US20220333087A1

US20220333087A1 - Flavonoid and anthocyanin bioproduction using microorganism hosts

Info

Publication number: US20220333087A1
Application number: US17/720,020
Authority: US
Inventors: Jingyi Li; Nicholas Brideau; Joshua Britton; Erik Holtzapple
Original assignee: Debut Biotechnology Inc
Current assignee: Debut Biotechnology Inc
Priority date: 2021-04-13
Filing date: 2022-04-13
Publication date: 2022-10-20
Also published as: US20220333123A1; AU2022256988A1; IL307636A; WO2022221392A1; CN117413066A; KR20230170713A; CA3215623A1; AU2022256988A9; US20220333118A1; BR112023021282A2; US20220333122A1; US20220333117A1; JP2024513971A; EP4323528A1; US12203104B2; EP4323528A4

Abstract

The invention is directed to methods involved in the production of flavonoids, anthocyanins and other organic compounds. The invention provides cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.

Description

I. RELATED APPLICATIONS

This application claims priority to U.S. Provisional Application No. 63/174,403, filed on Apr. 13, 2021. The content of U.S. Provisional Application No. 63/174,403 is hereby incorporated by reference in its entirety.

II. SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled DEBU-009-05-US-Sequence-Listing.txt, created on Mar. 21, 2022, last modified Apr. 13, 2022, and having a size of 448 KB. The content of the sequence listing is incorporated herein its entirety.

III. FIELD OF THE INVENTION

The invention related to materials (including engineered cells and cell lines) and methods involved in the production of flavonoids, anthocyanins and other organic compounds.

IV. BACKGROUND OF THE INVENTION

Flavonoids and anthocyanins are natural products produced in plants that find a variety of roles such as antioxidants, ultraviolet (UV) defense mechanisms, and colors. Over the past several years, the health benefits of flavonoids and anthocyanins have been widely demonstrated. These compounds are capable of scavenging radicals and can act as enzyme inhibitors and anti-inflammatory agents. With these recognized health and color benefits, much research has gone into understanding how these compounds are made in nature. Flavonoids and anthocyanins are synthesized from phenylpropanoid starter units and malonyl-Cofactor-A (malonyl-CoA) extender units that then undergo modifications to create many polyphenol compounds such as taxifolin, naringenin, and (+)-catechin. However, in most cases, these compounds are extracted or chemically manufactured.

V. SUMMARY OF THE INVENTION

To move away from agriculture and chemically derived products, we have created engineered cells for the bioproduction of flavonoids and anthocyanins. This approach provides a feasible route for the rapid, safe, economical, and sustainable production of a wide variety of important flavonoids.
Herein, a range of flavonoids and anthocyanins including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, and cyaninidin-3-glucoside are biomanufactured using a modified microbial host. Herein, the engineered cells include one or more genetic modifications that increase(s) flavonoid and anthocyanin bioproduction by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.
Provided herein are cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts. As nonlimiting examples, a genetic modification can be a modification for over-expressing or under-expressing one or more endogenous genes in the engineered host cell or can be a modification for expressing one or more non-native genes in the engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.
Also provided are cell cultures for producing one or more flavonoids or anthocyanins. The cell cultures include engineered cells as disclosed herein in a culture medium that includes a carbon source that can also be an energy source, such as glycerol, sugar, or an organic acid. In various embodiments, the culture medium can include at least one feed molecule such as but not limited to one or more organic acids or amino acids that can be converted into a flavonoid precursor (such as tyrosine, p-coumaroyl-CoA or malonyl-CoA). Examples of feed molecules include, but are not limited to, acetate, malonate, tyrosine, phenylalanine, pantothenate, coumarate, etc. In some embodiments, the feed molecules may be of reduced or low purity. For example, glycerol as a feed molecule may be crude glycerol, including a biomass comprising glycerol, for example, glycerol obtained as a byproduct of biodiesel processing. Alternatively, or in addition, the culture medium can include a supplemental compound that can be a cofactor or a precursor of a cofactor used by an enzyme that functions in a flavonoid pathway, such as, for examples, bicarbonate, biotin, thiamine, pantothenate, alpha-ketoglutarate, ascorbate, or 5-aminolevulinic acid.
Further provided are methods for producing flavonoids and anthocyanins that include culturing a cell engineered for the production of flavonoids or anthocyanins as provided herein under conditions in which the cell produces flavonoids or anthocyanins. In some examples, the methods include culturing the engineered cells in a culture medium that includes at least one feed molecule or supplement such as but not limited to: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid. The methods can further include recovering at least one of the flavonoids from culture medium, whole culture, or the cells.
In a first aspect, provided herein are cells engineered to produce one or more flavonoids or anthocyanins, wherein the cells include, in addition to nucleic acid sequences encoding either tyrosine ammonia lyase activity and/or phenylalanine ammonia lyase activity and cinnamate-4-hydroxylase activity, 4-coumarate-CoA ligase activity, chalcone synthase activity, chalcone isomerase activity, flavanone-3-hydroxylase activity, flavonoid 3′-hydroxylase activity or flavonoid 3′5′-hydroxylase activity, cytochrome P450 reductase activity, leucoanthocyanidin reductase activity, and dihydroflavonol-4-reductase activity, one or more genetic modifications for improving production of the flavonoids or anthocyanins. As set forth herein, a cell that is engineered to produce one or more of the flavonoids is engineered to include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase activity that can form 4-coumaric acid using tyrosine as substrate (e.g., tyrosine ammonia lyase TAL, EC: 4.3.1.25) or, alternatively or in addition, an exogenous nucleic acid sequence encoding phenylalanine ammonia lyase activity that can convert phenylalanine to trans-cinnamic acid and an exogenous nucleic acid sequence encoding cinnamate-4-hydroxylase activity that forms 4-coumaric acid from trans-cinnamic acid, an exogenous nucleic acid sequence encoding CoA ligase activity that forms p-coumaroyl-CoA from coumaric acid (e.g., 4-coumarate-CoA ligase, 4CL, EC:6.2.1.12), an exogenous nucleic acid sequence encoding polyketide synthase activity that forms naringenin chalcone using malonyl-CoA and p-coumaroyl-CoA as substrates (e.g., chalcone synthase, CHS, EC:2.3.1.74), an exogenous nucleic acid sequence encoding chalcone isomerase activity that forms naringenin from naringenin chalcone via its cyclase activity (e.g., chalcone-flavonone isomerase, CHI, EC:5.5.1.6), an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase activity that forms dihydrokaempferol from naringenin or forms taxifolin from eriodictyol (e.g., naringenin 3-dioxygenase, F3H, EC: 1.14.11.9), an exogenous nucleic acid sequence encoding flavonoid 3′-hydroxylase or flavonoid 3′5′-hydroxylase activity coupled with an exogenous nucleic acid sequence encoding cytochrome P450 reductase activity to form taxifolin or dihydromyricetin from dihydrokaempferol or to form eriodictyol or pentahydroxyflavone from naringenin (e.g., flavonoid 3′-monooxygenase, F3′H, EC: 1.14.13.21, EC: 1.14.14.82; cytochrome P450/NADPH-P450 reductase, EC:1.14.14.1; F3′5′H, EC:1.14.14.81), an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase activity that forms leucocyanidin from taxifolin, leucodelphinidin from dihydromyricetin, or leucopelargonidin from dihydrokaempferol (e.g., dihydroflavonol 4-reductase, EC:1.1.1), and an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase activity that forms catechin from leucocyanidin (e.g., leucoanthocyanidin reductase, LAR, EC:1.17.1.3). Optionally, a cell that is engineered to produce anthocyanins is further engineered to include an exogenous nucleic acid sequence encoding anthocyanin synthase activity that forms cyanidin from catechin or leucocyanidin, forms delphinidin from leucodelphinidin, or forms pelargonidin from leucopelargonidin (e.g., anthocyanin synthase, ANS, EC:1.14.20.4) and to include an exogenous nucleic acid sequence encoding glucosyltransferase activity that forms cyanidin-3-O-beta-D-glucoside from cyanidin, delphinidin-3-O-beta-D-glucoside from delphinidin, or pelagonidin-3-O-beta-D-glucoside from pelagonidin (e.g., anthocyanidin 3-O-glucosyltransferase, 3GT, EC:2.4.1.115). The cells provided herein that are engineered to produce flavonoids or anthocyanins are further engineered to increase the production of flavonoids or anthocyanins product, for example by increasing metabolic flux to a flavonoid or anthocyanin pathway, or by decreasing byproduct formation.
A cell engineered to produce a flavonoid is further engineered to increase the supply of precursor malonyl-CoA. One strategy for increasing malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC) activity. In various embodiments, the ACC enzyme, which in most eukaryotes, including fungi, is a large single chain polypeptide, and in plant and bacteria such as E. coli is a multi-subunit enzyme, is overexpressed in the host strain. Examples of acetyl-CoA carboxylase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACC genes of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, Ustilago maydis, and orthologs of these ACCs in other species having at least 50% amino acid identity to these ACCs.
Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). In some embodiments, acetyl-CoA synthase (ACS) that converts acetate and CoA to acetyl-CoA is over-expressed in the host cells. Cultures of engineered host cells that include overexpressed nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium, orthologs of these ACSs in other species having at least 50% amino acid identity to these ACSs.
Also considered, in further embodiments, is an engineered host cell that overexpresses a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Further, in E. coli, a variant of the Lpd subunit of PDH can be expressed that includes a mutation (E354K) that reduces inhibition of PDH by NADH.
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. In some embodiments, a cell engineered to produce a flavonoid, or an anthocyanin, is further engineered to increase the cell's supply of malonyl-CoA includes an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that generates malonyl-CoA from malonate. Examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. 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, for example, of Streptomyces coelicolor, or a malonate transporter encoded by DctPQM of Sinorhizobium medicae.
In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. Examples of malonate CoA-transferase that can be expressed in an engineered cell as provided herein include, without limitation, the alpha subunit (mdcA) of malonate decarboxylase from Acinetobacter calcoaceticus, Geobacillus sp, or a transferase having at least 50% identity to any of these or other naturally occurring malonate CoA-transferases.
In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA supply include upregulating endogenous pantothenate kinase (PanK) (EC:2.7.1.33) that produces CoA from pantothenate. Alternatively, or in addition, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33). Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.
Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) can be disrupted to reduce fatty acid biosynthesis. 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 acyl carrier protein (E. coli acpP).
Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption, or deletion of 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 aldehyde-alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), and enzyme acetate kinase phosphate acetyltransferase (ackA-pta).
Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, actetyl-CoA, and/or p-coumaryol-CoA. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and ybgC can be downregulated, disrupted, or deleted.
Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for one or more of the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to coumaric acid is the first committed step of the pathway. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. Strategies to increase tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the Phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins 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 effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. Strategies to increase heme supply include overexpression of the genes that synthesize the precursor ALA. In an E. coli host, ALA is formed from the 5-carbon skeleton of glutamate (the C5 pathway). 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 overexpressed include a mutated hemA (inserting two lysine residuals between Thr-2 and Leu-3 at N terminus of hemA gene from Salmonella typhimurium (EC:1.1.1.70). Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway (ALS is synthesized by the condensation of glycine and succinyl-CoA). Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Bradyrhizobium japonicum (EC: 2.3.1.37), ALAS of Rhodobacter capsulatus, or an ALAS having at least 50% sequence identity to a naturally occurring ALAS. Further, one or more of the downstream genes (e.g., in 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 flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.
In another aspect, provided herein are cell cultures that include engineered cells as provided herein in a culture medium, where the culture medium includes a carbon source that is also an energy source for the cells, where the carbon source can be, for example, glycerol, a sugar, or an organic acid, as nonlimiting examples. The culture medium can further include a feed molecule that is used to produce flavonoids or anthocyanins. A feed molecule can be, for example, acetate, malonate, tyrosine, pantothenate, coumarate, biotin, alpha-ketoglutarate, ascorbate, 5-aminolevulinic acid, succinate, or glycine. In some embodiments, the culture comprises a culture medium that includes a carbon source and at least one supplement that is a cofactor of an enzyme or is a precursor of an enzyme cofactor.
In yet another aspect, methods for producing flavonoids and anthocyanins that include incubating a culture of engineered host cell as provided herein to produce flavonoids or anthocyanins. The methods can further include recovering at least one of the flavonoids from the cells, the culture medium, or the whole culture.
In yet another aspect, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin 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 flavonoid or anthocyanin from glycerol occurs through enzymatic transformation. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs 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 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 genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (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 embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin 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 flavonoid or anthocyanin from a carbon source that can also be an energy source occurs 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 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 genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause increased metabolic flux to flavonoid precursors. In certain embodiments, one or more genetic modifications cause reduction in the formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (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 embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. In certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. Coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a plurality of engineered host cells, wherein each of the plurality of the engineered host cells comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs 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 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 genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (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 embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one the engineered host cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing a plurality of engineered host cells, wherein each of the plurality of the engineered host cell comprises one or more genetic modifications resulting production of flavonoid or anthocyanin 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 flavonoid or anthocyanin from a carbon source that can also be an energy source occurs 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 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 genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (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 embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one the engineered host cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.
In yet another aspect, the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the E. coli cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the E. coli cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.
In another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.
In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), delphinidin or gallocatechin to delphindin-3-glucoside (De3G), or afzelechin or pelargonidin to pelargonidin-3-glucoside (Pe3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method of increasing the transformation of cyanidin to cyanidin-3-glucoside (Cy3G), delphindin to delphindin-3-glucoside (De3G), or pelargonidin to pelagonidin-3-glucoside (Pe3G), comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.
In another aspect, the invention provides an engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b₅. In certain embodiments, cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.
In another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the
N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b₅. In certain embodiments, cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.

VI. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)

FIG. 1 shows the metabolic pathway of flavonoid and anthocyanin bioproduction in engineered cells and methods of preparing anthocyanins described herein.

FIG. 2 shows structures of the flavonoid and anthocyanin molecules that may be produced using engineered cells and methods of preparing anthocyanins described herein.

FIG. 3 shows HPLC spectra showing peaks corresponding to the molecules prepared using engineered cells and methods of preparing anthocyanins described herein.

FIG. 4 shows the pathway of flavonoid and anthocyanin bioproduction in engineered cells and methods of preparing anthocyanins described herein.

VII. DETAILED DESCRIPTION OF THE INVENTION

The present application provides engineered cells for producing one or more flavonoids, cultures that include the engineered cells, and methods of producing one or more flavonoids, or at least one anthocyanin. The terms “flavonoid”, “flavonoid product”, or “flavonoid compound” are used herein to refer to a member of a diverse group of phytonutrients found in almost all fruits and vegetables. As used herein, the terms “flavonoid”, “flavonoid product”, or “flavonoid compound” are used interchangeably to refer a molecule containing the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring. Flavonoids may include, but are not limited to, isoflavone type (e.g., genistein), flavone type (e.g., apigenin), flavonol type (e.g., kaempferol), flavanone type (e.g., naringenin), chalcone type (e.g., phloretin), anthocyanidin type (e.g., cyanidin), catechins, flavanones, and flavanonols. Flavonoid compounds of interest include, without limitation, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin, pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, and kaempferol. Anthocyanins are in the forms of anthocyanidin glycosides and acylated anthocyanins. Anthocyanin compounds of interest include, without limitation, cyanidin glycoside, delphinidin glycoside, pelargonidin glycoside, peonidin glycoside, and petunidin glycoside.
The terms ‘precursor’ or ‘flavonoid precursor’ as used herein may refer to any intermediate present in the biosynthetic pathway that leads to the production of catechins or anthocyanins. flavonoid precursors may include, but are not limited to tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.
Cells engineered for the production of a flavonoid or an anthocyanin can have one or multiple modifications, including, without limitation, the downregulation, disruption, or deletion of endogenous genes, the upregulation of an endogenous gene, and the introduction of exogenous genes.
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 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”.
A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to under-express or to 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 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.
The term “exogenous” or “heterologous” is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material that may be introduced on a vehicle such as a plasmid. Therefore, the term “endogenous” refers to a referenced molecule or activity that is naturally present in the host.
Genes or nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
The percent identity (% identity) between two sequences is determined when sequences are aligned for maximum homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Omega, and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide or amino acid sequence similarity or identity and can be useful in identifying orthologs of genes of interest. Additional sequences added to a polypeptide sequence, such as but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity.
A homolog is a gene or genes that have the same or identical functions in different organisms. Genes that are orthologous can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Paralogs are genes related by duplication within a genome, and can evolve new functions, even if these are related to the original one.
An engineered cell for producing flavonoids include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase (TAL) activity (alternatively or in addition, an exogenous nucleic acid encoding phenylalanine ammonia-lyase (PAL) activity and an exogenous nucleic acid encoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding chalcone isomerase (CHI) activity. Optionally, the engineered cell can further include an exogenous nucleic acid sequence encoding an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding flavonoid 3′-hydroxlase (F3′H) activity or flavonoid 3′,5′-hydroxylase (F3′5′H), an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase (DFR) activity, and/or an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase (LAR) activity.
Tyrosine ammonia-lyase (TAL) can be, for example, a member of the aromatic amino acid deaminase family that catalyzes the elimination of ammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosine ammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). Also considered for use in the engineered cells provided herein are TALs with SEQ ID NOS: 23-26, TALs listed in Table 1, TAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:1 that have the activity of a tyrosine ammonia lyase that produces p-coumaric acid from tyrosine.

TABLE 1

Tyrosine ammonia-lyase

	Organism	GenBank Accession Number

	Rhodotorula glutini	AGZ04575.1
	Flavobacterium johnsoniae	WP_012023194.1
	Herpetosiphon aurantiacus	ABX02653.1
	Rhodobacter capsulatus	ADE83766.1
	Saccharothrix espanaensis	AKE50820.1
	Trichosporon cutaneum	AKE50834.1

Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the non-oxidative deamination of L-phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is the Brevibacillus laterosporus phenylalanine ammonia-lyase (PAL; SEQ ID NO :2). Also considered for use in the engineered cells provided herein are PALs with SEQ ID NOS: 27-29, PAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 that have the activity of a phenylalanine ammonia lyase that produces trans-cinnamic acid from phenylalanine.
Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and catalyzes the formation of p-coumaric acid from trans-cinnamic acid. Considered for use in the engineered cells provided herein are C4H of Helianthus annuus L. (C4H; SEQ ID NO: 3), C4Hs with SEQ ID NOS: 30-32, and C4H homologs of other species, as well as variants of naturally occurring C4Hs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 the SEQ ID NO: 3 (C4H, Helianthus annuus L.) that have the activity of a C4H.
4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester. Considered for use in the engineered cells provided herein are 4CLs of Petroselinum crispum (SEQ ID NO: 4), 4CLs in Table 2, 4CLs with SEQ ID NOS: 33-36, and 4CL homologs of other species, as well as variants of naturally occurring 4CLs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID No: 4 (4CL, Petroselinum crispum) that have the activity of a 4CL.

TABLE 2

4-coumarate-CoA ligases

	Organism	GenBank Accession Number

	Petroselinum crispum	CAA31697.1
	Camellia sinensis	ASU87409.1
	Capsicum annuum	KAF3620173.1
	Castanea mollissima	KAF3954751.1
	Daucus carota	AIT52344.1
	Gynura bicolor	BAJ17664.1
	Ipomoea purpurea	AHJ60263.1
	Lonicera japonica	AGE10594.1
	Lycium chinense	QDL52638.1
	Nelumbo nucifera	XP_010265453.1
	Nyssa sinensis	KAA8540582.1
	Solanum lycopersicum	NP_001333770.1
	Striga asiatica	GER48539.1

The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS, SEQ ID NO: 5). Also considered for use in the engineered cells provided herein are the genes listed in Table 3, CHSs with SEQ ID: 37-40, and CHS homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 5 (CHS, Petunia x hybrida) that have the activity of a chalcone synthase.

TABLE 3

Chalcone synthases

	Organism	GenBank Accession Number

	Petunia hybrida	AAF60297.1
	Acer palmatum	AWN08245.1
	Callistephus chinensis	CAA91930.1
	Camellia japonica	BAI66465.1
	Capsicum annuum	XP_016566084.1
	Coffea arabica	XP_027118978.1
	Curcuma alismatifolia	ADP08987.1
	Dendrobium catenatum	ALE71934.1
	Garcinia mangostana	ACM62742.1
	Iochroma calycinum	AIY22758.1
	Iris germanica	BAE53636.1
	Lilium speciosum	BAE79201.1
	Lonicera caerulea	ALU09326.1
	Lycium ruthenicum	ATB56297.1
	Magnolia liliiflora	AHJ60259.1
	Matthiola incana	BBM96372.1
	Morus alba var. multicaulis	AHL83549.1
	Nelumbo nucifera	NP_001305084.1
	Nyssa sinensis	KAA8548459.1
	Paeonia lactiflora	AEK70334.1
	Panax notoginseng	QKV26463.1
	Ranunculus asiaticus	AYV99476.1
	Rosa chinensis	AEC13058.1
	Theobroma cacao	XP_007032052.2

Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ ID NO: 6), CHI of Table 4, CHIs with SEQ ID NOS: 41-44, and CHI homologs of other species, as well as variants of naturally occurring CHI having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 6 (CHI, Medicago sativa) that have the activity of a chalcone isomerase.

TABLE 4

Chalcone Isomerases

	Organism	GenBank Accession Number

	Medicago sativa	AGZ04578.1
	Dendrobium hybrid cultivar	AGY46120.1
	Abrus precatorius	XP_027366189.1
	Antirrhinum majus	BA032070.1
	Arachis duranensis	XP_015942246.1
	Astragalus membranaceus	ATY39974.1
	Camellia sinensis	XP_028119616.1
	Castanea mollissima	KAF3958409.1
	Cephalotus follicularis	GAV77263.1
	Clarkia gracilis subsp.	QPF47150.1
	sonomensis
	Dianthus caryophyllus	CAA91931.1
	Glycyrrhiza uralensis	AXO59749.1
	Handroanthus impetiginosus	PIN05040.1
	Lotus japonicus	CAD69022.1
	Morus alba	AFM29131.1
	Phaseolus vulgaris	XP_007142690.1
	Punica granatum	ANB66204.1
	Rhodamnia argentea	XP_030524476.1
	Spatholobus suberectus	TKY50621.1
	Trifolium subterraneum	GAU12132.1

A nucleic acid sequence encoding a CHI can in some embodiments be fused to a nucleic acid sequence encoding a CHS in an engineered cell as provided herein, such that the CHI activity is fused to the chalcone synthase activity, i.e., a fusion protein is produced in the engineered cell that has both condensing and cyclization activities.
Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substrates include (2S)-eriodictyol, (2S)-dihydrotricetin and (2S)-pinocembrin. Some F3H enzymes are bifunctional and also catalyzes as flavonol synthase (EC: 1.14.20.6). Considered for use in the engineered cells provided herein are F3H of Rubus occidentalis (SEQ ID NO: 7), F3Hs with SEQ ID NOS: 45-48, F3Hs listed in Table 5, and other F3H homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO:7 (F3H, Rubus occidentalis) that have the activity of a F3H.

TABLE 5

Flavanone 3-hydroxylases

	Organism	GenBank Accession Number

	Rubus occidentalis	ACM17897.1
	Abrus precatorius	XP_027347564.1
	Nyssa sinensis	KAA8547483.1
	Camellia sinensis	AAT68774.1
	Morelia rubra	KAB1219056.1
	Rosa chinensis	PRQ47414.1
	Malus domestica	AAD26206.1
	Vitis amurensis	ALB75302.1
	Iochroma ellipticum	AMQ48669.1
	Hibiscus sabdariffa	ALB35017
	Cephalotus follicularis	GAV71832

Flavonoid 3′-hydroxylases (F3′H) belongs to the cytochrome P450 family with systematic name of flavonoid, NADPH:oxygen oxidoreductase (3′-hydroxylating). In the flavonoid biosynthetic pathway, F3′H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. Considered for use in the engineered cells provided herein are F3′H of Brassica napus (F3′H; SEQ ID NO: 8), F3′H with SEQ ID NOS: 49-52, those listed in Table 6, and homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 F3′H. F3′H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. Cytochrome P450 reductases are diflavin oxidoreductases that supply electrons to F3′Hs. The P450 reductase can be from the same species as F3′H or different species from F3′H. Considered for use in the engineered cells provided herein are CPR of Catharanthus roseus (SEQ ID NO: 9), additional CPRs listed in Table 7, CPRs with SEQ ID NOS: 53-55, CPR homologs of other species, and variants of naturally occurring CPRs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 CPRs that have the activity of a CPR. In various embodiments, the N-terminal nucleic acid sequences in the genes of F3′H and/or CPR originated from eukaryotic cells can encode targeting leader peptides, which can be removed before introduction into prokaryotic host cells, if desired. In some embodiments, the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin).

TABLE 6

Flavonoid 3′-hydroxylases

	Organism	GenBank Accession Number

	Brassica napus	ABC58722.1
	Gerbera hybrid cultivar D1	ABA64468.1
	Cephalotus follicularis	GAV84063.1
	Theobroma cacao	XP_007037548.1
	Phoenix dactylifera	XP_008791304.2

TABLE 7

Cytochrome P450 reductases

	Organism	GenBank Accession Number

	Catharanthus roseus	CAA49446.1
	Brassica napus	XP_013706600.1
	Cephalotus follicularis	GAV59576.1
	Camellia sinensis	XP_028084858.1

A nucleic acid sequence encoding a F3′H can in some embodiments be fused to a nucleic acid sequence encoding a CPR in an engineered cell as provided herein, such that the F3′H activity is fused to the CPR activity.
In the cells engineered to produce dihydomyricetin, flavonoid 3′, 5′-hydroxylase (F3′5′H) can be used to convert dihydrokaempferol to dihydromyricetin or naringenin to pentahydroxyflavone, which is further converted to dihydromyricetin by a F3H. F3′5′H has the systematic name flavanone, NADPH:oxygen oxidoreductase and catalyzes the formation of 3′,5′-dihydroxyflavanone from flavanone. An exemplary F3′5′H is the Delphinium grandiflorum F3′5′H (SEQ ID NO: 10), Also considered for use in the engineered cells provided herein include F3′5′H with SEQ ID NOS:56-57, F3′5′H homologs of other species, and variants of naturally occurring F3′5′H having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NOS:10 that have the activity of a F3′5′H.
Dihydroflavonol 4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (Taxifolin, DHQ), or dihydromyricein (DHM) to reduce those compounds to the corresponding cis-flavan-3,4-diol (DHK to leucopelargonidin; Taxifolin to leucocyanidin; DHM to leucodelphinidin). An exemplary DFR is the Anthurium andraeanum DFR (SEQ ID NO: 11). Also considered for use in the engineered cells provided herein include DFRs in Table 8, DFRs with SEQ ID NOS: 58-61, and DFR homologs of other species, as well as variants of naturally occurring DFR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 11.

TABLE 8

Dihydroflavonol 4-reductases

	Organism	GenBank Accession Number

	Eustoma grandiflorum	BAD34461.1
	Anthurium andraeanum	AAP20866.1
	Camellia sinensis	AAT66505.1
	Morelia rubra	KAB1203810.1
	Dendrobium moniliforme	AEB96144.1
	Fragaria × ananassa	AHL46451.1
	Rosa chinensis	XP_024167119.1
	Acer palmatum	AWN08247.1
	Nyssa sinensis	KAA8531902.1
	Vitis amurensis	I82380.1
	Abrus precatorius	XP_027329642.1
	Angelonia angustifolia	AHM27144.1
	Pyrus pyrifolia	Q84KP0.1
	Theobroma cacao	XP_017985307
	Theobroma cacao	XP_007051597.2
	Brassica oleracea var. capitata	QKO29328.1
	Rubus idaeus	AXK92786.1
	Citrus sinensis	AAY87035.1
	Gerbera hybrida	P51105.1
	Cephalotus follicularis	GAV76940.1
	Ginkgo biloba	AGR34043.1
	Dryopteris erythrosora	QFQ61498.1
	Dryopteris erythrosora	QFQ61499.1
	Cephalotus follicularis	GAV76942.1

Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin. Considered for use in the engineered cells provided herein are LAR of Desmodium uncinatum (SEQ ID NO: 12), LARs with SEQ ID NOS: 62-65, and LAR homologs of other species, as well as variants of naturally occurring LAR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 12 (LAR, Desmodium uncinatum) that have the activity of a LAR.
Optionally, the cells are further engineered to include an anthocyanin synthase (ANS) which catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin, or leucodelphinidin to delphinidin. Considered for use in the engineered cells provided herein are ANS of Carica papaya (SEQ ID NO: 13), ANS with SEQ ID NOS: 66-69, and ANS homologs of other species, as well as variants of naturally occurring ANS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO:13 (ANS, Carica papaya) that have the activity of a ANS.
Optionally, the cells are further engineered to include a flavonoid-3-glucosyl transferase (3GT) to generate anthocyanins by transfer of a sugar moiety such as, without limitation, UDP-α-D-glucose to anthocyanidins to form glycosylated anthocyanins. Considered for use in the engineered cells provided herein are 3GT of Vitis labrusca (SEQ ID NO:14), 3GT with SEQ ID NOS: 70-73, and 3GT homologs of other species, as well as variants of naturally occurring 3GT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 SEQ ID NO: 14 (3GT, Vitis labrusca) that have the activity of a 3GT.
In various aspects, host cells may be engineered for enhanced production of flavonoids or anthocyanins 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 flavonoids or anthocyanins can be increasing yield, titer, or rate of production.
Thus, a host cell engineered for the production of a flavonoid or anthocyanin 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 increasing cell's malonyl-CoA supply that does not rely on the ACC step; expression or overexpression of at least one enzyme to increase tyrosine supply; expression or overexpression of at least one enzyme to increase CoA availability for synthesizing precursors malonyl-CoA or p-coumaryol-CoA; expression or overexpression at least one enzyme to increase heme biosynthesis; deletion or downregulation of at least one fatty acid synthesis enzyme; at least one alcohol dehydrogenase, 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. The foregoing list of modifications is nonlimiting.
Malonyl-CoA is the direct precursor for chalcone synthase to perform sequential condensations with p-coumaryol-CoA. 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 carboxylation of biotin using bicarbonate as a CO₂donor. 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 synthesis from acetyl-CoA. For example, Mucor circinelloides (SEQ ID NO: 15) 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, the genes listed in Table 9, genes with SEQ ID NOS: 74-76, naturally occurring orthologs of these ACCs, or variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 genes. Further, naturally occurring acetyl-CoA carboxylase genes can be further engineered to introduce single or multiple amino acid mutations to increase catalytic activity and/or remove feedback inhibition.

TABLE 9

Acetyl-CoA carboxylases

	Organism	GenBank Accession Number

	Lipomyces starkeyi	AJT60321.1
	Rhodotorula toruloides	GEM08739.1
	Ustilago maydis	XP_011390921.1
	Mucor circinelloides	EPB82652.1
	Kalaharituber pfeilii	KAF8466702.1
	Aspergillus fumigatus	KEY77072.1
	Rhodotorula diobovata	TNY18634.1
	Leucosporidium creatinivorum	ORY74050.1
	Microbotryum intermedium	SCV70467.1
	Mixia osmundae	GAA98306.1
	Puccinia graminis	KAA1079218.1
	Suillus occidentalis	KAG1764021.1
	Gymnopilus junonius	KAF8909366.1

Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is 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 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 a nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium (SEQ ID NO:16), and orthologs of these ACSs in other species having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 ACSs.
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 (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 sensitive to NADH inhibition than the native LPD.
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 a flavonoid or an anthocyanin as provided herein can include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from 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 product. Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 to express a malonyl-CoA synthetase. In Rhizobium trifolii, the math 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, for example of Streptomyces coelicolor (SEQ ID NO:18), of Rhizobiales bacterium (SEQ ID NO:80), of Rhizobium leguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ ID NO: 82), of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 synthetase and a malonate transporter can include a culture medium that includes malonate.
In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding 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 α subunit, is malonate CoA-transferase, and mdcD gene product, the β subunit, is a malonyl-CoA decarboxylase. The mdcE gene product, the γ subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the δ subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. When the α subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an a subunit to exchange the acetyl group for a malonyl group. As the 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 (SEQ ID NO: 19), mdcAs of Table 10, mdcAs with SEQ ID NOS: 84-87, or a transferase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally occurring malonate CoA-transferases.

TABLE 10

Malonate CoA-transferases (malonate decarboxylase subunit alpha)

	Organism	GenBank Accession Number

	Acinetobacter calcoaceticus	AAB97627.1
	Geobacillus sp.	QNU36929.1
	Acinetobacter johnsonii	WP_087014029.1
	Acinetobacter marinus	WP_092618543.1
	Acinetobacter rudis	WP_016655668.1
	Psychrobacter sp. G	WP_020444454.1
	Moraxella catarrhalis	WP_064617969.1
	Zoogloea sp.	MBL0283742.1
	Dechloromonas sp.	KAB2923906.1
	Stenotrophomonas rhizophila	WP_123729366.1
	Xanthomonas cucurbitae	WP_159407614.1

In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability 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-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 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, SEQ ID NO: 20), CoaX of Streptomyces sp. CL12509 (SEQ ID NO: 88), CoaX of Streptomyces cinereus (SEQ ID: 89), or CoaX of Kitasatospora kifunensis (SEQ ID NO: 90) Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.
Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavonoid biosynthesis for the precursor malonyl-CoA and thus limits flavonoid formation. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid 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. 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).
Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins 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.
Further, a cell engineered for the production of flavonoids or anthocyanins 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 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.
In further embodiments, a cell engineered for the production of flavonoids or anthocyanins can have one or more of fatty acid degradation genes downregulated, disrupted, or deleted to improve precursor supply to the flavonoid pathway. In E. 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, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from feedstock such as glucose or glycerol is necessary to make biological production economically viable. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The shikimate pathway is the central metabolic route leading to formation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), this pathway exclusively exists in plants and microorganisms. It starts with the condensation of intermediates of glycolysis and pentosephosphate-pathway, phosphoenolpyruvate (PEP), and erythrose-4-phosphate (E4P), respectively, which enter the pathway through a series of condensation and redox reactions via 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the central branch point metabolite chorismate is obtained via shikimate-3-phosphate under ATP hydrolysis and introduction of a second PEP. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. In E. coli three DAHP synthase isozymes exist (aroF, aroG, aroH), which are each feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), in contrast the two DAHP synthases of plants are not subject to feedback-inhibition. In plants and bacteria, the subsequent five steps are catalyzed by single enzymes. From the central intermediate chorismate the pathway branches off to anthranilate and prephenate leading to aromatic amino acid, para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA) synthesis, the latter being a precursor for folate metabolism. Strategies to increase L-tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the ppsA, aroG, and/or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.
Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins 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 effective strategy to increase flavonoid biosynthesis. 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 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 to convert ALA to heme. In an E. coli host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (G1tX), 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 overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemA with SEQ ID NOS: 91-93. 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 (SEQ ID:22), ALAS with SEQ ID NOS: 94-97, or an ALAS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, 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 flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.
Engineered cells that produce a flavonoid can be engineered to include multiple pathways to enhance flavonoid production. Those skilled in the art will recognize that the embodiments described herein can be combined in multiple ways. Examples of engineered cells having multiple genetic modifications are exemplary only and do not limit the scope of the invention.
Enzymes to be expressed or overexpressed in engineered cells according to the invention are set forth in Table 11.

Host Cells

A 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 cyanobacteria) cells.
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.
Nonlimiting examples of suitable microbial hosts for the bio-production of a flavonoid 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 or Pichia stipitis; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.

Culture Medium

In yet another aspect, methods for producing a flavonoid or an anthocyanin that include incubating a culture of an engineered host cell as provided herein to produce a flavonoid or an anthocyanin. The methods can further include recovering the flavonoid or anthocyanin from the culture medium, whole culture, or cells.
The culture comprises cells engineered for the production of flavonoids or anthocyanins 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, 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, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, alpha-ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.
Culture conditions can include aerobic, microaerobic or any combination alternating aerobic/microaerobic growth conditions. Further, culture conditions can include shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving a flavonoid product include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing flavonoid product. Exemplary growth procedures include, for example, 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 temperature can be from, for example, 20-37° C., depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH of the bioreactor can be controlled to range from 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for examples, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. To purify the flavonoids and/or anthocyanins to homogeneity the solution containing the flavonoids and/or anthocyanins was concentrated and the product purified via ion exchange or silica-based chromatography. The resulting solution was either lyophilized to yield the products in a solid form or was concentrated into a liquid solution.
In some embodiments, a method of producing a flavonoid or an anthocyanin comprises culturing an engineered cell disclosed herein in a culture medium to produce a flavonoid or an anthocyanin. In some embodiments, glycerol is used as a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In some embodiments, the method comprises isolating naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside. In some embodiments the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to about 99%, e.g., from about 50% to about 95% (for example from: about 50%, 55%, 60%, 65%, 70%, 75%, 80% to about: 85%, 90%, 95%, 97.5%, 99% or 99.9%). In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to: about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 55% to: about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 60% to: about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 65% to: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 70% to: about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 75% to: about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80% to about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 85% to: about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 90% to about 95%, or about 99%, or from about 95% to about 99% or greater.

VIII. EXAMPLES

Using The Modified Cell To Create Products

Example 1

Production of Naringenin in E. coli

An E. coli cell derived from MG1655 was engineered to overexpress ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) to produce naringenin when substrates tyrosine and coumaric acid were supplied in culture medium. ACC was expressed on a medium-copy plasmid (15-20 copies) while TAL, 4CL, CHS, and CHI were expressed on the chromosome. Cells of an OD 2.5 were cultured in a 48-well plate at 30 degree for 24 hours with a shaking speed of 600 RPM in minimal medium supplied with trace element, vitamins, 1 mM tyrosine,1 mM coumaric acid, and 2% glycerol. Cell cultures were extracted with DMSO at 1:1 ratio and centrifuged for 15 mins. The supernatant was analyzed for naringenin with HPLC. The cells produced 232 μM naringenin.
Variants of the foregoing host cell may be prepared using one or more of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) with one or more homologs of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), or CHI (SEQ ID NO: 6), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 enzymes.

Example 2

Production of Dihydrokaempferol in E. coli

An E. coli cell derived from MG1655 was engineered to overexpress F3H (SEQ ID NO: 7) on the chromosome to produce dihydrokaempferol when substrate naringenin was supplied in culture medium. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glycerol, trace elements, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with DMSO and centrifuged for 15 minutes. The supernatant was analyzed for dihydrokaempferol with HPLC. The cells produced 315 μM dihydrokaempferol.
Variants of the foregoing host cell may be prepared using a homolog of F3H (SEQ ID NO: 7), wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 enzyme.

Example 3

Production of Taxifolin in E. coli

An E. coli strain derived from MG1655 was engineered to overexpress F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) to produce taxifolin when the substrate naringenin was supplied in culture medium. F3H was overexpressed on the chromosome while F3′H and CPR were overexpressed on a medium-copy plasmid. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glucose, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with 50% DMSO and centrifuged for 15 minutes. The supernatant was analyzed for taxifolin with HPLC. The cells produced 500 μM taxifolin.
Variants of the foregoing host cell may be prepared using one or more of F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) along with one or more homologs of F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 enzymes.

Example 4

Production of Anthocyanidins and Anthocyanins

An E. coli strain derived from MG1655 was engineered to overexpress ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) to produce cyanidin-3-O-glucoside when the substrate (+)-catechin was supplied in culture medium. ANS and 3GT were overexpressed on the chromosome. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 1.0% glucose, 2.0 mM (+)-catechin, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were acidified with 2M HCL and extracted with 100% Ethanol. The supernatant was analyzed for cyanidin-3-O-glucoside by HPLC. The cells produced 50 mg/L cyanidin-3-O-glucoside.
Variants of the foregoing host cell may be prepared using one or both of ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) along with a homolog of ANS (SEQ ID NO: 13), 3GT (SEQ ID NO: 14), or both, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, 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 enzymes.

Analytical Methods

Example 5

Flavonoid Precursors and Flavonoids

For sampling naringenin, eriodictyol, dihydrokaempferol and taxifolin, extraction of total flavonoids from E. coli were performed on whole cell broth. 500 μL of whole cell broth was vortexed for 30 seconds with 500 μL of DMSO (dimethyl sulfoxide) and centrifuged for 15 minutes. For HPLC analysis, 50 μL of supernatant was transferred to an HPLC vial.
The HPLC method was as follows: An Agilent 1200 HPLC was fitted with an Ascentis C18 Column 150 mm×4.6 mm, 3μm, equipped with a R-18 (3 μm) guard column. The column was heated to 30 ° C. with the sample block being maintained at 25 ° C. For each sample, 5 μL was injected and the product was eluted at a flow rate of 1.5 mL/min using 0.1% phosphoric acid in water (solvent A), acetonitrile (solvent B), and methanol (solvent C) with the following gradient:


Time	A (%)	B (%)	C (%)

0	85	10	5
2.5	85	10	5
7.5	70	25	5
12.5	50	45	5
15	85	10	5

The run time was a total of 15 minutes with naringenin, eriodictyol, dihydrokaempferol and taxifolin eluting at 12.50, 11.56, 10.20, and 8.85 minutes respectively. A diode array detector (DAD) was used for the detection of the molecule of interest at 288 nm.

Example 6

Anthocyanidins and Anthocyanins

For sampling (+)-catechin, cyanidin, and cyanidin-3-glucoside the reaction fluid was acidified with 13 M HCl (1:40 v/v), and extracted with 100% ethanol followed by mixing, centrifugation and filtration through a 0.45 μm filter. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a LiChrospher RP-8 Column 250 mm×4.6 mm, 5 μm, equipped with a LiChrospher 100 RP-8 (5 μm) LiChroCART 4-4 guard column. The column was heated to 25° C. with the sample block being maintained at 25° C. For each sample, 10 μL was injected and the product was eluted at a flow rate of 1.0 ml/min using 0.1% phosphoric acid in water (solvent A) and acetonitrile (solvent B) with the following gradient: 90% A to 10% A for 12 min, 90% A for 0.5 min, and 90% A for 3.5 min for column equilibration. The run time was a total of 16 minutes with cyanidin-3-glycoside eluting at 6.95 mins and cyanidin eluting at 8.9 minutes. A diode array detector (DAD) was used for the detection of the molecule of interest at either 280 nm or 530 nm.

Example 7

Flavonoid Production

The example provides a combination of modifications to the E. coli host genome including deletions and overexpression of enzymes from other organisms to recapitulate the bioproduction pathway described in FIG. 4. Accordingly, the invention provides an engineered host cell that comprises one or more genetic modifications (as shown in FIG. 4 and described in this Example 7 and herein above in this application) that result in production of flavonoid or anthocyanin 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 flavonoid or anthocyanin from a carbon source that can also be an energy source occurs 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, and (v) 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, and (v) any combination thereof. As shown in FIG. 4, in certain embodiments, one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors. As shown in FIG. 4, in certain embodiments, one or more of the genetic modifications cause reduction of formation of byproducts. As shown in FIG. 4, in certain embodiments, one or more genetic modifications are selected from: (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.
As shown in FIG. 4, in certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.
As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.
As shown in FIG. 4, in certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof.
As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof.
As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof.
The compositions as described above, can be used in methods described herein for increasing the production of flavonoids or anthocyanins. Such methods involve providing any of the compositions described above to result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (such as shown in part or in whole in FIG. 4).
In yet another aspect, it is envisioned that the pathway illustrated in FIG. 4 can be carried out using a plurality of engineered host cells, as opposed to a single host cell as described above. In such embodiments, the plurality of the engineered host cells have one or more genetic modifications that result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (as shown in FIG. 4).
Aspects of the invention are now described with reference herein to FIG. 4.
Step 1: conversion of pyruvate to acetate. poxB is deleted to reduce carbon loss and eliminate the byproducts.
Step 2: conversion of pyruvate to lactate. ldhA is deleted to reduce carbon loss and eliminate the byproducts.
Step 3: conversion of Acetyl-CoA to acetate. ackA-pta is deleted to reduce carbon loss and eliminate the byproducts.
Step 4: conversion of Acetyl-CoA to ethanol (EtOH). adhE is deleted to reduce carbon loss and eliminate the byproducts.
Step 5: conversion of acetyl-CoA to a substrate for the tricarboxylic acid cycle (TCA).
Step 6: conversion of acetyl-CoA to mal-CoA. Heterologous ACC is expressed to increase the concentration of available mal-CoA. Heterologous ACC may be obtained from fungal species. Accordingly, embodiments of the invention provide an engineered host cell that comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.
Step 7: conversion of mal-CoA to malonyl-ACP (acyl carrier protein). malonyl-coA-ACP transacylase (fabD) is downregulated to increase carbon flux.
Step 8: conversion of malonyl-ACP to 3-ketyoacyl-ACP. beta-ketoacyl-ACP synthase II (fabF) is downregulated to increase carbon flux.
Step 9: conversion to mal-CoA to naringenin chalcone; conversion of coumaryl-CoA to naringenin chalcone. A heterologous CHS is overexpressed.
Step 10: conversion to naringenin chalcone to naringenin. A heterologous CHI is overexpressed.
Steps 11, 12, and 13: conversion of naringenin to dihydrokaempferol (DHK); conversion of naringenin to eriodictyol (EDL); conversion of eriodictyol (EDL) to dihydroquercetin (DHQ); conversion of (DHK) to dihydroquercetin (DHQ); conversion of dihydrokaempferol (DHK) to dihydromyricetin (DHM); conversion of pentahydroxyflayaone (PHF) to dihydromyricein (DHM). Heterologous F3′5′H, F3H, F3H, and/or CPR are overexpressed. Accordingly, as shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b₅. In certain embodiments, cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
As shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b₅. In certain embodiments, cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
Step 14: conversion of dihydroquercetin (DHQ) to leucocyanidin (LC); conversion of dihydrokaempferol (DHK) to leucopelargonidin (LP); and conversion of dihydromyricetin (DHM) to leucodelphinidin (LD). Heterologous DFR is overexpressed.
Step 15: conversion of leucocyanidin (LC) to catechin; conversion of leucodelphinidin (LD) to gallocatechin; and conversion of leucopelargonidin (LP) to afzelechin. Heterologous LAR is overexpressed.
Step 16: conversion of catechin to cyanidin; conversion of leucocyanidin (LC) to catechin; conversion to leucodelphinidin (LD) to delphinidin; conversion of gallocatechin to delphinidin; conversion of leucopelargonidin (LP) to pelargonidin; or conversion of afzelechin to pelargonidin. Heterologous ANS is overexpressed. Step 16 could be carried in vivo or in a cell-free medium. Accordingly, as shown in FIG. 4, in another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof.
In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.
Step 17: conversion of pelargonidin to callistephin; conversion of delphinidin to myrtillin (De3G); conversion of cyanidin to Cy3G. Heterologous 3GT was overexpressed in E. coli. Step 17 could be carried in vivo or as a cell-free reaction.
Step 18: conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine.
Step 19: conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA is overexpressed to upregulate tyrosine.
Step 20: conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). aroG variant is overexpressed to upregulate tyrosine.
Step 21: conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) to dehydroquinate (DHQ).
Step 22: conversion of dehydroquinate (DHQ) to 3-dehydroshikimate (DHS).
Step 23: conversion of 3-dehydroshikimate (DHS) to shikimic acid (SHK). aroE is overexpressed to upregulate tyrosine.
Step 24: conversion of shikimic acid (SHK) to shikimate-3-phosphate (S3P).
Step 25: conversion of shikimate-3-phosphate (S3P) to 5-enolpyruvylshikimate-3-phosphate (EPSP).
Step 26: conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismic acid (CHA).
Step 27: conversion of chorismic acid (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variant is overexpressed.
Step 28: conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine; conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Accordingly, as shown in FIG. 4, embodiments of the invention provide an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene.
As shown in FIG. 4, in another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene.
Step 29: conversion of tyrosine to coumaric acid. A heterologous TAL is overexpressed.
Step 30: conversion of courmaric acid to coumaryl-CoA. A heterologous 4CL is overexpressed.
Step 31: conversion of glutamate (Glut) to glutamyl-tRNA.
Step 32: conversion of glutamyl-tRNA to glutamate semialdehyde (GSA). hemA is overexpressed to upregulate ALA.
Step 33: conversion of glutamate semialdehyde (GSA) to δ amino levulinic acid (ALA). hemL is overexpressed to upregulate ALA.
Step 34: conversion of δ amino levulinic acid (ALA) to porphobilinogen (PBG).
Step 35: conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB).
Step 36: conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III (UPPIII).
Step 37: conversion of uroporphyrinogen III (UPPIII) to coproporphyrinogen III (CPPIII).
Step 38: conversion of coproporphyrinogen III (CPPIII) to protoporphyrinogen IX (PPPIX).
Step 39: conversion of protoporphyrinogen IX (PPPIX) to protoporphyrin IX, which is subsequently covered to heme.
Step 40: conversion of prephenate (PPA) to phenylpyruvate (POPP).
Step 41: conversion of phenylalanine (Phe) to cinnamate. Heterologous PAL and/or TAL are overexpressed.
Step 42: conversion of cinnamate to coumaric acid. Heterologous C4H/CPR are overexpressed.

TABLE 11

Enzyme Sequences:

Enzyme:	Sequence:	SEQ ID:

Tyrosine ammonia-	MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTDEEIVRMGAS	1
lyase (TAL)	ARTIEEYLKSDKPIYGLTQGFGPLVLFDADSELEQGGSLISHLGT
Saccharothrix	GQGAPLAPEVSRLILWLRIQNMRKGYSAVSPVFWQKLADLWNKGF
espanaensis	TPAIPRHGTVSASGDLQPLAHAALAFTGVGEAWTRDADGRWSTVP
Accession:	AVDALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHRSALRLV
ABC88669.1	RACAVLSARLATLLGANPEHYDVGHGVARGQVGQLTAAEWIRQGL
	PRGMVRDGSRPLQEPYSLRCAPQVLGAVLDQLDGAGDVLAREVDG
	CQDNPITYEGELLHGGNFHAMPVGFASDQIGLAMHMAAYLAERQL
	GLLVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRIRQLVF
	PASLTTLPTNGWNQDHVPMALNGANSVFEALELGWLTVGSLAVGV
	AQLAAMTGHAAEGVWAELAGICPPLDADRPLGAEVRAARDLLSAH
	ADQLLVDEADGKDFG

Phenylalanine	MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH	2
ammonia-lyase	ISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK
(PAL)	AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL
Brevibacillus	EVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK
laterosporus LMG	VLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL
15441	AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA
Accession:	SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS
WP_003337219.1	IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNG
	GNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPN
	LIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRS
	TEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRD
	SKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK
	KEIHDQNVND

Cinnamate-4-	MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLPVPIFGN	3
hydroxylase (C4H)	WLQVGDDLNHRNLTDLAKKFGEIFLLRMGQRNLVVVSSPDLAKEV
Helianthus annuus	LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV
L.	PFFTNKVVQQYRYGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQL
Accession:	MMYNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQSFEYNYG
QJC72299.1	DFIPILRPFLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV
	DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAIETTLWSI
	EWGIAELVNHPEIQAKLRNELDTKLGPGVQVTEPDLHKLPYLQAV
	IKETLRLRMAIPLLVPHMNLHDAKLGGYDIPAESKILVNAWWLAN
	NPEQWKKPEEFRPERFFEEESKVEANGNDFRYLPFGVGRRSCPGI
	ILALPILGITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKH
	STIVAKPRAL

4-coumarate-CoA	MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCL	4
ligase (4CL)	INGATGETFTYSQVELLSRKVASGLNKLGIQQGDTIMLLLPNSPE
Petroselinum	YFFAFLGASYRGAISTMANPFFTSAEVIKQLKASQAKLIITQACY
crispum	VDKVKDYAAEKNIQIICIDDAPQDCLHFSKLMEADESEMPEVVIN
Accession:	SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMH
P14912.1	SEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKFDIVPFLEL
	IQKYKVTIGPFVPPIVLAIAKSPVVDKYDLSSVRTVMSGAAPLGK
	ELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGA
	CGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPE
	STRTTIDEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAP
	AELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRTNGFTTTEE
	EIKQFVSKQVVFYKRIFRVFFVDAIPKSPSGKILRKDLRARIASG
	DLPK

Chaicone synthase	MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITN	5
(CHS)	SEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPS
Petunia x hybrida	LDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV
Accession:	DMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
AAF60297.1	NKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAIIIG
	SDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKD
	VPGLISKNIEKSLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEI
	KLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKASAKEGLGT
	TGEGLEWGVLFGFGPGLTVETVVLHSVAT

Chalcone isomerase	MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERGLTIEGNFI	6
(CHI)	KFTAIGVYLEDIAVASLAAKWKGKSSEELLETLDFYRDIISGPFE
Medicago sativa	KLIRGSKIRELSGPEYSRKVMENCVAHLKSVGTYGDAEAEAMQKF
Accession:	AEAFKPVNFPPGASVFYRQSPDGILGLSFSPDTSIPEKEAALIEN
P28012.1	KAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN

Flavanone 3-	MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFSNEIPIISL	7
hydroxylase (F3H)	SGIDEVEGRRAEICNKIVEACEDWGVFQIVDHGVDAKLISEMTRL
Rubus occidentalis	ARDFFALPPEEKLRFDMSGGKKGGFIVSSHLQGEAVQDWREIVTY
Accession:	FSYPVRHRDYSRWPDKPEGWRAVTQQYSDELMGLACKLLEVLSEA
ACM17897.1	MGLEKEALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDPGTI
	TLLLQDQVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHFLSNG
	RFKNADHQAVVNSNHSRLSIATFQNPAQEAIVYPLKVREGEKPIL
	EEPITYTEMYKKKMSKDLELARLKKLAKEQQPEDSEKAKLEVKQV
	DDIFA

Flavonoid 3′	MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIGNLPHM	8
hydroxylase (F3′H)	GPKPHQTLAAMVTTYGPILHLRLGFADVVVAASKSVAEQFLKVHD
Brassica napus	ANFASRPPNSGAKHMAYNYQDLVFAPYGQRWRMLRKISSVHLFSA
Accession:	KALEDFKHVRQEEVGTLMRELARANTKPVNLGQLVNMCVLNALGR
ABC58723.1	EMIGRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFVPALDCL
	DLQGVAGKMKRLHKRFDAFLSSILEEHEAMKNGQDQKHTDMLSTL
	ISLKGTDFDGEGGTLTDTEIKALLLNMFTAGTDTSASTVDWAIAE
	LIRHPEIMRKAQEELDSVVGRGRPINESDLSQLPYLQAVIKENFR
	LHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWS
	DPLTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAGLSLGL
	RTIQLLTATLVHGFEWELAGGVTPEKLNMEETYGITLQRAVPLVV
	HPKLRLDMSAYGLGSA

Cytochrome P450	MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPAVMAMLLE	9
reductase (CPR)	NKELVMILTTSVAVLIGCVVVLIWRRSSGSGKKVVEPPKLIVPKS
Catharanthus	VVEPEEIDEGKKKFTIFFGTQTGTAEGFAKALAEEAKARYEKAVI
roseus	KVIDIDDYAADDEEYEEKFRKETLAFFILATYGDGEPTDNAARFY
Accession:	KWFVEGNDRGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVAEQ
Q05001	GGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDNLLRDEDDTTVS
	TTYTAAIPEYRVVFPDKSDSLISEANGHANGYANGNTVYDAQHPC
	RSNVAVRKELHTPASDRSCTHLDFDIAGTGLSYGTGDHVGVYCDN
	LSETVEEAERLLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCT
	LRTALTRYADLLNTPKKSALLALAAYASDPNEADRLKYLASPAGK
	DEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFAAIAPRLQPRFYS
	ISSSPRMAPSRIHVTCALVYEKTPGGRIHKGVCSTWMKNAIPLEE
	SRDCSWAPIFVRQSNFKLPADPKVPVIMIGPGTGLAPFRGFLQER
	LALKEEGAELGTAVFFFGCRNRKMDYIYEDELNHFLEIGALSELL
	VAFSREGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDAKGMAR
	DVHRTLHTIAQEQGSMDSTQAEGFVKNLQMTGRYLRDVW

Flavonoid 3′, 5′-	MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKGWPVVGA	10
hydroxylase	LPMLGNMPHAALADLSRRYGPIVYLKLGSRGMVVASTPDSARAFL
(F3′5′H)	KTQDLNFSNRPTDAGATHIAYNSQDMVFADYGPRWKLLRKLSSLH
Delphinium	MLGGKAVEDWAVVRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAM
grandiflorum	ANMLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFNVGDFIP
Accession:	SIAWMDLQGIVRGMKRLHKKFDALLDKILREHTATRRERKEKPDL
BAO66642	VDVLMDNRDNKSEQERLTDTNIKALLLNLFSAGTDTSSSTIEWAL
	TEMIKNPSIFGRAHAEMDQVIGRNRRLEESDIPKLPYLQAICKET
	FRKHPSTPLNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNV
	WENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRICAGTRMG
	IVLVEYILGSLVHAFEWKLRDGETLNMEETFGIALQKAVPLAAVV
	TPRLPPSAYVV

Dihydroflavonol 4-	MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATVRDPSNMKKVK	11
reductase (DFR)	HLLDLPGAANRLTLWKADLVDEGSFDEPIQGCTGVFHVATPMDFE
Anthurium	SKDPESEMIKPTIEGMLNVLRSCARASSTVRRVVFTSSAGTVSIH
andraeanum	EGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK
Accession:	NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNP
AAP20866.1	VQFVHLDDLCNAHIFLFECPDAKGRYICSSHDVTIAGLAQILRQR
	YPEFDVPTEFGEMEVFDIISYSSKKLTDLGFEFKYSLEDMFDGAI
	QSCREKGLLPPATKEPSYATEQLIATGQDNGH

Leucoanthocyanidin	MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYPTFLLVRP	12
reductase (LAR)	GPVSPSKAVIIKTFQDKGAKVIYGVINDKECMEKILKEYEIDVVI
Desmodium	SLVGGARLLDQLTLLEAIKSVKTIKRFLPSEFGHDVDRTDPVEPG
uncinatum	LTMYKEKRLVRRAVEEYGIPFTNICCNSIASWPYYDNCHPSQVPP
Accession:	PMDQFQIYGDGNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRP
Q84V83.1	SSNCYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENIIPESI
	VSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKFRSLDDCYED
	FVPMVHDKIHAGKSGEIKIKDGKPLVQTGTIEEINKDIKTLVETQ
	PNEEIKKDMKALVEAVPISAMG

Anthocyanin	MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFDEEKKD	13
dioxygenase (ANS)	EGPQVPTIDLRDIDSDDQQVRQRCRDELKKAAVDWGVMHLVNHGI
Carica papaya	PDHLIDRVKKAGQAFFELPVEVKEKYANDQASGNIQGYGSKLANN
Accession:	ASGQLEWEDYYFHLIFPEEKRDLAIWPNNPADYIEVTSEYARQLR
XP_021901846.1	RLVSKILGVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCPQ
	PELALGVEAHTDISALTFILHNMVPGLQLFYEGKWVTAKCVPNSI
	VMHVGDTIEILSNGKYKSILHRGLVNKEKVRISWAVFCEPPKEKI
	ILKPLPETVSENEPPLFPPRTFAQHIQHKLFRKNQENLEAK

Anthocy anidin-3-	MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPHAVFSFFST	14
O-glycotransferase	SESNASIFHDSMHTMQCNIKSYDVSDGVPEGYVFTGRPQEGIDLF
(3GT)	MRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGVA
Vitis labrusca	WLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS
Accession:	KVRFRDLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFEEL
ABR24135	DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKP
	TSVVYISFGTVTTPPPAELVALAEALEASRVPFIWSLRDKARMHL
	PEGFLEKTRGHGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESV
	AGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSC
	FDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTLVDLV
	SKPKDV

Acetyl-CoA	MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHTVISKVLIAN	15
carboxylase (ACC)	NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIR
Mucor	MADNFVEVPGGSNNNNYANVELIVDVAERTAVHAVWAGWGHASEN
circinelloides	PRLPEMLAKSKHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTM
1006PhL	GWSGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKAAEKIGF
Accession:	PVMIKASEGGGGKGIRMVKDGSNFAQLFAQVQGEIPGSPIFIMKL
EPB82652.1	AGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIA
	KPDVFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDDQFYFLELNP
	RLQVEHPTTEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPN
	SASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAGFKPSS
	GIMQELNFRSSTNVWGYFSVVSAGGLHEYADSQFGHIFAYGENRQ
	QARKNMVIALKELSIRADFRSTVEYIIRLLETPDFEENTINTGWL
	DMLISKKLTAERPDTMLAVFCGAVTKAHMASLDCFQQYKQSLEKG
	QVPSKGSLKTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQ
	VGIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGKTCLLEKE
	SDPTQLRSPSPGKLVNLLVENGDHLNAGDAYAEIEVMKMYMPLIA
	TEDGHVQFIKQAGATLEAGDIIGILSLDDPSRVKHALPFNGTVPA
	FGAPHITGDKPVQRFNATKLTLQHILQGYDNQALVQTVVKDFADI
	LNNPDLPYSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP
	AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFARYRNGLTE
	HAYSNYVELMEAYYDVEILFNQQREEEVILSLRDQHKDDLDKVLA
	VTLSHAKVNIKNNVILMLLDLINPVSTGSALDKYFTPILKRLSEI
	ESRATQKVTLKARELLILCQLPSYEERQAQMYQILKNSVTESVYG
	GGSEYRTPSYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEV
	YCRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANGIDSNKRIA
	SYSDLTFLLNKTEEEPIRTGAMTACNSLADLQAELPRILTAFEEE
	PLPPMLQRNAAPKEERMENILNIAVRADEDMDDTAFRTKICEMIT
	ANADVFRQAHLRRLSVVVCRDNQWPDYYTFRERENYQEDETIRHI
	EPAMAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDCRFF
	IRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHEYKNS
	DCNHLFINFIPTFAIEADDVEHALKDFVDRHGKRLWKLRVTGAEI
	RFNVQSKKPDAPIIPMRFTVDNVSGFILKVEVYQEVKTEKSGWIL
	KSVNKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPE
	LFRQSVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVLQEIDR
	APGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDITFKIGSFGVAE
	DQVFYKASELARALGIPRIYLSANSGARIGLADELISQFRAAWKD
	ASNPTAGFKYLYLTPAEYDVLAQQGDAKSVLVEEIQDEGETRLRI
	TDVIGHTDGLGVENLKGSGLIAGATSRAYDDIFTITLVTCRSVGI
	GAYLVRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQLGGT
	QIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVRNAPVSMRLGAD
	PIDRDIEYTPPKGPSDPRFFLAGKSENGKWLSGFFDQDSFVETLS
	GWARTVVVGRARLGGIPMGVVSVETRTVENIVPADPANSDSTEQV
	FMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQR
	DMYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGAWVVVDPT
	INKDMMEMYADNNARGGVLEPEGIVEIKYRKPALLATMERLDATY
	ASLKKQLAEEGKTDEEKAALKVQVEAREQELLPVYQQISIQFADL
	HDRAGRMKAKGVIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVT
	ATSAAPMPREQMLDLVKQWFTNDNETVNFEDADELVSEWFEKRAS
	VIDQRISKLKSDATKEQIVSLGNADQEAVIEGFSQLIENLSEDAR
	AEILRKLNSRF

Acetyl-CoA	MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTFWGEQGKI	16
synthase (ACS)	LDWITPYQKVKNTSFAPGNVSIKWYEDGTLNLAANCLDRHLQENG
Salmonella	DRTAIIWEGDDTSQSKHISYRELHRDVCRFANTLLDLGIKKGDVV
typhimurium	AIYMPMVPEAAVAMLACARIGAVHSVIFGGFSPEAVAGRIIDSSS
Accession:	RLVITADEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRTGS
NP_463140.1	DIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPLFILYTSGSTGK
	PKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSY
	LLYGPLACGATTLMFEGVPNWPTPARMCQVVDKHQVNILYTAPTA
	IRALMAEGDKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE
	KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFFGVQPALVD
	NEGHPQEGATEGNLVITDSWPGQARTLFGDHERFEQTYFSTFKNM
	YFSGDGARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHP
	KIAEAAVVGIPHAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRK
	EIGPLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTS
	TLADPGVVEKLLEEKQAIAMPS

Malonyl-CoA	MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAAAGATAGRI	17
synthase (matB)	GGAGRVAVWATPAMETGVAVVAALLAGVAAVPLNPKSGDKELAHI
Streptomyces	LSDSAPSLVLAPPDAELPPALGALERVDVDVRARGAVPEDGADDG
coelicolor	DPALVVYTSGTTGPPKGAVIPRRALATTLDALADAWQWTGEDVLV
Accession:	QGLPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAARELNDGAT
WP_011028356	MLFGVPTMYHRIAETLPADPELAKALAGARLLVSGSAALPVHDHE
	RIAAATGRRVIERYGMTETLMNTSVRADGEPRAGTVGVPLPGVEL
	RLVEEDGTPIAALDGESVGEIQVRGPNLFTEYLNRPDATAAAFTE
	DGFFRTGDMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENAL
	LEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPPALGTLADHV
	AARLAPHKRPRVVRYLDAVPRNDMGKIMKRALNRD

Malonate	MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA	18
transporter (matC)	DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR
Streptomyces	VALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL
coelicolor	LMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS
Accession:	LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG
NP_626686.1	SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTP
	ARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRA
	VGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAA
	VLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAAL
	AVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA
	VPALAWLVLVVPGFG

Malonate CoA-	MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARAIELLETVIAS	19
transferase (MdcA)	GDRVCLEGNNQKQADFLSKCLSQCNPDAVNDLHIVQSVLALPSHI
Acinetobacter	DVFEKGIASKVDFSFAGPQSLRLAQLVQQQKISIGSIHTYLELYG
calcoaceticus	RYFIDLTPNICLITAHAADREGNLYTGPNTEDTPAIVEATAFKSG
Accession:	IVIAQVNEIVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQ
AAB97627.1	ITEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIELLLPTYAA
	SLGLKGQICTNWALNPHPTLIPAIESGFVDSVHSFGSEVGMEDYI
	KERPDVFFTGSDGSMRSNRAFSQTAGLYACDSFIGSTLQIELQGN
	SSTATVDRISGFGGAPNMGSDPHGRRHASYAYTKAGREATDGKLI
	KGRKLVVQTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMIYG
	EDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTPVGLKRDAAK
	VEELRQRGIIQRPEDLGIDPTQVSRDLLAAKSVKDLVKWSGGLYS
	PPSRFRNW

Pantothenate kinase	MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVEQLTSQQAL	20
(CoaX)	PVRACRLVSVRSEQETSQLVARLEQLFPVSALVASSGKQLAGVRN
Pseudomonas	GYLDYQRLGLDRWLALVAAHHLAKKACLVIDLGTAVTSDLVAADG
aeruginosa	VHLGGYICPGMTLMRSQLRTHTRRIRYDDAEARRALASLQPGQAT
Accession:	AEAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGGDAELVRD
Q9HWCL1	ELAGARIMPDLVFVGLALACPIE

glutamyl-tRNA	MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSLLAQPMVQG	21
reductase (hemAm)	GVVLSTCNRTELYLSVEEQDNLQEALIRWLCDYHNLNEDDLRNSL
Salmonella	YWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHL
typhimurium	NASALRRMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFE
Accession:	SLSTVTVLLVGAGETIELVARHLREHKVQKMIIANRTRERAQALA
AAA88610.1	DEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKS
	RRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQSIISHNLAQ
	RQAAAVEAETIVEQEASEFMAWLRAQGASETIREYRSQSEQIRDE
	LTTKALSALQQGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDG
	DDERLNILRDSLGLE

5-aminolevulinic	MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGK	22
acid synthase	QDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNISGTT
(ALAS)	AYHRRLEAEIADLHGKEAALVFSSAYIANDATLSTLRLLFPGLII
Rhodobacter	YSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPK
capsulatus	LIAFESVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMYGPR
Accession:	GAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASAKMVDAVRSY
CAA37857	APGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLK
	MRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQP
	INFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCA

Tyrosine ammonia-	MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCAR	23
lyase (TAL)	AHARLEHAIAEQRHIYGITTGFGPLANRLIGADQGAELQQNLIYH
Rhodobacter	LATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAVLN
capsulatus SB 1003	AGFAPEVPAQGTVGASGDLTPLAHMVLALQGRGRMIDPSGRVQEA
Accession:	GAVMDRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEAARAID
ADE84832.1	AALRHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALD
	GAGRVCRTLTAARRLTAADLRPEDHPAQDAYSLRVVPQLVGAVWD
	TLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALAS
	DALNAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQAGLQSGF
	MGAQVTATALLAEMRANATPVSVQSLSTNGANQDVVSMGTIAARR
	ARAQLLPLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDR
	IRAVSPGLRADRPLAGHIEAVAQGLRHPSAAADPPA

Tyrosine ammonia-	MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQATKTVVIDG	24
lyase (TAL)	HNMKVGDVVAVARHGAKVELAASVAGPVQASVDFKESKKHTSIYG
Trichosporon	VTTGFGGSADTRTSDTEALQISLLEHQLCGYLPTDPTYEGMLLAA
cutaneum	MPIPIVRGAMAVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVP
Accession:	LRGTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLTAPEA
XP_018276715	IAKYKLKTVRLASKEGLGLVNGTAVSAAAGALALYDAECLAMMSQ
	TNTALTVEALDGHVGSFAPFIQEIRPHVGQIEAAKNIRHMLSNSK
	LAVHEEPELLADQDAGILRQDRYALRTSAQWIGPQLEMLGLARQQ
	IETELNSTTDNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQN
	LGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCKGLDIHCGA
	YCAELGFLANPMSNHVQSTEMHNQSVNSMAFASARKTMEANEVLS
	LLLGSQMYCATQALDLRVMEVKFKMAIVKLLNDTLTKHFSTFLTP
	EQLAKLNTTAAITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDAL
	MVNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADGRNDLEPA
	EYLGQTRAVYEAIRSDLGVKVRRGDVAEGKSGKSIGSNVARIVEA
	MRDGRLMGAVSKMFF

Tyrosine ammonia-	MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSG	25
lyase (TAL)	NKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSP
Flavobacterium	VCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHG
johnsoniae	GVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPI
Accession:	QVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINEL
WP_012023194	VQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHL
	YSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEF
	NSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLA
	ERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAES
	QMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAI
	EMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPF
	VQKVKDHLINN

Tyrosine ammonia-	MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILAQIEASCAY	26
lyase (TAL)	INQAVKEHQPVYGVTTGFGGMANVIISPEEAAELQNNAIWYHKTG
Herpetosiphon	AGKLLPFTDVRAAMLLRANSHMRGASGIRLEIIQRMVTFLNANVT
aurantiacus	PHVREFGSIGASGDLVPLISITGALLGTDQAFMVDFNGETLDCIS
DSM 785	ALERLGLPRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLALA
Accession:	LEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWAADHMLELLQGS
ABX04526.1	QLSRNELDGSHDYRDGDLIQDRYSLRCLPQFLGPIIDGMAFISHH
	LRVEINSANDNPLIDTASAASYHGGNFLGQYIGVGMDQLRYYMGL
	MAKHLDVQIALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTAN
	SIMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLARQTIQT
	LQQYIAITLMFGVQAVDLRTHKLAGHYNAAELLSPLTAKIYHAVR
	SIVKHPPSPERPYIWNDDEQVLEAHISALAHDIANDGSLVSAVEQ
	TLSGLRSIILFR

Phenylalanine	MHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAMECSHLEEIKRM	27
ammonia-lyase	VDTYQNATQVMIEGATLTVPQVAAIARRPEVHVVLDAANARSRVD
(PAL)	ESSNWVLDRIMGGGDIYGVTTGFGATSHRRTQQGVELQRELIRFL
Physcomitrella	NAGVLSKGNSLPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKL
patens	LNAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVTEDGR
Accession:	EVSALEALRIAGVEKPFELAPKEGLALVNGTAVGSALASTVCYDA
XP_001758374.1	NIMVLLAEVLSALFCEVMQGKPEFADPLTHKLKHHPGQMEAAAVM
	EWVLDGSSFMKAAAKFNETDPLRKPKQDRYALRTSPQWLGPQVEV
	IRNATHAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSMDN
	MRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSGGPNPSLDYGMK
	GAEIAMASYLSEINYLANPVTTHVQSAEQHNQDVNSLGLVSARKT
	EEAMEILKLMSATFLVGLCQAIDLRHVEETMQSAVKQVVTQVAKK
	TLFMGSDGSLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLME
	KLRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSRVVPAVR
	EAYDTKGLSLVPNRIQDCRTYPLYKLVRGDLKTQLLSGQRTVSPG
	QEIEKVFNAISAGQLVAPLLECVQGWTGTPGPFSARASC

Phenylalanine	MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNELLEFLQ	28
ammonia-lyase	KGRDQLENKLKENVAIYGINTGFGGNGDLIIPFDKLDYHQSNLLD
(PAL)	FLTCGTGDFFNDQYVRGIQFIIIIALSRGWSGVRPMVIQTLAKHL
Dictyostelium	NKGIIPQVPMHGSVGASGDLVPLSYIANVLCGKGMVKYNEKLMNA
discoideum AX4	SDALKITSIEPLVLKSKEGLALVNGTRVMSSVSCISINKFETIFK
Accession:	AAIGSIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNKYFN
XP_644510.1	TSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRCAPQILGIISEN
	ISNAKIVIKREILSVNDNPLIDPYYGDVLSGGNFMGNHIARIMDG
	IKLDISLVANHLHSLVALMMHSEFSKGLPNSLSPNPGIYQGYKGM
	QISQTSLVVWLRQEAAPACIHSLTTEQFNQDIVSLGLHSANGAAS
	MLIKLCDIVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIK
	IIPILENDRRTDIDVREITNAILQDKLDFFNLNL

Phenylalanine	MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH	29
ammonia-lyase	ISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK
(PAL)	AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL
Brevibacillus	EVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK
laterosporus	VLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL
LMG 15441	AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA
Accession:	SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS
WP_003337219.1	IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNG
	GNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPN
	LIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRS
	TEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRD
	SKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK
	KEIHDQNVND

Cinnamate-4-	MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGPIPVPVFGN	30
hydroxylase (C4H)	WLQVGDDLNHRNLTEMAKKFGEVFMLRMGQRNLVWSSPDLAKEVL
Rubus sp. SSL-2007	HTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVP
Accession:	FFTNKVVQQYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQLM
ABX74781.1	MYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSRLAQSFEYNYGD
	FIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLSSTQATT
	NEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSIE
	WGIAELVNHPEIQKKLRDELDTVLGRGVQITEPEIQKLPYLQAVV
	KETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANN
	PAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGII
	LALPILGITLGRLVQNFELLPPPGQTQLDTTEKGGQFSLHILKHS
	PIVMKPRT

Cinnamate-4-	MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPVPVFGN	31
hydroxylase (C4H)	WLQVGDDLNHRNLTDMAKKFGDVFMLRMGQRNLVVVSSPDLAKEV
Fragaria vesca	LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV
Accession:	PFFTNKVVQQYRHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQL
XP_004294725.1	MMYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLAQSFEYNYG
	DFIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLASTQVT
	TNEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSI
	EWGIAELVNHPEIQKKLRDELDTVLGHGVQVTEPELHKLPYLQAV
	VKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLAN
	NPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGI
	ILALPILGVTLGRLVQNFEMLPPPGQTQLDTTEKGGQFSLHILKH
	STIVMKPRA

Cinnamate-4-	MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVPVFGN	32
hydroxylase (C4H)	WLQVGDDLNHRNLTEYAKKFGDVFLLRMGQRNLVVVSSPELAKEV
Solanum tuberosum	LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV
Accession:	PFFTNKVVQQYRGGWESEAASVVEDVKKNPESATNGIVLRKRLQL
ABC69046.1	MMYNNMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFEYNYG
	DFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVDERKKLANTKSM
	DSNALKCAIDHILEAQQKGEINEDNVLYIVENFNVAAIETTLWSI
	EWGIAELVNHPHIQKKLRDEIDTVLGPGMQVTEPDMPKLPYLQAV
	IKETLRLRMAIPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLAN
	NPAHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGRRSCPGI
	ILALPILGITLGRLVQNFEMLPPPGQSKLDTSEKGGQFSLHILKH
	STIVMKPRSF

4-coumarate-CoA	MGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKVSDRACL		33
ligase (4CL)	INGATGETFSYAQVELISRRVASGLNKLGIHQGDTMMILLPNTPE
Daucus carota	YFFAFLGASYRGAVSTMANPFFTSPEVIKQLKASQAKLIITQACY
Accession:	VEKVKEYAAENNITVVCIDEAPRDCLHFTTLMEADEAEMPEVAID
AIT52344.1	SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIH
	SEDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIVPFLEL
	IQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVRTVMSGAAPLGK
	ELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGA
	CGTVVRNAEMKIVDPETHASLPRNQSGEICIRGDQIMKGYLNDPE
	STKTTIDEEGWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAP
	AEIEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGSTTTEE
	EIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKILRKELRARIASG
	DLPK

4-coumarate-CoA	MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGSRPCLI		34
ligase (4CL)	NGSTGEILTYDQVELASRRVGSGLHRLGIRQGDTIMLLLPNSPEF
Striga asiatica	VLAFLGASHIGAVSTMANPFFTPAEVVKQAAASRAKLIVTQACHV
Accession:	DKVRDYAAEHGVKVVCVDGAPPEECLPFSEVASGDEAELPAVKIS
GER48539.1	PDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIH
	SDDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIVPFLEL
	IQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRTVMSGAAPLGR
	ELEDAVRLKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKSGA
	CGTVVRNAEMKIVDTETGASLGRNQPGEICIRGDQIMKGYLNDPE
	STERTIDKEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAP
	AELEALLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTITED
	EIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKDLRARLAAG
	VPN

4-coumarate-CoA	MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISEFSSRPC		35
ligase (4CL)	LINGANNQIYTYADVELNSRKVAAGLHKQFGIQQKDTIMILLPNS
Capsicum annuum	PEFVFAFLGASYLGAISTMANPLFTPAEVVKQVKASNAEIIVTQA
Accession:	CHVNKVKDYALENDVKIVCIDSAPEGCVHFSELIQADEHDIPEVQ
KAF3620179.1	IKPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLY
	IHSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFDIVPFL
	ELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLSSVRTVMSGAAPL
	GKELEDTVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKS
	GACGTVVRNAEMKIVDPDTGNSLHRNQSGEICIRGDQIMKGYLND
	PEATAGTIDKEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQV
	APAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVRSNGSTIT
	EDEVKEFISKQVIFYKRIKRVFFVDAVPKSPSGKILRKDLRAKLA
	AGFPN

4-coumarate-CoA	MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSSKPCLI		36
ligase (4CL)	NGSTGKVYTYSDVELTSRKVAAGFHNLGIQQRDTIMLLLPNCPEF
Camellia sinensis	VFAFLGASYLGAIITMANPFFTPAETIKQAKASNSKLIITQSSYT
Accession:	SKVLDYSSENNVKIICIDSPPDGCLHFSELIQSNETQLPEVEIDS
ASU87409.1	NEVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHS
	EDMMMCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKLI
	QRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMSGAAPLGKE
	LEDAVRAKFPHAKLGQGYGMTEAGPVLAMCLAFAKKPFEIKSGAC
	GTVVRNAEMKIVDPDAGFSLPRNQPGEICIRGDQIMKGYLNDPEA
	TERTIDKQGWLHTGDIGYIDDDDELFIVDRLKELIKYKGFQVAPA
	ELEALLLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVTENE
	IKKYISEQVVFYKKINRVYFVDAIPKAPSGKILRKDLRARLAAGI
	PS

Chaicone synthase	MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTYPDYYFRITN	37
(CHS)	SEHKTELKEKFKRMCEKSMIKTRYMHLTEEILKENPNMCAYMAPS
Capsicum annuum	LDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV
Accession:	DMPGCDYQLAKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
XP_016566084.1	NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAIIMG
	SDPIPGVERPLFQLVSAAQTLLPDSEGAIDGHLREVGLTFHLLKD
	VPGLISKNIEKSLVEAFQPLGISDWNSLFWIAHPGGPAILDQVEL
	KLGLKPEKLKATREVLSNYGNMSSACVLFILDEMRKASTKEGLGT
	SGEGLEWGVLFGFGPGLTVETVVLHSVAI

Chaicone synthase	MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYPDYYFRITK	38
(CHS)	SEHKAELKEKFQRMCDKSMIKKRYMYLTEEILKENPSMCEYMAPS
Rosa chinensis	LDARQDMVVVEIPKLGKEAATKAIKEWGQPKSKITHLVFCTTSGV
Accession:	DMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
AEC13058.1	NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVG
	SDPLPEVEKPLFELVSAAQTILPDSDGAIDGHLREVGLTFHLLKD
	VPGLISKNIEKSLNEAFKPLNITDWNSLFWIAHPGGPAILDQVEA
	KLGLKPEKLEATRHILSEYGNMSSACVLFILDEVRRKSAANGHKT
	TGEGLEWGVLFGFGPGLTVETVVLHSVAA

Chaicone synthase	MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQADYPDYYFR	39
(CHS)	ITNSDHMTDLKDKFKRMCEKSMITKRHMYLTEEILKENPKMCEYM
Morus alba var.	APSLDARQDIVVVEVPKLGKEAAAKAIKEWGQPKSKITHLIFCTT
multicaulis	SGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDL
Accession:	AENNKGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDGAAAV
AHL83549.1	ILGADPDTSVERPIFELVSAAQTILPDSEGAIDGHLREVGLTFHL
	LKDVPGLISKNIEKSLVEAFTPIGISDWNSIFWIAHPGGPAILDQ
	VEAKLGLKQEKLSATRHVLSEYGNMSSACVLFILDEVRKKSVEEG
	KATTGEGLEWGVLFGFGPGLTVETIVLHSLPAV

Chaicone synthase	MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQADYPDYYFRI	40
(CHS)	TKSEHLTELKEKFKQMCDKSMIRKRYMYLTEEILKENPNICAFMA
Dendrobium	PSLDARQDIVVTEVPKLAREASARAIKEWGQPKSRITHLIFCTTS
catenatum	GVDMPGADYQLTRLLGLRPSVNRIMLYQQGCFAGGTVLRLAKDLA
Accession:	ENNAGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAII
ALE71934.1	VGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLREMGLTFHLL
	KDVPGLISKNIQKSLVETFKPLGIHDWNSIFWIAHPGGPAILDQV
	EIKLGLKEEKLASSRNVLAEYGNMSSACVLFILDEMRRRSAEAGQ
	ATTGEGLEWGVLFGFGPGLTVETVVLRSVPIAGAV

Chaicone isomerase	MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTIEGNFIK	41
(CHI)	FTAIGVYLEDVAVASLATKWKGKSSEELLETLDFYRDIISGPFEK
Trifolium pratense	LIRGSKIRELSGPEYSRKVTENCVAHLKSVGTYGDAEVEAMEKFV
Accession:	EAFKPINFPPGASVFYRQSPDGILGVSISIHFFP
PNX83855.1

Chaicone isomerase	MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGVRGLTIEGNF	42
(CHI)	IKFTGIGVYLEDQAVASLATKWKGKSSEELVESLDFFRDIISGPF
Abrus precatorius	EKLIRGSKIRQLSGPEYSKKVMENCVAHMKSVGTYGDAEAAGIEE
Accession:	FAQAFKPVNFPPGASVFYRQSPDGVLGLSFSQDATIPEEEAAVIK
XP_027366189.1	NKPVSAAVLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN

Chaicone isomerase	MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGERGLTIDGK	43
(CHI)	FIKFTGIGVYLEDKAVPSLAGKWKDKSSQQLLQTLHFYRDIISGP
Arachis duranensis	FEKLIRGSKILALSGVEYSRKVMENCVAHMKSVGTYGDAEAEAIQ
Accession:	QFAEAFKNVNFKPGASVFYRQSPLGHLGLSFSQDGNIPEKEAAVI
XP_015942246.1	ENKPLSSAVLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQ
	HN
Chaicone isomerase	MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGERGLEIQGK	44
(CHI)	FIKFTAIGVYLEGDAVASLAVKWKGKSKEELTDSVEFFRDIVTGP
Cephalotus	FEKFTQVTTILPLTGQQYSEKVSENCVAFWKSVGIYTDAEAKAIE
follicularis	KFIEVFKEETFPPGSSILFTQSPNGALTIAFSKDGVIPEVGKAVI
Accession:	ENKLLAEGLLESIIGKHGVSPVAKQCLATRLSELL
GAV77263.1

Flavanone 3-	MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV	45
hydroxylase (F3H)	KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF
Abrus precatorius	ESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT
Accession:	EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE
XP_027329642.1	NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQ
	GQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQK
	YPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA
	VETCKEKRLLSETAEISGTTQK

Flavanone 3-	MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLERGYIVRATVR	46
hydroxylase (F3H)	DPANLKKVKHLLDLPKADTNLTLWKADLNEEGSFDEAIEGCSGVF
Camellia sinensis	HVATPMDFESKDPENEVIKPTINGVLSIIRSCTKAKTVKRLVFTS
Accession:	SAGTVNVQEHQQPVFDENNWSDLHFINKKKMTGWMYFVSKTLAEK
AAT66505.1	AAWEAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRNE
	GHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSSHDATIHD
	LAKLMREKWPEYNVPTEFKGIDKDLPVVSFSSKKLIGMGFEFKYS
	LEDMFRGAIDTCREKGLLPHSFAENPVNGNKV

Flavanone 3-	MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYIVRATVRDPA	47
hydroxylase (F3H)	NMKKVKHLQELEKADKNLTLWKADLTEEGSFDEAIKGCSGVFHVA
Nyssa sinensis	TPMDFESKDPENEVIKPTINGVLSIVRSCVKAKTVKRLVFTSSAG
Accession:	TVNLQEHQQLVYDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAW
KAA8531902.1	EATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEAHY
	SIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSHDATIYDLAK
	MIREKWPEYNVPTELKGIEKDLQTVSFSSKKLIGMGFEFKYSLED
	MYKGAIDTCREKGLLPYSTHETPANANANANANVKKNQNENTEI

Flavanone 3-	MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV	48
hydroxylase (F3H)	KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF
Rosa chinensis	ESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE
Accession:	ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE
XP_024167119.1	NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQ
	GQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREK
	YPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGA
	VDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG

Flavonoid 3′	MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIGNLPHM	49
hydroxylase (F3′H)	GQMPHHSLAAMARTYGPLMHLRLGFVDVIVAASASVASQLLKTHD
Cephalotus	ANFSSRPHNSGAKYIAYNYQDLVFAPYGPRWRMLRKISSVHLFSG
follicularis	KALDDYRHVRQEEVAVLIRALARAESKQAVNLGQLLNVCTANALG
Accession:	RVMLGRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIGDFIPA
GAV84063.1	LDWLDLQGVAAKMKNLHKRFDTFLTGLLEEHKKMLVGDGGSEKHK
	DLLSTLISLKDSADDEGLKLTDTEIKALLLNMFTAGTDTSSSTVE
	WAIAELIRHPKILAQVLKELDTVVGRDRLVTDLDLPQLTYLQAVI
	KETFRLHPSTPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARD
	PKEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAGRRICAG
	MSLGLRMVQLLTATLVHAFDWDLTSGLMPEDLSMEEAYGLTLQRA
	EPLMVHPRPRLSPNVY

Flavonoid 3′	MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPIIGNLPH	50
hydroxylase (F3′H)	MGPVPHHSLAALAKVYGPLMHLRLGFVDVVVAASASVAAQFLKVH
Theobroma cacao	DANFSSRPPNSGAKYVAYNYQDLVFAPYGPRWRMLRKISSVHLFS
Accession:	GKALDDFRHVRQDEVGVLVRALADAKTKVNLGQLLNVCTVNALGR
EOY22049.1	VMLGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNIGDFIPAL
	EWLDLQGVQAKMKKLHKRFDRFLSAILEEHKIKARDGSGQHKDLL
	STFISLEDADGEGGKLTDTEIKALLLNMFTAGTDTSSSTVEWAIA
	ELIRHPKILAQVRKELDSVVGRDRLVSDLDLPNLTYFQAVIKETF
	RLHPSTPLSLPRMASESCEINGYHIPKGATLLVNVWAIARDPDEW
	KDPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRICAGMSLG
	LRMVQLLAATLVHAFDWELADGLMPEKLNMEEAFGLTLQRAAPLM
	VHPRPRLSPRAY

Flavonoid 3′	MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPWPVVGNLP	51
hydroxylase (F3′H)	HLGTIPHHSLAAMAKKYGPLMHLRLGFVDVVVAASASVAAQFLKT
Gerbera hybrida	HDANFADRPPNSGAKHIAYNYQDLVFAPYGPRWRMLRKICSVHLF
Accession:	STKALDDFRHVRQEEVAILARALVGAGKSPVKLGQLLNVCTTNAL
ABA64468.1	ARVMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIGDFIPVLDW
	LDLQGVTKKMKKLHAKFDSFLNTILEEHKTGAGDGVASGKVDLLS
	TLISLKDDADGEGGKLSDIEIKALLLNLFTAGTDTSSSTIEWAIA
	ELIRNPQLLNQARKEMDTIVGQDRLVTESDLGQLTFLQAIIKETF
	RLHPSTPLSLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW
	ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRICVGMSLG
	LRMVQLLTATLIHAFDWELADGLNPKKLNMEEAYGLTLQRAAPLV
	VHPRPRLAPHVYETTKV

Flavonoid 3′	MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGPRGWPVLG	52
hydroxylase (F3′H)	NLPQLGPKPHHTLHALSRAHGPLFRLRLGSVDVVVAASAAVAAQF
Phoenix dactylifera	LRAHDANFSNRPPNSGAEHIAYNYQDLVFAPYGPGWRARRKLLNV
Accession:	HLFSGKALEDLRPVREGELALLVRALRDRAGANELVDLGRAANKC
XP_008791304.2	ATNALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAGVFNVGDF
	VPGIGWLDLQGVVRRMKELHRRYDGFLDGLIAAHRRAAEGGGGGG
	KDLLSVLLGLKDEDLDFDGEGAKLTDTDIKALLLNLFTAGTDTTS
	STVEWALSELVKHPDILRKAQLELDSVVGGDRLVSESDLPNLPFM
	QAIIKETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNVWA
	IARDPAVWRDPLEFRPARFLPDGGCEGMDVKGNDFGIIPFGAGRR
	ICAGMSLGIRMVQFMTATLAHAFHWDLPEGQMPEKLDMEEAYGLT
	LQRATPLMVHPVPRLAPTAYQS

Cytochrome P450	MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF	53
reductase (CPR)	LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFY
Camellia sinensis	GTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQK
Accession:	LKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY
XP_028084858	GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIE
	DDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDP
	LSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKT
	MASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF
	LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGT
	QTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
	KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGV
	FSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD
	FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTT
	TSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHL
	EFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSV
	HADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA
	LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFP
	SAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGP
	SPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDP
	SIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR
	RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAA
	QVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE
	VIVKKLQMEGRYLRDVW

Cytochrome P450	MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF	54
reductase (CPR)	LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFY
Cephalotus	GTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQK
follicularis	LKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY
Accession:	GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIE
GAV59576.1	DDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDP
	LSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKT
	MASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF
	LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGT
	QTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
	KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGV
	FSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD
	FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTT
	TSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHL
	EFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSV
	HADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA
	LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFP
	SAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGP
	SPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDP
	SIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR
	RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAA
	QVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE
	VIVKKLQMEGRYLRDVW

Cytochrome P450	MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAELSSMLIE	55
reductase (CPR)	NRQFAMIISTSIAVLIGCIVMLLWRRSGGSGSSKRAETLKPLVLK
Brassica napus	PPREDEVDDGRKKVTIFFGTQTGTAEGFAKALGEEARARYEKTRF
Accession:	KIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFY
XP_013706600.1	KWFTEGDDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQ
	GAQRLVHVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVT
	PYTAAVLEYRVSIHNSADALNEKNLANGNGHAVFDAQHPYRANVA
	VRRELHTPESDRSCTHLEFDIAGSGLTYETGDHVGVLSDNLNETV
	EEALRLLDMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALT
	RYACLLSSPKKSALLALAAHASDPTEAERLKHLASPAGKDEYSKW
	VVESQRSLLEVMAEFPSAKPPLGVFFAAVAPRLQPRFYSISSSPK
	IAETRIHVTCALVYEKMPTGRIHKGVCSTWMKSAVPYEKSENCCS
	APIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVES
	GVELGPSVLFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREF
	GPTKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKGMARDVHRSL
	HTIAQEQGSMDSTKAESFVKNLQMSGRYLRDVW

Flavonoid 3′, 5′-	MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPGPKGWPF	56
hydroxylase	VGALPLLGTMPHVALAQMAKKYGPVMYLKMGTCGMVVASTPDAAR
(F3′5′H)	AFLKTLDLNFSNRPPNAGATHLAYNAQDMVFADYGPRWKLLRKLS
Cephalotus	NLHMLGGKALEDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLT
follicularis	YAMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTSAGYFNIGD
Accession:	FIPSIAWMDLQGIERGMKKLHKRFDALLTKMFEEHMATAHERKGN
GAV62131	PDLLDIVMANRDNSEGERLTTTNIKALLLNLFSAGTDTSSSIIEW
	SLAEMLKNPSILKRAHEEMDQVIGRNRRLEESDIKKLPYLQAICK
	ESFRKHPSTPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP
	EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGRRICAGTR
	MGIVLVEYILGTLVHSFDWSLPHGVKLNMDEAFGLALQKAVPLAA
	IVSPRLAPTAYVV

Flavonoid 3′, 5′-	MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGALPFI	57
hydroxylase	GPMPHSGLALLARRYGPIMFLKMGIRRVVVASSSTAARTFLKTFD
(F3′5′H)	SHFSDRPSGVISKEISYNGQNMVFADYGPKWKLLRKVSSLHLLGS
Dendrobium	KAMSRWAGVRRDEALSMIQFLKKHSDSEKPVLLPNLLVCAMANVI
moniliforme	GRIAMSKRVFHEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGW
Accession:	LDPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQGNPDLLDLV
AEB96145	VASEVKGEDGEGLCEDNIKGFISDLFVAGTDTSAIVIEWAMAEML
	KNPSILRRAQEETDRVIGRHRLLDESDIPNLPYLQAICKEALRKH
	PPTPLSIPHYASEPCEVEGYHIPGETWLLVNIWAIGRDPDVWENP
	LVFDPERFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMVMV
	QYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPKAVPLAVMATPR
	LPAAAYGLL

Dihydroflavonol 4-	MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVRDPDNEKKV	58
reductase (DFR)	KHLVELPKAKTHLTLWKADLSDEGSFDEAIHGCTGVFHVATPMDF
Acer palmatum	ESKDPENEVIKPTINGVLGIMKACKKAKTVKRLVFTSSAGTVDVE
Accession:	EHKKPVYDENSWSDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEE
AWN08247.1	NSIDFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAIIKQ
	GNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATILELSKFLRER
	YPEYDLPTEYKGVDDSLENVVFCSKKILDLGFQFKYSLEDMFTGA
	VETCREKGLIPLTNIDKKHVAAKGLIPNNSDEIHVAAAEKTTATA

Dihydroflavonol 4-	MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV	59
reductase (DFR)	KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF
Abrus precatorius	ESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT
Accession:	EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE
XP_027329642.1	NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQ
	GQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQK
	YPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA
	VETCKEKRLLSETAEISGTTQK

Dihydroflavonol 4-	MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRATVRDPTNLKK	60
reductase (DFR)	VKPLLDLPRSNELLSIWKADLDGIEGSFDEVIRGSIGVFHVATPM
Dendrobium	NFQSKDPENEVIQPAINGLLGILRSCKNAGSVQRVIFTSSAGTVN
moniliforme	VEEHQAAAYDETCWSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFV
Accession:	KDNHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHYSIL
AEB96144.1	KQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDSTIYGLAEMLK
	NRYPTYAIPHKFKEIDPDIKCVSFSSKKLMELGFKYKYTMEEMFD
	DAIKTCREKKLIPLNTEEIVLAAEKFEEVKEQIAVK

Dihydroflavonol 4-	MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV	61
reductase (DFR)	KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF
Rosa chinensis	ESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE
Accession:	ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE
XP_024167119.1	NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQ
	GQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREK
	YPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGA
	VDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG

Leucoanthocyanidin	MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTTFLLVRS	62
reductase (LAR)	LDKGAIPSKSKTINSLHDKGAILIHGVIEDQEFVEGILKDHKIDI
Camellia sinensis	VISAVGGANILNQLTIVKAIKAVGTIKRFLPSEFGHDVDRANPVE
Accession:	PGLAMYKEKRMVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV
XP_028127206.1	IPPLDRFQIYGDGTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHF
	RPSCNFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAENIIPQ
	SIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPDETFRTMDECF
	DDFVMKMDRWN

Leucoanthocyanidin	MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGKSTYILS	63
reductase (LAR)	RAAARCPSKARAIKALEDQGAISIHGSVNDQEFMEKTLKEHEIDI
Coffea arabica	VISAVGGGNLLEQVILIRAMKAVGTIKRFLPSEFGHDVDRAEPVE
Accession:	PGLTMYNEKRRVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV
XP_027097479.1	SPPLDQFQIYGDGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHF
	RPSCNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEENIIPQ
	SVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYPDEPFQTMDECF
	NEFAGKIEEDKKHVVGTKGKNIAHRLVDVLTAPKLCA

Leucoanthocyanidin	MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEASLDSGRPTY	64
reductase (LAR)	ILARSSSNSPSKASTIKFLQDRGATVIYGSITDKEFMEKVLKEHK
Theobroma cacao	IEVVISAVGGGSILDQFNLIEAIRNVDTVKRFLPSEFGHDTDRAD
Accession:	PVEPGLTMYEQKRQIRRQIEKSGIPYTYICCNSIAAWPYHDNTHP
ADD51357.1	ADVLPPLDRFKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKT
	VHFQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQMAKEMR
	IPQSVVAALTHDIFINGCQINFSLDKPTDVEVCSLYPDTPFRTIN
	ECFEDFAKKIIDNAKAVSKPAASNNAIFVPTAKPGALPITAICT

Leucoanthocyanidin	MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLPTYVLVR	65
reductase (LAR)	PGPSRPSKSDTIKSLKDRGAIILHGVMSDKPLMEKLLKEHEIEIV
Fragaria x	ISAVGGATILDQITLVEAITSVGTVKRFLPSEFGHDVDRADPVEP
ananassa	GLTMYLEKRKVRRAIEKSGVPYTYICCNSIASWPYYDNKHPSEVV
Accession:	PPLDQFQIYGDGTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFR
ABH07785.2	PSSNLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAENRIPES
	IVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEESFRTLDECFN
	DFLVKVGGKLETDKLAAKNKAAVGVEPMAITATCA

Anthocyanin	MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRLNEELTSM	66
dioxygenase (ANS)	GNVFEEEKKEEGSQVPTIDIKDIASEDPEVRGKAIQELKRAAMEW
Chenopodium	GVMHLVNHGISDELIDRVKVAGQTFFELPVEEKEKYANDQASGNV
quinoa	QGYGSKLANSASGRLEWEDYYFHLSYPEDKRDLSIWPETPADYIP
Accession:	AVSEYSKELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQF
XP_021735950.1	KINYYPKCPQPELALGVEAHTDVSALTFILHNMVPGLQLFYEGKW
	VTAKCVPNSIIMHIGDTIEILSNGKYKSILHRGLVNKEKVRISWA
	VFCEPPKEKIILKPLPDLVSDEEPARYPPRTFAQHVQYKLFRKTQ
	GPQTTITKN

Anthocyanin	MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDDSLGDALEE	67
dioxygenase (ANS)	IKKTEEGPQIPIVDLRGFDSGDEKERLHCMEEVKEAAVEWGVMHI
Iris sanguinea	VNHGIAPELIERVRAAGKGFFDLPVEAKERYANNQSEGKIQGYGS
Accession:	KLANNASGQLEWEDYFFHLIFPSDKVDLSIWPKEPADYTEVMMEF
QCI56004.1	AKQLRVVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKINYY
	PKCPQPELALGVEAHTDVSSLSFILHNGVPGLQVFHGGRWVNARL
	VPGSLVVHVGDTLEILSNGRYKSVLHRGLVNKEKVRISWAVFCEP
	PKEKIVLEPLAELVDKRSPAKYPPRTFAQHIQHKLFKKAQEQLAG
	GVHIPEAIQN

Anthocyanin	MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGDVFEEEKK	68
dioxygenase (ANS)	LEGPQIPVVDLMGLEWENEEVFKKVEEDMKKAASEWGVMHIFNHG
Magnolia sprengeri	ISMELMDRVRIAGKAFFDLPIEEKEMYANDQASGKIAGYGSKLAN
Accession:	NASGQLEWEDYFFHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQL
AHU88620.1	RGLVTKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYYPKCP
	QPDLALGVEAHTDVSALTFILHNMVPGLQVFFDDKWVTAKCIPGA
	LVVHIGDSLEILSNGKYRSILHRGLVNKEKVRISWAIFCEPPKEK
	VVLQPLPELVSEAEPARFTPRTFSQHVRQKLFKKQQDALENLKSE

Anthocyanin	MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIGNVFEEEK	69
dioxygenase (ANS)	KEGPEVPTIDLTEIESEDEVVRARCHETLKKAAQEWGVMNLVNHG
Prosopis alba	IPEELLNQLRKAGETFFSLPIEEKEKYANDQASGKIQGYGSKLAN
Accession:	NASGQLEWEDYFFHLVFPEDKCDLSIWPRTPSDYIEVTSEYARQL
XP_028787846.1	RGLATKILGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPICP
	QPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQWITAKCVPNS
	IFMHIGDTVEILSNGRYKSILHRGLVNKEKVRISWAVFCEPPKEK
	IILKPLPELVTDDEPARFPPRTFAQHIQHKLFRKCQEGLSK

Anthocy anidin-3-	MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPNTHFSFLN	70
O-glycotransferase	TSQSNNSIFSSDVYNRQPNLKAHNVWDGVPEGYVFVGKPQESIEL
(3GT)	FVKAAPETFRKGVEAAVAETGRKVSCLVTDAFFWFAAEIAGELGV
Cephalotus	PWVPFWTAGPCSLSTHVYTDLIRKTIGVGGIEGREDESLEFIPGM
follicularis	SQVVIRDLQEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEE
Accession:	LDLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLPWLDQQK
GAV66155.1	VASVAYISFGTVMAPSPPELVALAEALEASKIPFIWSLGEKLKVH
	LPKGFLDKTRTHGIVVPWAPQSDVLENGAVGVFITHCGWNSLLES
	IAGGVPMICRPFFGDQRLNGRMVQDVWEIGVTATGGPFTTEGVMG
	DLDLILSQARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALLNL
	VRLSI

Anthocy anidin-3-	MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRRLAAASPNT	71
O-glycotransferase	LFSFFSTSQSNNSLFSNTITNLPRNIKVFDVADGVPDGYVFAGKP
(3GT)	QEDIELFMKAAPHNFTTSLDTCVAHTGKRLTCLITDAFLWFGAHL
Prunus cerasifera	AHDLGVPWLPLWLSGLNSLSLHVHTDLLRHTIGTQSIAGRENELI
Accession:	TKNVNIPGMSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN
AKV89253.1	AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAASPPLPEAP
	TAADDVTGCLSWLDKQKAASSVVYVSFGSVARPPEKELLAMAQAL
	EASGVPFLWSLKDSFKTPLLNELLIKASNGMVVPWAPQPRVLAHA
	SVGAFVTHCGWNSLLETIAGGVPMICRPFFGDQRVNARLVEDVLE
	IGVTVEDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLLAQQP
	VEPKGSSAQNFKLLLDVISGSTKV

Anthocy anidin-3-	MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFSFFNSAVS	72
O-glycotransferase	NSTLFNNGVLDSYDNIRVYHVWDGTPQGQAFTGSHFEAVGLFLKA
(3 GT)	SPGNFDKVIDEAEVETGLKISCLITDAFLWFGYDLAEKRGVPWLA
Scutellaria	FWTSAQCALSAHMYTHEILKAVGSNGVGETAEEELIQSLIPGLEM
baicalensis	AHLSDLPPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEID
Accession:	PIITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLDSQTR
A0A482AQV3	PKSVVYISFGTVITPPENELAALSEALETCNYPFLWSLNDRAKKS
	LPTGFLDRTKELGMIVPWAPQPRVLAHRSVGVFVTHCGWNSILES
	ICSGVPLICRPFFGDQKLNSRMVEDSWKIGVRLEGGVLSKTATVE
	ALGRVMMSEEGEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEI
	INAPQSS

Anthocy anidin-3-	MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPHAVFSFFST	73
O-glycotransferase	SQSNASIFHDSMHTMQCNIKSYDISDGVPEGYVFAGRPQEDIELF
(3GT)	TRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGLA
Vitis vinifera	WLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS
Accession:	KVRFRDLQEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL
P51094	DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKP
	TSVVYISFGTVTTPPPAEVVALSEALEASRVPFIWSLRDKARVHL
	PEGFLEKTRGYGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESV
	AGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSC
	FDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFITLVDLV
	SKPKDV

Acetyl-CoA	MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHSVITKVLIC	74
carboxylase (ACC)	NNGIAAVKEIRSIRKWAYETFGDERAIEFTVMATPEDLKVNADYI
Ustilago maydis	RMADQYVEVPGGSNNNNYANVDLIVDVAERAGVHAVWAGWGHASE
521	NPRLPESLAASKHKIIFIGPPGSAMRSLGDKISSTIVAQHADVPC
Accession:	MPWSGTGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAEKIG
XP_011390921.1	YPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLGEVPGSPVFVMK
	LAGQARHLEVQLLADQYGNAISIFGRDCSVQRRHQKIIEEAPVTI
	APEDARESMEKAAVRLAKLVGYVSAGTVEWLYSPESGEFAFLELN
	PRLQVEHPTTEMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDP
	RGNEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDTGFKPG
	MGALTELNFRSSTSTWGYFSVGTSGALHEYADSQFGHIFAYGADR
	SEARKQMVISLKELSIRGDFRTTVEYLIKLLETDAFESNKITTGW
	LDGLIQDRLTAERPPADLAVICGAAVKAHLLARECEDEYKRILNR
	GQVPPRDTIKTVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRT
	LVQLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSKTCLIEQ
	ENDPTQIRSPSPGKLVRFLVDSGDHVKANQAIAEIEVMKMYLPLV
	AAEDGVVSFVKTAGVALSPGDIIGILSLDDPSRVQHAKPFAGQLP
	DFGMPVIVGNKPHQRYTALVEVLNDILDGYDQSFRMQAVIKELIE
	TLRNPELPYGQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEF
	PAARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYAGGLKA
	HEGNVLASFLQKYYEVESQFTGEADVVLELRLQADGDLDKVVALQ
	TSRNGINRKNALLLTLLDKHIKGTSLVSRTSGATMIEALRKLASL
	QGKSTAPIALKAREVSLDADMPSLADRSAQMQAILRGSVTSSKYG
	GDDEYHAPSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALCT
	YVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQLPRSASSLKER
	ERQVSISDLSMMDNNRRARPIRELRTGAMTSCADVADIPELLPKV
	LKFFKSSAGASGAPINVLNVAVVDQTDFVDAEVRSQLALYTNACS
	KEFSAARVRRVTYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIE
	PALAYQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADTRFF
	VRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEVALGQPEYRTA
	DASHIFMSFIYQLDVSLVDVQKAIAGFLERHGTRFFRLRITGAEI
	RMILNGPNGEPRPIRAFVTNETGLVVRYETYEETVADDGSVILRG
	IEPQGKDATLNAQSAHFPYTTKVALQSRRSRAHALQTTFVYDFID
	VLGQAVRASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRAPG
	MNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQAGSFGPVEDRF
	FAAASKLARELGVPRLYISANSGARIGLATEALDLFKVKFVGDDP
	AKGFEYIYLDDESLQAVQAKAPNSVMTKPVQAADGSVHNIITDII
	GKPQGGLGVECLSGSGLIAGETSRAKDQIFTATIITGRSVGIGAY
	LARLGERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQIM
	YKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGPLPIMPTTDSWD
	RAVTYQPPRGPYDPRWLINGTKAEDGTKLTGLFDEGSFVETLGGW
	ATSVVTGRARLGGIPVGVIAVETRTLERVVPADPANPNSTEQRIM
	EAGQVWYPNSAYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMY
	DEILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVVVDSAIND
	NGMIEMSADVNSARGGVLEASGLVEIKYRADKQRATMERLDSVYA
	KLSKEAAEATDFTAQTTARKALAEREKQLAPIFTAIATEYADAHD
	RAGRMLATGVLRSALPWENARRYFYWRLRRRLTEVAAERTVGEAN
	PTLKHVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLAASKQL
	KAQYILAQISTLDPELRAQLAASLK

Acetyl-CoA	MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHTVISKVLIAN	75
carboxylase (ACC)	NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIR
Hesseltinella	MADNYIEVPGGTNNNNYANVELIVDVAERTGVHAVWAGWGHASEN
vesiculosa	PRLPEMLAKSKNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTM
Accession:	GWSGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLASAEKIGF
ORX57605.1	PVMIKASEGGGGKGIRKVEDPSTFKQAFAQVQGEIPGSPIFIMKL
	AGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIA
	KPDIFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDEKFYFLELNP
	RLQVEHPTTEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN
	SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPDAGFKPSG
	GVMQELNFRSSTNVWGYFSVVSSGAMHEYADSQFGHIFAYGENRQ
	QARKNMVIALKELSIRGDFRTTVEYIIRLLETPDFTDNTINTGWL
	DMLISKKLTAERPDTMLAVFCGAVTKAHLASVECWQQYKNSLERG
	QIPSKESLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKTQ
	VGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMIDGKTCLLEKE
	SDPTQLRSPSPGKLVSLLLENGDHIRTGQAYAEIEVMKMYMPLVA
	SEDGHVQFIKQVGATLEAGDIIGILSLDDPSRVKHALPFTGQVPK
	YGLPHLTGDKPHQRFTHLKQTLEYVLQGYDNQGLIQTIVKELSEV
	LNNPELPYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKGFP
	AVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIAASYSNGLKQ
	HEHSVYVDLMEQYYNVEVLFNSNQSRDEEVILALRDQHKDDLEKV
	INIILSHAKVNIKNNLILMLLDIIYPATSSEALDRCFLPILKHLS
	EIDSRGTQKVTLKAREYLILCQLPSLEERQSQMYNILKSSVTESV
	YGGGTEYRTPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAAL
	EVYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSAGGGFNNQR
	IASYSDLTFLLNKTEEEPIRTGAMVALKTLEELEAELPRIMTAFE
	EEPLPPMLMKQPPPDKTEERMENILNISIQGQDMEDDTLRKNMTT
	LIQAHSDAFRKAALRRITLVVCRDNQTPDYYTFRERNGYEEDETI
	RHIEPALAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
	RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHDY
	KNSDCNHLFINFIPTFAIEADEVETALKDFVDRHGKRLWKLRVTG
	AEIRFNIQSKRPDAPVIPLRFTVDNVSGYILKVDVYQEVKTDKNG
	WILKSVGKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYD
	FPELFRQAIHNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA
	IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDITFKIGSFG
	VQEDLVFYKASEYARELGVPRVYLSANSGARIGLADELISRFHVA
	WKDEDQPGSGFEYLYLLPEEYDALIQQGDAQSVLVQEVQDKGERR
	FRITDIIGHTDGLGVENLRGSGLIAGATSRAYDDIFTITLVTCRS
	VGIGAYLVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNLQL
	GGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPECRGAPLPMRA
	GADPIDREIEYLPPKGPSDPRFFLAGKQENGKWLSGFFDHGSFVE
	TLSGWARTVVVGRARLGGIPMGVVAVETRTVENIVPADPANADSQ
	EQVVMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSG
	GQRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGELRGGAWVVV
	DSTINEDMMEMYADTQARGGVLEPEGIVEIKYRRPQLLATMERLD
	PVYSDLKRRLAALDDSQKEQADELIAQVEAREQALLPVYQQVAIQ
	FADLHDRSGRMEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIR
	KMDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVAWFDAKNLLLD
	QRIAKLKSEKLKDHVVQLASVDQDAVVEGFSKLMESLSVDQRKEV
	LHKLATRF

Acetyl-CoA	MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVFMHGLGSST	76
carboxylase (ACC)	SFWEAPFSRSNLSSRFRLIRYDFDGHGLSPVSLLDAADDGAMIPL
Rhodotorula	VDLVEDLAAVMEWTGVDKVAGIVGHSMSGLVASTFAAKYPQKVEK
toruloides	LVLLGAMRSLNPTVQTNMLKRADTVLESGLSAIVAQVVSAALSDK
NBRC10032	SKQDSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDYSTIKAK
Accession:	TLVVSGESDYLSNKETTEALVNDIPGAKEVQMDGVGHWHAVEDPA
GEM08739.1	GLAKILDGFFLQGKFSGEAKAVNGSHAVDETPKKPKYDHGRVVKY
	LGGNSLESAPPSNVADWVRERGGHTVITKILIANNGIAAVKEIRS
	VRKWAYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVEVPGG
	TNNNNYANVDVIVDVAERAGVHAVWAGWGHASENPRLPESLAASK
	HKIVFIGPPGSAMRSLGDKISSTIVAQHAEVPCMDWSGQGVDQVT
	QSLEGYVTVADDVYQQACVHDADEGLARASRIGYPVMIKASEGGG
	GKGIRKVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLEVQV
	LADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAKPDTFEQMEKS
	AVRLAKLVGYVSAGTVEFLYSAADDKFAFLELNPRLQVEHPTTEM
	VSGVNLPAAQLQVAMGVPLHRIRDIRTLYGKAPNGSSEIDFEFEN
	PESAKTQRKPSPKGHVVAVRITAENPDAGFKPSMGTLQELNFRSS
	TNVWGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKNMVVALK
	ELSIRGDFRTTVEYLIKLLETDAFEQNTITTAWLDSLISARLTAE
	RPDTTLAIICGAVTKAHLASEANIAEYKRILEKGQSPPKELLATV
	VPLEFVLEDVKYRATASRSSPSSWSIYVNGSNVSVGIRPLADGGL
	LILLDGRSYTCYAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSP
	GKLVRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQFIKQ
	PGATLEAGDILGILSLDDPSRVHHAKPFDGQLPALGLPSIIGTKP
	HQRFAYLKDVLSNILMGYDNQAIMQSSIKELISVLRNPELPYGEA
	NAVLSTLSGRIPAKLEQTLRQYIDSAHESGAEFPSAKCRKAIDTT
	LEQLRPAEAQTVRNFLVAFDDIVYRYRSGLKHHEWSTLAGIFAAY
	AETEKPFSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKNSL
	VLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKATTKVALKARE
	VLIHIQLPSLDERLGQLEQILKASVTPTVYGEPGHDRTPRGEVLK
	DVIDSRFTVFDVLPSFFQHQDQWVSLAALDTYVRRAYRSYNLLNI
	EHIEADAAEDEPATVAWSFRMRKAASESEPPTPTTGLTSQRTASY
	SDLTFLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFPDSNK
	GKLQQQPAASSSQEQWNVINVALTVPASAQVDEDALRADFAAHVN
	AMSAEIDARGMRRLTLLICREGQYPSYYTVRKQDGTWKELETIRD
	IEPALAFQLELGRLSNFHLEPCPVENRQVHIYYATAKGNSSDCRF
	FVRALVRPGRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRRA
	ADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRFWRLRVTGAE
	IRIVLEDAQGNIQPIRAIIENVSGFVVKYEAYREVTTDKGQVILK
	SIGPQGALHLQPVNFPYPTKEWLQPKRYKAHVVGTTYVYDFPDLF
	RQAIRKQWKAVGKTAPAELLVAKELVLDEFGKPQEVARPPGTNNI
	GMVGWIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFYAV
	TQLARQLGLPRVYLSANSGARLGIAEELVDLFSVAWADSSRPEKG
	FKYLYLTAEKLGELKNKGEKSVITKRIEDEGETRYQITDIIGLQE
	GLGVESLKGSGLIAGETSRAYDDIFTITLVTARSVGIGAYLVRLG
	QRAVQVEGQPIILTGAGALNKVLGREVYSSNLQLGGTQIMYKNGV
	SHLTAANDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSID
	YTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSFQETLSGWAQT
	VVVGRARLGGIPMGAIAVETRTIERIIPADPANPLSNEQKIMEAG
	QVWYPNSSFKTGQAIFDFNREGLPLIIFANWRGFSGGQQDMFDEV
	LKRGSLIVDGLSAYKQPVFVYIVPNGELRGGAWVVLDPSINAEGM
	MEMYVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTYHALRV
	KSTDASLSPTDAAQAKTELAAREKQLMPIYQQVALQFADSHDKAG
	RILSKGCAREALEWSNARRYFYARLRRRLAEEAAVKRLGEADPTL
	SRDERLAIVHDAVGQGVDLNNDLAAAAAFEQGAAAITERVKLARA
	TTVASTLAQLAQDDKEAFAASLQQVLGDKLTAADLARILA

Malonyl-CoA	MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVARSGRMANVL	77
synthase (matB)	VARGVKPGDRVAAQAEKSVAALVLYLATVRAGAVYLPLNTAYTLH
Rhodopseudomonas	ELDYFIGDAEPKLVVCDPAKREGIAALAQKVGAGVETLDAKGQGS
palustris	LSEAAAQASVDFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNL
Accession:	ASNSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLFARASM
WP_011661926.1	IFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQSDGLTKEAARHM
	RLFISGSAPLLADTHREWASRTGHAVLERYGMTETNMNTSNPYDG
	ARVPGAVGPALPGVSLRVVDPETGAELSPGEIGMIEVKGPNVFQG
	YWRMPEKTKAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITG
	GFNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAVVVRDKG
	ASVDEAAVLGALQGQLAKFKMPKRVLFVDDLPRNTMGKVQKNVLR
	EAYAKLYAK

Malonyl-CoA	MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCSARITHVL	78
synthase (matB)	VKLGVSPGDRVAVQVEKSAQALMLYLGCLRAGAVYLPLNTAYTPA
Rhizobium	ELEYFLGDATPKLVVVSPCAAEQLEPLARRVGTRLLTLGVNGDGS
sp. BUS003	LMDMASLEPVEFADIERKADDLAAILYTSGTTGRSKGAMLTHDNL
Accession:	LSNAQTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAGGAI
NKF42351.1	YLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQDERLNKANTRHM
	RLFISGSAPLLAETHRLFEEYTGHAILERYGMTETNMITSNPCDG
	ARVPGTVGYALPGVSVRITDPVSGEPLAAGEPGMIEVKGPNVFQG
	YWNMPDKTKEEFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISG
	GYNIYPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVGQPK
	AHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNTMGKVQKNVLR
	DRYRDLYLK

Malonyl-CoA	MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMTGTARYANVL	79
synthase (matB)	VGLGVKPGDRVAVQVEKSAGAIFLYLACVRAGAVFLPLNTAYTLT
Ochrobactrum sp.	EIEYFLGDAEPALVVCDPARRDGITEVAKKTGVPAVETLGKGQDG
3-3	SLFDKAAAAPETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDN
Accession:	LASNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTILVAGAS
WP_114216069.1	MLFLPKFDADKVFELMPRATTMMGVPTFYVRLVQDARLTREATKH
	MRLFISGSAPLLAETHKLFREKTGVSILERYGMTETNMNTSNPYD
	GDRVAGTVGFPLPGVALRVADPETGAAIPQGEIGVIEVKGPNVFS
	GYWRMPEKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLVIS
	GGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGEGVTAVVVAEK
	GASLDEATIIKTLEQRLARYKLPKRVIVVDDLPRNTMGKVQKNLL
	RDAYKGLYGG

Malonate	MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA	80
transporter (matC)	DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR
Rhizobiales	VALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL
bacterium	LMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS
Accession:	LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG
MBN8942514.1	SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTP
	ARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRA
	VGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAA
	VLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAAL
	AVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA
	VPALAWLVLVVPGFG

Malonate	MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMIIGMKT	81
transporter (matC)	NEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRLVRGR
Rhizobium	IGLIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV
leguminosarum	MMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS
Accession:	FFFNLAIAVLVFFVFGGARVMKHDPASLGPLPELHPEGVSASIRG
AAC83457.1	HGGTPAKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIGAL
	VFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSWSTVLLIAGIIT
	YVGVMEKAGTVDYVANGISSLGMPLLVALLLCFTGAIVSAFASST
	ALLGAIIPLAVPFLLQGHISAIGVVAAIAISTTIVDTSPFSTNGA
	LVVANAPDDSREQVLRQLLIYSALIAIIGPIVAWLVFVVPGLV

Malonate	MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLIIGMKP	82
transporter (matC)	ADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWLVERSVRMVRGR
Agrobacterium vitis	VGWIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV
Accession:	LMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS
WP_180575084.1	FFFNLAIAVLIFFIFGGLSILKQRSSVKGPLPELHPEGISASIKG
	HGGTPAKPFREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL
	VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTVLLISGIIT
	YVGVMEKAGTIDYVAHGISSLGMPLLVALLLCFTGAIVSAFASST
	ALLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGA
	LVVANAPDDQRDKVMRQMLIYSALIALIGPVIAWLVFVVPGII

Malonate	MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLIIGMKT	83
transporter (matC)	SDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRMVRGH
Neorhizobium sp.	VAWIPWVMFVVAAITGFGALGPAAVAILAPVALSFAVQYRIHPVM
Accession:	MGLMVIHGAQAGGFSPISVYGGITNQIVAKAGLPFAPTSLFLSSF
WP_105370917.1	FFNLAIAVLVFFVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGH
	GGTPAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGIGALV
	FKFNVGLVAITVAVALALISPKTQKAAVDKVSWSTVLLIAGIITY
	VGVLEKAGTVNYVANGISSLGMPLLVALLLCFTGAIVSAFASSTA
	LLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGAL
	VVANAPDETREQVLRQLLIYSALIAIIGPVVAWLVFVVPGLV

Malonate CoA-	MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALLETVIDRGDR	84
transferase (MdcA)	VCLEGNNQKQADFLSKSLSSCNPDIVNGLHIVQSVLALPSHIDVF
Moraxella	ERGIASKVDFSFAGPQSLRLAQLVQAQKITIGAIHTYLELYGRYF
catarrhalis	IDLTPNVALITAHAADKRGNLYTGANTEDTPAIVEATTFKSGIVI
Accession:	AQVNEIVDELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITE
WPO64617969.1	IQILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTYAESLG
	LKGEICTHWALNPHPTLIPAIESGFIHSVHSFGSEVGMENYVKAR
	SDVFFTGADGSMRSNRAFSQTAGLYACDLFIGSTLQIDLQGNSST
	ATADRIAGFGGAPNMGSDPHGRRHASYAYMKAGREAVDGSPIKGR
	KLVVQMVETYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDDV
	THIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLGRDDTMVAR
	LRERKVIQRPEDLGINPMHATRDLLAAKSVKDLVRWSDRLYEPPS
	RFRNW

Malonate CoA-	MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEIPVDRIIDLL	85
transferase (MdcA)	EAVIQPGDRVCLEGNNQKQADFLSESLADCDPARINHLSMVQSVL
Dechloromonas	ALPSHVDLFERGLATRLDFSFSGPQGARLAKLVQEQRIEIGAIHT
aromatica	YLELFGRYFMDLTPNVALIAAQAADAEGNLYLGPNTEDTPAIVEA
Accession:	TAFKGGIVIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPLF
WP_011289741.1	TRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIGFDTAAIELL
	LPTYAADLGLKGKICTHWALNPHPTLIPAIEAGFVESVHCFGSEV
	GMDDYISARSDIFFTGADGSMRSNRAFSQTAGLYACDMFIGSTLQ
	MDLAGNSSTATLGRITGFGGAPNMGSDPHGRRHASPAWLKAGREA
	YGPQAIRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASMGSDLP
	PIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAIRGVAGFTPVGM
	ARDKGTVENLRDRGIIRRPEDLGIDPRQASRDLLAARSIKDLVRC
	SGGLYAPPSRFRNW

Malonate CoA-	MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVVALLEAVIEPG	86
transferase (MdcA)	DRVCLEGNNQKQADFLARCLTEVDPARVHDLHMVQSVLSLAAHLD
Pseudomonas	VFERGIAKRLDFSFSGPQAARLAGLVSEGRIEIGAIHTYLELFGR
cissicola	YFIDLTPRIALVTAQAADRHGNLYTGPNTEDTPVIVEATAFKGGI
Accession:	VIAQVNEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPAQI
WP_078590875.1	SEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIELLLPTYAQS
	LGLKGKICRHWALNPHPALIPAIESGFVQSVHSFGSELGMENYIA
	ARPDIFFTGADGSMRSNRALSQTAGLYACDMFIGSTLQIDLQGNS
	STATRDRIAGFGGAPNMGSDARGRRHASAAWLKAGREAATPGEMP
	RGRKLVVQMVETFREHMAPAFVDRLDAWELAERANMPLPPVMIYG
	DDVSHVLTEEGIANLLLCRTPEEREQAIRGVSGYTAVGLGRDKRM
	VENLRDRGVIKRPDDLGIRPRDATRDLLAARTVKDLVRWSGGLYD
	PPKRFRNW

Malonate CoA-	MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTEKIVEALTA	87
transferase (MdcA)	LIFPGDRVVIEGNNQKQASFLSKALSQVNPEKVNGLHIIMSSVSR
Geobacillus	PEHLDLFEKGIARKIDFSYAGPQSLRMSQMLEDGKLVIGEIHTYL
subterraneus	ELYGRLFIDLTPSVALVAADKADASGNLYTGPNTEETPTLVEATA
Accession:	FRDGIVIAQVNELADELPRVDIPGSWIDFVVAADHPYELEPLFTR
WP_184319829.1	DPRLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIELLLP
	TYGESLGLKGKICKHWALNPHPTLIPAIETGWVESIHCFGGEVGM
	EKYIAARPDIFFTGKDGNLRSNRTLSQVAGQYAVDLFIGSTLQID
	RDGNSSTVTNGRLAGFGGAPNMGHDPRGRRHSSPAWLDMITSDHP
	AAKGRKLVVQMVETFQKGNRPVFVESLDAIEVGRSARLATTPIMI
	YGEDVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLERDP
	RKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSIEELVEWSEGL
	YEPPARFRSW

Pantothenate kinase	MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADELAVLLQG	88
(CoaX)	LMGTHPLLGMELGEGIDGIAICSTVPAVLHELREVSRRYYGDVPA
Streptomyces sp.	ILVEPGVKTGVPILMDNPKEVGTDRIINAVAAQHLYGGPAIVVDF
CLI2509	GTATTFDAVSARGEYTGGVIAPGIEISVEALGLRGAQLRKIELAR
Accession:	PRSVIGKSTVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVTV
WP_095682415.1	IATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERNAGRR

Pantothenate kinase	MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQAPAELLKGLTF	89
(CoaX)	RLERLNPDFIGVSSVLGQAVNNHVAESLERLQKPFEFAQVHAKHA
Streptomyces	LMSSDYNPAQLGVDRWLQMLGIIEPSKKQCVIGCGTAVTIDLVDQ
cinereus	GHHLGGYIFPSIYLQRESLFSGTRQISIIDGTFDSIDSGTNTQDA
Accession:	VHHGIMLSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI
WP_188874884.1	RQDLVLAGLQRFFAAKNNTKNQN

Pantothenate kinase	MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTADELAVLMQG	90
(CoaX)	LMGRQPGGAGRERVDGLAICSSVPAVLHELREVTRRYYGDLPAVL
Kitasatospora	VAPGVKTGVHVLMDNPKEVGADRIVNALAANHLYGGPCIVVDFGT
kifunensis	ATTFDAINERGDYVGGAIAPGIEISVEALGVRGAQLRKIELAKPR
Accession:	NVIGKNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPEDVQVI
WP_184936930.1	ATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYERNTAS

glutamyl-tRNA	MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQALPMVQGGV	91
reductase (hemA)	VLSTCNRTEIYLSVEEQDNLREALIRWLCEYHNLNEEDLRNSLYW
Citrobacter	HQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHQNA
freundii	SALERMFQKSFSVAKRVRTETDIGSSAVSVAFAACTLARQIFESL
Accession:	STVTVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQVLADE
NTY05430.1	VGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKNRR
	NQPMLLVDIAVPRDVEPEVGKLSNAYLYSVDDLQSIISHNLAQRK
	AAAVEAETIVEQEASEFMAWLRAQGASDTIREYRSQSEQIRDELT
	AKALAALQQGGDAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDS
	ERLNILRDSLGLE

glutamyl-tRNA	MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQPLVQGGV	92
reductase (hemA)	VLSTCNRTELYLSVEQQENLQEQLVKWLCDYHHLSADEVRKSLYW
Pseudomonas	HQDNAAVSHLMRVASGLDSLVVGEPQILGQVKKAFAESQHGQAVS
reactans	GELERLFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL
Accession:	SDVSVLLVGAGETIELVARHLREHKVRHMMIANRTRERAQVLASE
NWA43040.1	VGAEVITLQDIDARLADADIIISSTASPLPIIGKGMVERALKARR
	NQPMLMVDIAVPRDIEPEVGKLANAYLYSVDDLHSIIQNNMAQRK
	AAAVQAESIVEQESSNFMAWLRSQGAVEIIRDYRSRADLVRAEAE
	AKALAAIAQGADVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDV
	ERLQILRDSLGLDQQ

glutamyl-tRNA	MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQQPAVRGGV	93
reductase (hemA)	VLSTCNRTELYLSMEDKENSHEQLIRWLCQYHQIEPNELQSSIYW
Gammaproteobacteria	HQDNQAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQNYDSLS
Accession:	SELERLFQKSFSVAKRVRTETQIGANAVSVAFAACTLARQIFESL
WP_193016510.1	SSLTILLVGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE
	VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVERALKKRR
	NQPMLLVDIAVPRDIEQDVEKLNNVYLYSVDDLEAIIQHNREQRQ
	AAAVQAEHIVQQESGQFMDWLRAQGAVGAIREYRDSAETLRAEMT
	EKAITLIQNGADAEKVIQQLSHQLMNRLIHTPTKSLQQAASDGDI
	ERLNLLRESLGITHN

5-aminolevulinic	MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITIPPNATVEMC	94
acid synthase	PHAKAARDAARIAEDLAAAAASKQQPAKKAGGCPFHAAQAQAQAK
(ALAS)	PAAAPKETVATADKKGKSPRAAGGFDYEKFYEEELDKKHQDKSYR
Schizophyllum	YFNNINRLAARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLETM
commune H4-8	HRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLHRKEGALVFTS
Accession:	CYVANDATLSTLGSKMPGCVIFSDRMNHASMIQGIRHSGTKKVIF
XP_003036856.1	EHNDLADLEKKLAEYPKETPKIIAFESVYSMCGSIGPIKEICDLA
	EKYGAITFLDEVHAVGLYGPRGAGVAEHLDYDLHKAAGDSPDAIP
	GTVMDRVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIFT
	TSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREVKRRFAELDIP
	VVPGPSHIVPVLVGDAALAKQASDKLLAEHDIYVQAINYPTVARG
	EERLRITVTQRHTLEQMDHLIGAVDQVFNELNINRVQDWKRLGGR
	ASVGVPGGQDFVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAV
	VAARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQSSGLKL
	PVEEMTMGQTIAVAA

5-aminolevulinic	MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTALTERATK	95
acid synthase	CPVMGPALNVRSKEIVAGYASVAANSDVALIHKEKGVFPPPGATV
(ALAS)	EMCPHASAARAAARMADDLAAAAEKKKGHFTSAAPRDEAAQAAAA
Crassisporium	GCPFHVKAAADAAAARKAAAAPAPVKAKEDGGFNYESFYVNELDK
junariophilum	KHQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCANDYLGMGN
Accession:	NPVVLETMHRTLDKYGHGAGGTRNIAGNGAMHLSLEQELATLHRK
KAF8165006.1	PAALVFSSCYVANDATLSTLGAKLPGCIFFSDTMNHASMIQGMRH
	SGAKRVLFKHNDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGP
	IKEICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDYDAHVAA
	GESPHPIKGSVMDRVDIITGTLGKAYGAVGGYIAGSDDFVDMIRS
	YAPGFIFTTSLPPATVAGARASVVYQKHYVGDRQLKQVNVREVKR
	RFAELDVPVVPGPSHIVPVLVGDAALAKAASDKLLAEHNIYVQSI
	NYPTVARGEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA
	DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLDGTSPRTLRNG
	EAAVVDAMAVGQARAVFDNLLGPISGKLQSERSVLASSTPAAANP
	ARPAARKVVKMKTGGVPMSEDIPLPPPDVSASA

5-aminolevulinic	MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSISILTER	96
acid synthase	ATKCPVMGPALNVRSKEITAGYASVAGSSEVDQIHKQQGVTVPVN
(ALAS)	ATVEMCPHASAARAAARMADDLAAAAAQKKVGSGASSAKAAAAGC
Dendrothele	PFHKSVAAGASASTASKPSAPIHKASVPGGFDYDNFYNNELEKKH
bispora CBS	KDKSYRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLGMGNNP
962.96	VVLETMHRTLDKYGHGAGGTRNIAGNGALHLGLEQELAALHRKEA
Accession:	ALVFSSCYVANDATLSTLGSKLPGCILFSDKMNHASMIQGMRHSG
THV05492.1	AKKVIFNHNDLEDLENKLKQYPKETPKIIAFESVYSMCGSIGPIK
	EICDLAEKYGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAAGK
	SPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSMDFVDTIRSYA
	PGFIFTTSLPPATVSGAQASVAYQKEYLGDRQLKQVNVREVKRRF
	AELDIPVIPGPSHILPVLVGDAALAKAASDKLLTDHDIYVQSINY
	PTVAVGEERLRITVTPRHTLEQMDKLVRAVNQVFTELNINRISDW
	KVAGGRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQRFLVD
	AQGVTAARGRFDTLLGPMSGSLQANPTLPLVD
	DELKVPLPTLVAAAA

5-aminolevulinic	MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHALWHSPKGKR	97
acid synthase	DVVIWCSNDYLGMGQHPKVVGAMVETATRVGTGAGGTRNIAGTHH
(ALAS)	PLVQLEAELADLHGKEASLLFTSGYVSNQTGIATIAKLIPNCLIL
Bradyrhizobium	SDELNHNSMIEGIRQSGCERVVFRHNDLADLEEKLKAAGPNRPKL
japonicum	IACESLYSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMYGPRG
Accession:	GGIAERDGVMHRIDILEGTLAKAFGCLGGYIAANGQIIDAVRSYA
AOAOA3YXD2	PGFIFTTALPPAICSAATAAIRHLKTSNWERERHQDRAARVKAIL
	NAAGLPVMSSDTHIVPLFIGDAEKCKQASDLLLEQHGIYIQPINY
	PTVAKGTERLRITPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKS
	LAAE

Cytochrome b5	MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLEEHPGGEEV	98
Petunia x hybrida,	LIESAGKDATKEFQDIGHSKAAKNLLFKYQIGYLQGYKASDDSEL
Accession:	ELNLVTDSIKEPNKAKEMKAYVIKEDPKPKYLTFVEYLLPFLAAA
AAD10774.1	FYLYYRYLTGALQF

TABLE 12

Glossary of abbreviations

	Abbreviation	Full Name

	3GT	anthocyanidin-3-O-glycotransferase
	4CL	4-coumarate-CoA ligase
	ACC	acetyl-CoA carboxylase
	ACOT	acyl-CoA thioesterase
	acpP	acyl carrier protein
	ACS	acetyl-CoA synthase
	adhE	aldehyde-alcohol dehydrogenase
	ADP	adenosine diphosphate
	ALA	5-aminolevulinic acid
	ALAS	ALA synthase
	ANS	anthocyanin dioxygenase
	aroG	DAHP synthase
	aroK	shikimate kinase
	aroL	shikimate kinase
	ATP	adenosine triphosphate
	C3G	cyanidin-3-O-glycoside
	C4H	cinnimate-4-hydroxylase
	CHI	chalcone isomerase
	CHS	chalcone synthase
	CoA	coenzyme A
	CPR	cytochrome P450 Reductase
	DAD	diode array detector
	DAHP	deoxy-d-arabino-heptulosonate-7-phosphate
	DctPQM	a malonate transporter
	DFR	dihydroflavonol 4-reductase
	DHK	dihydrokaempferol
	DHM	dihydromyricein
	DHQ	dihydroquercetin
	DMSO	dimethyl sulfoxide
	E4P	erythrose-4-phosphate
	F3′H	flavonoid 3′ hydroxylase
	F3H	flavanone 3-hydroxylase
	fabB	beta-ketoacyl-ACP synthase I
	fabD	malonyl-coA-ACP transacylase
	fabF	beta-ketoacyl-ACP synthase II
	FadA	3-ketoacyl-CoA thiolase
	FadB	fatty acid oxidation complex subunit alpha
	FadE	acyl-CoA dehydrogenase
	GltX	glutamyl-tRNA synthetase
	hemA	glutamyl-tRNA reductase
	hemL	glutamate-1-semialdehyde aminotransferase
	HPLC	high performance liquid chromatography
	ldhA	lactate dehydrogenase
	LAR	leucoanthocyanidin reductase
	matB	malonyl-CoA synthase
	matC	malonate transporter
	mdcA	malonate coA-transferase
	mdcC	acyl-carrier protein, subunit of mdc
	mdcD	malonyl-CoA decarboxylase, subunit of mdc
	mdcE	co-decarboxylase, subunit of mdc
	pABA	para-aminobenzoic acid
	PAL	phenylalanine ammonia-lyase
	PanK	pantothenase kinase
	Pdh	pyruvate dehydrogenase
	PEP	phosphoenolpyruvate
	pHBA	para-hydroxybenzoic acid
	PHE	phenylalanine
	pheA	chorismate mutase/prephenate dehydrogenase
	poxB	pyruvate dehydrogenase
	ppsA	phosphoenolpyruvate synthase
	TAL	tyrosine ammonia-lyase
	TCA	tricarboxylic acid cycle
	tesA	thioesterase I
	tesB	thioesterase II
	tktA	transketolase
	TRP	tryptophan
	TYR	tyrosine
	TyrA	chorismate mutase
	tyrR	transcriptional regulator
	ybgC	a thioesterase
	yciA	a thioesterase
	ydiB	QUIN/shikamate dehydrogenase
	ackA-pta	Acetate kinase-phosphate acetyltransferase

Claims

1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H).

2. The cell of claim 1, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK).

3. The engineered host cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.

4. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is truncated to remove the N-terminal leader sequence.

5. The engineered host cell of claim 1, wherein the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.

6. The engineered host cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).

7. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).

8. The engineered host cell of claim 1, wherein the flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.

9. The engineered host cell of claim 1, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.

10. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57.

11. The engineered host cell of claim 1, wherein the engineered host cell further comprises cytochrome b₅.

12. The engineered host cell of claim 11, wherein the cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.

13. The engineered host cell of claim 1, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.

14. A method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H).

15. The method of claim 14, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) are naringenin and/or dihydrokaempferol (DHK).

16. The method of claim 14, wherein the flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.

17. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is truncated to remove the N-terminal leader sequence.

18. The method of claim 14, wherein the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.

19. The method of claim 14, wherein the flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).

20. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).

21. The method of claim 14, wherein the flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.

22. The method of claim 14, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.

23. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from the group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57.

24. The method of claim 14, wherein the engineered host cell further comprises cytochrome b₅.

25. The method of claim 24, wherein the cytochrome b₅has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.

26. The method of claim 14, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.