 37396 / 182 ELLVLPDGFLEKAGDKGKMVQWSPQEQVLAHPSVACFV THCGWNSTMESLTSGMPVVAFPQWGDQVTDAVYLCDV K L

[0473] V L I D V I T E

[0474] L Q C T

[0475] V I N A T G G V

[0476]

[0477] 86 Patent Application DEBU-033 / 01WO 37396 / 182 5 AUI41 Rhodiola MGSEPLVHVFLVSFPGQGHVNPLLRLGKRLASKGLLVTF 123 rosea TTPESIGHQMRKANKIVDGQPNPVGDGFLRFEFFEDGWD S P S H V L A

[0478] V D K D G E

[0479] T K S H M S I L N I

[0480] G L

[0481]

[0482] 87 Patent Application DEBU-033 / 01WO 37396 / 182 PTAADPDVPVELPGLPVMAMVELPFMVRPEYAQCLWGD TLRAQVGAIKRTVSWVLVNSFYELERSAVDALRAHTTVK L F A S

[0483] L Y R K T P P L V

[0484] F L H V Y G

[0485] L S S W P

[0486]

[0487] 88 Patent Application DEBU-033 / 01WO 37396 / 182 SIVCFVTHCGWNSTMEAVSSGVPTVCCPQWGDQVTDAV YMIDVLKTGVRLCRGETEERVVPREEVAERLREITKGEK

[0488] R P S V

[0489] A F C L D A

[0490] H F H H F Q D A E

[0491]

[0492] 89 Patent Application DEBU-033 / 01WO 37396 / 182 15 XP_03 Punica MGSESSLVHVFLVSFPGQGHVNPLLRLGKRLASKGLLVT 138211 granatum FTTPESIGKQMRKASNISDQPAPVGDGFIRFEFFEDGWDE C Y V K

[0493]

[0494] The Table below provides exemplary sequences for sucrose synthase enzymes in accordance with the methods of invention.

[0495] Sucrose Synthase Enzymes

[0496] A A I T K R R Y Q S A T

[0497]

[0498] 90 Patent Application DEBU-033 / 01WO 37396 / 182 17 P30298. Oryza MAAKLARLHSLRERLGATFSSHPNELIALFSRYVNQGKGM 2 sativa LQRHQLLAEFDALIEADKEKYAPFEDILRAAQEAIVLPPW V L L A H P P K Q E V

[0499] E E H

[0500] L G V E R Y L IS E

[0501] I N

[0502]

[0503] 91 Patent Application DEBU-033 / 01WO 37396 / 182 RHLSAKLFHDKESLHPLLEFLRLHSVKGKTLMLNDRIQNP DALQHVLRKAEEYLGTVPPETPYSEFEHKFQEIGLERGWG S K R G V Q C F V E

[0504] P Q R E N R L L N K N K

[0505] N Q M

[0506]

[0507] 92 Patent Application DEBU-033 / 01WO 37396 / 182 LGTEHCHILRVPFRTENGIVRKWISRFEVWPYLETYTDDV AHEIAGELQANPDLIIGNYSDGNLVACLLAHKMGVTHCTI A N Y

[0508] V F F L K F V A A Q

[0509] K V K L I

[0510]

[0511] 93 Patent Application DEBU-033 / 01WO 37396 / 182 QEIAGTKNTVGQYESHGAFTLPGLYRVVHGIDVFDPKFNI VSPGADMTIYFPYSEETRRLTALHGSIEEMLYSPDQTDEHV V R E M

[0512] D L P I C P I S D V

[0513] D P V R Q L R

[0514]

[0515] 94 Patent Application DEBU-033 / 01WO 37396 / 182 EFNPKVTQNWISRFEIWPYLETFAIDAERELRAEFGHVPDL IIGNYSDGNLVAFLLARRLKVTQCNIAHALEKSKYLFSNLY S D F E

[0516]

[0517] The Table below provides exemplary sequences for enzymes involved in generation and / or recycling of UDP-glucose the reaction intermediates in accordance with the methods of invention.

[0518] SEQ ID Gene Accessio

[0519] Sequence

[0520] Q D

[0521] V A S L D

[0522] A S L M S

[0523] D I

[0524]

[0525] 95 Patent Application DEBU-033 / 01WO 37396 / 182 AINYLFQHRPQWGKDVAVGKTLVSSAMIDRVVNDLGRKLVE VPVGFKWFVDGLFDGSFGFGGEESAGASFLRFDGTPWSTDK I K K G L T I

[0526] M A V V F

[0527] G D V G N G Q I R V

[0528] A K K

[0529]

[0530] 96 Patent Application DEBU-033 / 01WO 37396 / 182 MFAEDLKRTEKMTVDDVFEQSAQKMREQGMSEIAISQFRH AYHVWASEKESAWIREDTVEPLHGVRSFHDVYKTIDHDKAV F K

[0531] F V L V I L

[0532] D A L

[0533] A

[0534] W R R

[0535] R E K T

[0536] C F T

[0537] F

[0538]

[0539] 9 Patent Application DEBU-033 / 01WO 37396 / 182 EGRDAAGKGGAIHATMSNMNPRSARIVALTKPTETEQGQW YFQRYVATFPTSGEFVLFDRSWYNRAGVEPVMGFCTPEQYE D W S

[0540]

[0541] The Table below provides exemplary sequences for monooxygenase enzyme.

[0542] SEQ NCBI Organis Sequence

[0543] ID Access m

[0544] P R R G E D R R L T P L H P

[0545] K D P

[0546] I

[0547]

[0548] 98 Patent Application DEBU-033 / 01WO 37396 / 182 AENVGRFWQKANAILDRMIADPSGEGWMYETVRQHFRHP DIVPESYMRSMMMAILVAAHETTTLATANAFRLLLERRDA T G L

[0549] V H G L G V L H D K G P P F Y

[0550] G I

[0551]

[0552] Patent Application DEBU-033 / 01WO 37396 / 182 AVQLIQKWERLNVNEYIDVPADMTRLTLDTIGLCGFNYRFN SFYRDQPHPFIVSMVRALDESMNRLQRANPNDSLYEEERLQ E L L A E L L L K E

[0553] R D R E R I D V F I P G D F

[0554]

[0555] 100 Patent Application DEBU-033 / 01WO 37396 / 182 GCRNPHHDYLYQEELASFEREGLVTLHTAFSRVEGKEKRYV QHLIRQDAGHLIRLLDQGGRLYICGDGSRMAPDVEAALRSA

[0556] F F F D P L K I D K Y G D

[0557] A I E L T V K R I A

[0558]

[0559] 101 Patent Application DEBU-033 / 01WO 37396 / 182 DSGLTVRVAADQTVLAALRAAGVDVPSDCEEGLCGTCEVP VLDGEVDHRDVVLTKAERAAGTRMMTCCSRACGDRITLRL

[0560] K G I N Q L L A E L L L K E

[0561] K Q I I E S

[0562] N G R A G

[0563]

[0564] 102 Patent Application DEBU-033 / 01WO 37396 / 182 DTVHATTPHLASGACIGIEDALVLADELALGMGDVPAALAA FEARRWERCRMVVENSARLGAIEIEGGDKEEHARIMRESLL

[0565] N V V P D

[0566] R G L V A

[0567] S K V D E

[0568] S T E G

[0569] S V

[0570]

[0571] Patent Application DEBU-033 / 01WO 37396 / 182 australic RLGCTFTAIEQDAEGVEVSFTDGSRGRYDLVIGADGLYSKV a REGVFPAAPKPRYSGQAVWRAVLPRPAEVTTAMMWMGPK E I A M

[0572] Q R R T R A

[0573] R H G L I A

[0574] R H T P L

[0575]

[0576] The Table below provides exemplary sequences for PKSIII, PKSII, and PKSII accessory enzymes.

[0577] 104 Patent Application DEBU-033 / 01WO 37396 / 182 57 ACC7 Polygon MANVLQEIRNSQKATGPATVLAIGTAVPPTCYPQADYPDFY 6752 um FRVCKSEHMTQLKKKMQYICDRSGIRQRYMFHTEENLGKN D Q H Q E

[0578] S P A L L

[0579] Y P K Q A A

[0580] F Y I N N R D

[0581]

[0582] 105 Patent Application DEBU-033 / 01WO 37396 / 182 61 C4NF Aloe MGSIAESSPLMSRENVEGIRKAQRAEGTATVMAIGTAHPPHI 91.1 arboresc FPQDTYADFYFRATNSEHKVELKKKFDRICKKTMIGKRYFN A L F G

[0583] K H V L

[0584] F L I C H L L

[0585] F Y I N N K E

[0586]

[0587] 106 Patent Application DEBU-033 / 01WO 37396 / 182 65 QNJ44 Polygon MANVLQEIRNSQKATGPATVLAIGTAVPPTCYPQADYPDFY 868 um FRVCKSEHMTQLKKKMQYICDRSGIRQRYMFHTEENLGKN D Q E A

[0588] E W G Q F

[0589] K S A P E I F

[0590] T S Q F E S

[0591]

[0592] 107 Patent Application DEBU-033 / 01WO 37396 / 182 70 CBH3 Strepto MSSFSIDDLKRILREGAGATAELDGDILDASFDDLGYDSLAL 2807.1 myces LETGSRIGREYGLEFEDTAFADVETPRDLVGVVNAQLSAPA

[0593] L I L S G S K

[0594] R N T

[0595] F P

[0596] D K L

[0597]

[0598] The Table below provides exemplary sequences for cyclase / aromatase enzyme.

[0599] 75 AAG3 Strepto MSGRKTFLDLSFATRDTPSEATPVVVDLLDHVTGATVLGLS K

[0600]

[0601] 108 Patent Application DEBU-033 / 01WO 37396 / 182 VYGRTREYLQLEKLNNLGALPGATGYDISCFPVAVAGTGA GWTRVVAVFEQEEED

[0602] E L A

[0603] T V

[0604] E

[0605] D Q T V R

[0606] V

[0607] A T A

[0608] F T R V

[0609] E A

[0610]

[0611] 109 Patent Application DEBU-033 / 01WO 37396 / 182 82 WP_1 Longim MTRFVDLSVRIQPTPSEATPVTIHPLSHRDGARVLGLEPADF 89057 ycelium PDEMAISNETVTLTTHTGTHMDAPLHYGPFSAGLPAKSIDEV A E I

[0612] M D L

[0613] E T D

[0614]

[0615] The Table below lists exemplary acetyl-CoA carboxylase (ACC), acetyl-CoA synthetase (ACS), malonyl-CoA synthetase (MCS), malonate transporter (matC), malonate-CoA transferase (mdcA), pantothenate kinase (PanK), glutamyl-tRNA reductase (hemA), and 5-aminolevulinic 5 acid synthase (ALAS) enzymes.

[0616] 85 ACC EPB82 Mucor MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHE GHTVISKVLIANNGMAAMKEIRSVRKWAYETF V

[0617] EI G I P L T

[0618]

[0619] 110 Patent Application DEBU-033 / 01WO 37396 / 182 KTQVGIRDLSDGGLLISIDGKSHTTYSRDEVQAT RMMVDGKTCLLEKESDPTQLRSPSPGKLVNLLV V S E E I T A S R E V E I Q K Q E L R L R W M G F N E

[0620]

[0621] 111 Patent Application DEBU-033 / 01WO 37396 / 182 WFEKRASVIDQRISKLKSDATKEQIVSLGNADQ EAVIEGFSQLIENLSEDARAEILRKLNSRF

[0622] G D F S D L Q R E A S L Q R N A Q I L K F V S E F C S

[0623]

[0624] 112 Patent Application DEBU-033 / 01WO 37396 / 182 RRSRAHALQTTFVYDFIDVLGQAVRASWRKVA ASKIPGDVIKSAVELVFDEQENLREVKRAPGMN A A II L W L P G I A

[0625] S H E A P I M V S Q L H T

[0626]

[0627] 113 Patent Application DEBU-033 / 01WO 37396 / 182 GYDNQGLIQTIVKELSEVLNNPELPYSELSASMS VLSGRIPGRLEQQLHDLINQAHAQNKGFPAVDI A Q S I I A I R Y L P Y T I P G K G P E L V I A

[0628]

[0629] 114 Patent Application DEBU-033 / 01WO 37396 / 182 88 ACC GEM0 Rhodotorul MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAP 8739.1 a AIVFMHGLGSSTSFWEAPFSRSNLSSRFRLIRYDF

[0630] S V

[0631] A I D R F E D V A F A L D E S E F E Y

[0632]

[0633] 115 Patent Application DEBU-033 / 01WO 37396 / 182 RADFAAHVNAMSAEIDARGMRRLTLLICREGQ YPSYYTVRKQDGTWKELETIRDIEPALAFQLEL R E V A E K E Y Y V K E I A R L L K E R

[0634] D

[0635] T L A

[0636]

[0637] 116 Patent Application DEBU-033 / 01WO 37396 / 182 FKNMYFSGDGARRDEDGYYWITGRVDDVLNVS GHRLGTAEIESALVAHPKIAEAAVVGIPHAIKGQ L S

[0638] L V

[0639] T I F S S K

[0640] L D T D K L V V A

[0641] M L G L

[0642]

[0643] 117 Patent Application DEBU-033 / 01WO 37396 / 182 SVRITDPVSGEPLAAGEPGMIEVKGPNVFQGYW NMPDKTKEEFRSDGYFTTGDIGVMETDGRISIV I

[0644] D D

[0645] K T A P R S G I K

[0646] F F F

[0647] E T G I A V

[0648] F F F

[0649] E T

[0650]

[0651] 118 Patent Application DEBU-033 / 01WO 37396 / 182 AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICG VLTYVGVLEEMGTITWAGEGVGGIGVPLLAAV I A V

[0652] A A

[0653] M P S V

[0654] Q A

[0655] T F

[0656] G Q H V W M V

[0657]

[0658] 119 Patent Application DEBU-033 / 01WO 37396 / 182 ANAPDETREQVLRQLLIYSALIAIIGPVVAWLVF VVPGLV

[0659] E A

[0660] L D A G K R S

[0661] R A L S T G I T K D

[0662] N D E L R A E

[0663]

[0664] 120 Patent Application DEBU-033 / 01WO 37396 / 182 SMRSNRAFSQTAGLYACDMFIGSTLQMDLAGN SSTATLGRITGFGGAPNMGSDPHGRRHASPAWL V T A

[0665] I T A A S A L M

[0666] I K

[0667] E T S T A E

[0668] A L

[0669]

[0670] 121 Patent Application DEBU-033 / 01WO 37396 / 182 DDAEARRALASLQPGQATAEAVERGCLLMLRG FVREQYAMACELLGPDCEIFLTGGDAELVRDEL

[0671] S I L D

[0672] R A K E D

[0673] F P V L I

[0674] D

[0675] S S E A S

[0676] R S

[0677]

[0678] 122 Patent Application DEBU-033 / 01WO 37396 / 182 GHQNASALERMFQKSFSVAKRVRTETDIGSSAV SVAFAACTLARQIFESLSTVTVLLVGAGETIELV E E L

[0679] L Q H

[0680] A V L Q

[0681] N S

[0682] S R L D D I A

[0683] F V

[0684] F K E A S

[0685]

[0686] 123 Patent Application DEBU-033 / 01WO 37396 / 182 PTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLL WARCA

[0687] E A T I E F H P F A P

[0688] V R

[0689] L C R D F V L T L V

[0690]

[0691] 124 Patent Application DEBU-033 / 01WO 37396 / 182 115 ALAS THV0 Dendrothel MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSP 5492.1 e bispora RFPSISILTERATKCPVMGPALNVRSKEITAGYAS A A

[0692] F E E G E I H P A Q L

[0693] F E

[0694] A A A II N

[0695]

[0696] Incorporation by Reference

[0697] References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, publicly accessible databases, have been 5 made throughout this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes.

[0698] Equivalents

[0699] 125 Patent Application DEBU-033 / 01WO 37396 / 182 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 5 that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.

[0700] 126

Claims

Patent Application DEBU-033 / 01WO 37396 / 182 CLAIMS1. A method for cell-free production of kermesic acid, 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 kermesic acid.

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

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

4. The method of claim 3, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56.

5. The method of claim 4, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56.

6. The method of claim 4, wherein the reaction medium further comprises NADPH and / or NADH and optionally further comprises components for regeneration of NADPH and / or NADH.

7. The method of claim 6, wherein the reaction medium further comprises glucose.

8. The method of claim 7, wherein the reaction medium further comprises glucose dehydrogenase (GDH).

9. The method of claim 1, wherein the reaction medium is agitated to introduce oxygen in the reaction medium.127Patent Application DEBU-033 / 01WO 37396 / 18210. The method of claim 1, wherein the reaction medium further comprises oxygen.

11. The method of claim 1, wherein the monooxygenase enzyme is produced in an engineered host cell.

12. The method of claim 11, wherein the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells.

13. The method of claim 11, wherein the one or more enzymes are introduced in the host cell by integration into genome of the host cell or on a plasmid.

14. The method of claim 13, 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.

15. The method of claim 14, 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 kermesic acid.

16. The method of claim 15, wherein the one or more enzymes is purified from the cell lysate for production of kermesic acid.

17. The method of claim 16, wherein the one or more enzymes is immobilized on a solid support for cell-free production of kermesic acid from flavokermesic acid.

18. The method of claim 16, wherein the one or more enzymes is in a solution for cell-free production of kermesic acid from flavokermesic acid.

19. The method of any one of claims 1-18, wherein the cell-free medium further comprises:buffer, flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, GDH, and / or water.128Patent Application DEBU-033 / 01WO 37396 / 182 20. The method of claim 19, wherein the buffer is a phosphate buffer.

21. A method for production of kermesic acid in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for conversion of a substrate to kermesic acid.

22. The method of claim 21, wherein the substrate is flavokermesic acid.

23. The method of claim 22, wherein the one or more genetic modifications comprise expression and / or overexpression of monooxygenase enzyme.

24. The method of claim 23, wherein the engineered host cell further comprises one or more genetic modifications for reduction of use of precursors for production of kermesic acid.

25. The method of claim 21, wherein the engineered host cell is E. coli.

26. The method of claim 25, wherein the engineered host cell is cultured in a medium comprising flavokermesic acid.

27. The method of claim 25, wherein the engineered host cell comprises further genetic modifications for production of flavokermesic acid.

28. The method of claim 25, wherein the one or more genetic modifications comprise overexpression of monooxygenase enzyme.

29. The method of claim 28, wherein the one or more genetic modifications comprise overexpression of glucose dehydrogenase.

30. The method of claim 25, wherein the medium further comprises NADPH and / or NADH.

31. A method for production of flavokermesic acid in an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications for transformation of129Patent Application DEBU-033 / 01WO 37396 / 182 one or more substrates to flavokermesic acid and / or kermesic acid through one or more intermediates.

32. The method of claim 31, wherein the one or more substrates is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof.

33. The method of claim 32, wherein the one or more substrate is glucose, glycerol, or any combination thereof.

34. The method of claim 33, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

35. The method of claim 34, wherein the one or more genetic modifications lead to increase in metabolic flux to precursors or cofactors for production of flavokermesic acid.

36. The method of claim 33, wherein the one or more intermediates are selected from the group consisting of acetyl-CoA, malonyl-CoA, an octaketide, and flavokermesic acid anthrone (FKA).

37. The method of claim 33, wherein the one more genetic modifications are selected from overexpression of polyketide synthase (PKS), aromatase, and / or cyclases.

38. The method of claim 37, wherein glucose or glycerol are transformed to acetyl-CoA and malonyl-CoA by enzymatic transformation through one or more enzymes.

39. The method of claim 38, wherein acetyl-CoA and malonyl-CoA are transformed to an octaketide.

40. The method of claim 39, wherein the transformation is mediated by polyketide synthase (PKS).130Patent Application DEBU-033 / 01WO 37396 / 18241. The method of claim 40, wherein the octaketide is transformed to flavokermesic acid anthrone (FKA).

42. The method of claim 41, wherein the transformation is mediated by one or more cyclases or aromatases.

43. The method of claim 42, wherein flavokermesic acid anthrone (FKA) is transformed to flavokermesic acid.

44. The method of claim 43, wherein said transformation is mediated by an oxygenase enzyme.

45. The method of claim 43, wherein said transformation is mediated by monooxygenase enzyme.

46. The method of claim 31, wherein the one or more genetic modifications cause reduction of formation of byproducts.

47. The method of claim 46, wherein the one or more genetic modifications comprise downregulation and / or deletion of one or more genes selected from the group consisting of yahK, guaC, yqjH, queF, curA, gdhA, gnd, dkgB, yeaE, yjgB and yqhD.

48. The method of claims 35 or 46, wherein the one or more genetic modifications are selected from the group consisting of overexpression of ACC, ACS, MCS, matC, mdcA, panK, hemA, and / or ALAS.

49. The method of claim 31, wherein the engineered host cell is E. coli.

50. The method of claim 33, wherein the medium further comprises NADPH and / or NADH.

51. A method of cell free production of carminic acid, wherein the method comprises providing one or more enzymes in a cell-free medium, wherein the one or more enzymes131Patent Application DEBU-033 / 01WO 37396 / 182 result in transformation of flavokermesic acid to carminic acid through one or more intermediates.

52. The method of claim 51, wherein the one or more intermediates are selected from the group consisting of kermesic acid and C-glucosylated flavokermesic acid.

53. The method of claim 52, wherein the one or more enzymes are selected from the group consisting of monooxygenase, C-glucosyltransferase, glucose dehydrogenase (GDH), sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and / or nucleoside diphosphate kinase (NDK).

54. The method of claim 52, wherein the one or more enzymes are selected from the group consisting of monooxygenase and / or C-glucosyltransferase.

55. The method of claim 54, wherein the cell-free medium further comprises glucose dehydrogenase (GDH) enzyme.

56. The method of claim 52, wherein the cell-free medium comprises flavokermesic acid and monooxygenase enzyme.

57. The method of claim 56, wherein UDP-glucose and CGT are added to the cell-free medium.

58. The method of claim 56, wherein the cell-free medium comprises UDP-glucose, monooxygenase and / or C-glucosyltransferase.

59. The method of claim 58, wherein UDP-glucose is synthesized in the cell-free medium by the one or more enzymes.

60. The method of claim 59, wherein the UDP-glucose is synthesized from one or ingredients selected from the group consisting of: sucrose, glucose, UTP, UDP, ATP, glucose-6- phosophate, glucose-1-phosphate, and / or polyphosphate.132Patent Application DEBU-033 / 01WO 37396 / 18261. The method of claim 60, wherein the one or more enzymes is selected from the group consisting of sucrose synthase (SuSy), glucokinase (GLK), hexokinase (HK), phosphoglucomutase (PGM), polyphosphate kinase (PPK), UTP—glucose-1-phosphate uridylyltransferase (UGP), and nucleoside diphosphate kinase (NDK).

62. The method of claim 51, wherein the cell-free medium is agitated to include oxygen from the atmosphere.

63. The method of claim 62, wherein the cell-free medium further comprises NADPH / NADH.

64. The method of claim 63, wherein the cell-free medium further comprises NADPH regeneration system.

65. The method of claim 64, wherein the NADPH regeneration system comprises glucose and glucose dehydrogenase (GDH).

66. The method of claim 51, wherein the one or more enzymes is produced in an engineered host cell.

67. The method of claim 66, wherein the engineered host cell is selected from a group consisting of: bacteria, yeast, and / or fungal cells.

68. The method of claim 67, wherein the one or more enzymes are introduced in the host cell by integration into genome of the host cell or on a plasmid.

69. The method of claim 68, 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.133Patent Application DEBU-033 / 01WO 37396 / 182 70. The method of claim 69, 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 carminic acid.

71. The method of claim 70, wherein the one or more enzyme is purified from the cell lysate for production of carminic acid.

72. The method of claim 71, wherein the one or more enzyme is immobilized on a solid support for cell-free production of carminic acid from flavokermesic acid.

73. The method of claim 71, wherein the one or more enzyme is in a solution for cell-free production of carminic acid from flavokermesic acid.

74. The method of any one of claims 51-74, wherein the cell-free medium further comprises:buffer, flavokermesic acid, magnesium chloride, cell lysate, sucrose, glucose, oxygen, GDH, and / or water.

75. The method of claim 74, wherein the buffer is a phosphate buffer.

76. The method of claim 75, wherein reaction is conducted in a bubble column reactor, wherein the one or more enzymes are in a solution.

77. The method of claim 76, wherein reaction is conducted in a packed bed reactor, wherein the one or more enzymes are immobilized.

78. The method of any of claims 51, wherein the method results in titer value of produced carminic acid is up to about 5000 times higher than methods for cell-based production of carminic acid.

79. The method of claim 28, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%,134Patent Application DEBU-033 / 01WO 37396 / 182 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56.

80. The method of claim 28, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56.

81. The method of claim 56, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56.

82. The method of claim 56, wherein the monooxygenase enzyme is an engineered monooxygenase enzyme for conversion of flavokermesic acid to kermesic acid, and optionally the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56.

83. A cell-free composition for production of kermesic acid and / or carminic acid, wherein the cell-free composition comprises a monooxygenase enzyme, wherein the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56.

84. A cell-free composition for production of kermesic acid and / or carminic acid, wherein the cell-free composition comprises a monooxygenase enzyme, wherein the monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56.135Patent Application DEBU-033 / 01WO 37396 / 182 85. An engineered host cell for production of kermesic acid, wherein the engineered host cell comprises one or more genetic modifications, wherein the genetic modification is an overexpression of monooxygenase enzyme, and said monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 39-56.

86. An engineered host cell for production of kermesic acid, wherein the engineered host cell comprises one or more genetic modifications, wherein the genetic modification is an overexpression of monooxygenase enzyme, and said monooxygenase enzyme is an enzyme that has at least 90%, 95%, 96%, 97%, 98%, or 99% amino acid sequence identity from the enzymes provided in SEQ ID NOS: 48-56.136

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