US20220333117A1

US20220333117A1 - Flavonoid and anthocyanin bioproduction using microorganism hosts

Info

Publication number: US20220333117A1
Application number: US17/720,006
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: CA3215623A1; IL307636A; WO2022221392A1; US20220333118A1; JP2024513971A; US20220333122A1; EP4323528A1; AU2022256988A1; KR20230170713A; US20220333123A1; CN117413066A; AU2022256988A9; BR112023021282A2; US20220333087A1

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-009-01-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'S′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′S′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'S′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'S′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'S′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'S′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×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×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 α 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 (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, 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-aminolevulinic 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-aminolevulinic 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, 3 μm, 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 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 (AC S) 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'S′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'S′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'S′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'S′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'S′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-	MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD	1
lyase (TAL)	EEIVRMGASARTIEEYLKSDKPIYGLTQGFGPLVLFDA
Saccharothrix	DSELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNM
espanaensis	RKGYSAVSPVFWQKLADLWNKGFTPAIPRHGTVSAS
Accession:	GDLQPLAHAALAFTGVGEAWTRDADGRWSTVPAVD
ABC88669.1	ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR
	SALRLVRACAVLSARLATLLGANPEHYDVGHGVARG
	QVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCA
	PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL
	LHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGL
	LVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRI
	RQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEAL
	ELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGI
	CPPLDADRPLGAEVRAARDLLSAHADQLLVDEADGK
	DFG

Phenylalanine	MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK	2
ammonia-lyase	GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV
(PAL)	TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD
Brevibacillus	EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
laterosporus LMG	ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE
15441	REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC
Accession:	LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ
WP_003337219.1	KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD
	SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK
	WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA
	MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
	RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS
	VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH
	LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD
	RALDGDIEKVVQLIRSGNLKKEIHDQNVND

Cinnamate-4-	MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLP	3
hydroxylase (C4H)	VPIFGNWLQVGDDLNHRNLTDLAKKFGEIFLLRMGQ
Helianthus annuus	RNLVVVS SPDLAKEVLHTQGVEFGSRTRNVVFDIFTG
L.	KGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYR
Accession:	YGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQLMM
QJC72299.1	YNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQS
	FEYNYGDFIPILRP
	FLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV
	DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAI
	ETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQ
	VTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHD
	AKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRP
	ERFFEEESKVEANGNDFRYLPFGVGRRSCPGIILALPIL
	GITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKHS
	TIVAKPRAL

4-coumarate-CoA	MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKV	4
ligase (4CL)	GDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQ
Petroselinum	QGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTS
crispum	AEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIIC
Accession:	IDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPY
P14912.1	SSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYM
	HSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKF
	DIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLS
	SVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGM
	TEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIV
	DPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTI
	DEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVA
	PAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRT
	NGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSG
	KILRKDLRARIASGDLPK

Chalcone synthase	MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTY	5
(CHS)	PDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLT
Petunia x hybrida	EEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAA
Accession:	QKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKL
AAF60297.1	LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
	GARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGA
	GAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHL
	REVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNS
	LFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNY
	GNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVL
	FGFGPGLTVETVVLHSVAT

Chalcone isomerase	MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERG	6
(CHI)	LTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELL
Medicago sativa	ETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENC
Accession:	VAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASV
P28012.1	FYRQSPDGILGLSFSPDTSIPEKEAALIENKAVSSAVLE
	TMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN

Flavanone 3-	MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFS	7
hydroxylase (F3H)	NEIPIISLSGIDEVEGRRAEICNKIVEACEDWGVFQIVD
Rubus occidentalis	HGVDAKLISEMTRLARDFFALPPEEKLRFDMSGGKKG
Accession:	GFIVSSHLQGEAVQDWREIVTYFSYPVRHRDYSRWPD
ACM17897.1	KPEGWRAVTQQYSDELMGLACKLLEVLSEAMGLEKE
	ALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDP
	GTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVV
	NLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQ
	NPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSK
	DLELARLKKLAKEQQPEDSEKAKLEVKQVDDIFA

Flavonoid 3′	MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIG	8
hydroxylase (F3′H)	NLPHMGPKPHQTLAAMVTTYGPILHLRLGFADVVVA
Brassica napus	ASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDL
Accession:	VFAPYGQRWRMLRKISSVHLFSAKALEDFKHVRQEE
ABC58723.1	VGTLMRELARANTKPVNLGQLVNMCVLNALGREMI
	GRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFV
	PALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAM
	KNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKA
	LLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEE
	LDSVVGRGRPINESDLSQLPYLQAVIKENFRLHPPTPLS
	LPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDP
	LTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAG
	LSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEET
	YGITLQRAVPLVVHPKLRLDMSAYGLGSA

Cytochrome P450	MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPA	9
reductase (CPR)	VMAMLLENKELVMILTTSVAVLIGCVVVLIWRRSSGS
Catharanthus	GKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGT
roseus	AEGFAKALAEEAKARYEKAVIKVIDIDDYAADDEEYE
Accession:	EKFRKETLAFFILATYGDGEPTDNAARFYKWFVEGND
Q05001	RGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVA
	EQGGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDN
	LLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANG
	HANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRS
	CTHLDFDIAGTGLSYGTGDHVGVYCDNLSETVEEAER
	LLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTA
	LTRYADLLNTPKKSALLALAAYASDPNEADRLKYLAS
	PAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFA
	AIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGR
	IHKGVCSTWMKNAIPLEESRDCSWAPIFVRQSNFKLP
	ADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGT
	AVFFFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSR
	EGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDA
	KGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQM
	TGRYLRDVW

Flavonoid 3′, 5′-	MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKG	10
hydroxylase	WPVVGALPMLGNMPHAALADLSRRYGPIVYLKLGSR
(F3′5′H)	GMVVASTPDSARAFLKTQDLNFSNRPTDAGATHIAYN
Delphinium	SQDMVFADYGPRWKLLRKLSSLHMLGGKAVEDWAV
grandiflorum	VRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAMAN
Accession:	MLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFN
BAO66642	VGDFIPSIAWMDLQGIVRGMKRLHKKFDALLDKILRE
	HTATRRERKEKPDLVDVLMDNRDNKSEQERLTDTNI
	KALLLNLFSAGTDTSSSTIEWALTEMIKNPSIFGRAHA
	EMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHPSTP
	LNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNVW
	ENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRIC
	AGTRMGIVLVEYILGSLVHAFEWKLRDGETLNMEETF
	GIALQKAVPLAAVVTPRLPPSAYVV

Dihydroflavonol 4-	MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATV	11
reductase (DFR)	RDPSNMKKVKHLLDLPGAANRLTLWKADLVDEGSFD
Anthurium	EPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNV
andraeanum	LRSCARASSTVRRVVFTSSAGTVSIHEGRRHLYDETS
Accession:	WSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK
AAP20866.1	NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPH
	YSILNPVQFVHLDDLCNAHIFLFECPDAKGRYICSSHD
	VTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKL
	TDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYA
	TEQLIATGQDNGH

Leucoanthocyanidin	MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYP	12
reductase (LAR)	TFLLVRPGPVSPSKAVIIKTFQDKGAKVIYGVINDKEC
Desmodium	MEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIK
uncinatum	RFLPSEFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEY
Accession:	GIPFTNICCNSIASWPYYDNCHPSQVPPPMDQFQIYGD
Q84V83.1	GNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRPSSN
	CYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENII
	PESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKF
	RSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQT
	GTIEEINKDIKTLVETQPNEEIKKDMKALVEAVPISAM
	G

Anthocyanin	MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFD	13
dioxygenase (ANS)	EEKKDEGPQVPTIDLRDIDSDDQQVRQRCRDELKKAA
Carica papaya	VDWGVMHLVNHGIPDHLIDRVKKAGQAFFELPVEVK
Accession:	EKYANDQASGNIQGYGSKLANNASGQLEWEDYYFHL
XP_021901846.1	IFPEEKRDLAIWPNNPADYIEVTSEYARQLRRLVSKIL
	GVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCP
	QPELALGVEAHTDISALTFILHNMVPGLQLFYEGKWV
	TAKCVPNSIVMHVGDTIEILSNGKYKSILHRGLVNKEK
	VRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQ
	HIQHKLFRKNQENLEAK

Anthocyanidin-3-	MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPH	14
O-glycotransferase	AVFSFFSTSESNASIFHDSMHTMQCNIKSYDVSDGVPE
(3GT)	GYVFTGRPQEGIDLFMRAAPESFRQGMVMAVAETGR
Vitis labrusca	PVSCLVADAFIWFAADMAAEMGVAWLPFWTAGPNS
Accession:	LSTHVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFR
ABR24135	DLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFE
	ELDDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCL
	QWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASR
	VPFIWSLRDKARMHLPEGFLEKTRGHGMVVPWAPQA
	EVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFF
	GDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQI
	LSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTL
	VDLVSKPKDV

Acetyl-CoA	MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHT	15
carboxylase (ACC)	VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF
Mucor	TVMATPEDLKANAEYIRMADNFVEVPGGSNNNNYAN
circinelloides	VELIVDVAERTAVHAVWAGWGHASENPRLPEMLAKS
1006PhL	KHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTMGW
Accession:	SGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKA
EPB82652.1	AEKIGFPVMIKASEGGGGKGIRMVKDGSNFAQLFAQV
	QGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFG
	RDCSVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLG
	KLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPT
	TEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNS
	ASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAG
	FKPSSGIMQELNFRSSTNVWGYFSVVSAGGLHEYADS
	QFGHIFAYGENRQQARKNMVIALKELSIRADFRSTVE
	YIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTML
	AVFCGAVTKAHMASLDCFQQYKQSLEKGQVPSKGSL
	KTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQV
	GIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGK
	TCLLEKESDPTQLRSPSPGKLVNLLVENGDHLNAGDA
	YAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDII
	GILSLDDPSRVKHALPFNGTVPAFGAPHITGDKPVQRF
	NATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLP
	YSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP
	AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFAR
	YRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVIL
	SLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLI
	NPVSTGSALDKYFTPILKRLSEIESRATQKVTLKAREL
	LILCQLPSYEERQAQMYQILKNSVTESVYGGGSEYRTP
	SYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEVY
	CRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANG
	IDSNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLAD
	LQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNI
	AVRADEDMDDTAFRTKICEMITANADVFRQAHLRRL
	SVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMA
	YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
	RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE
	IVSHEYKNSDCNHLFINFIPTFAIEADDVEHALKDFVD
	RHGKRLWKLRVTGAEIRFNVQSKKPDAPIIPMRFTVD
	NVSGFILKVEVYQEVKTEKSGWILKSVNKIPGAMHM
	QPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQ
	SVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVL
	QEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDI
	TFKIGSFGVAEDQVFYKASELARALGIPRIYLSANSGA
	RIGLADELISQFRAAWKDASNPTAGFKYLYLTPAEYD
	VLAQQGDAKSVLVEEIQDEGETRLRITDVIGHTDGLG
	VENLKGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYL
	VRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQL
	GGTQIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVR
	NAPVSMRLGADPIDRDIEYTPPKGPSDPRFFLAGKSEN
	GKWLSGFFDQDSFVETLSGWARTVVVGRARLGGIPM
	GVVSVETRTVENIVPADPANSDSTEQVFMEAGGVWFP
	NSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRD
	MYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGA
	WVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKY
	RKPALLATMERLDATYASLKKQLAEEGKTDEEKAAL
	KVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKG
	VIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATS
	AAPMPREQMLDLVKQWFTNDNETVNFEDADELVSE
	WFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIE
	GFSQLIENLSEDARAEILRKLNSRF

Acetyl-CoA	MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTF	16
synthase (ACS)	WGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGT
Salmonella	LNLAANCLDRHLQENGDRTAIIWEGDDTSQSKHISYR
typhimurium	ELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAV
Accession:	AMLACARIGAVHSVIFGGFSPEAVAGRIIDSSSRLVITA
NP_463140.1	DEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRT
	GSDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPL
	FILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYH
	PGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEG
	VPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEG
	DKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE
	KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFF
	GVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLF
	GDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITG
	RVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIP
	HAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIG
	PLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNL
	GDTSTLADPGVVEKLLEEKQAIAMPS

Malonyl-CoA	MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAA	17
synthase (matB)	AGATAGRIGGAGRVAVWATPAMETGVAVVAALLAG
Streptomyces	VAAVPLNPKSGDKELAHILSDSAPSLVLAPPDAELPPA
coelicolor	LGALERVDVDVRARGAVPEDGADDGDPALVVYTSGT
Accession:	TGPPKGAVIPRRALATTLDALADAWQWTGEDVLVQG
WP_011028356	LPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAAREL
	NDGATMLFGVPTMYHRIAETLPADPELAKALAGARL
	LVSGSAALPVHDHERIAAATGRRVIERYGMTETLMNT
	SVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDG
	ESVGEIQVRGPNLFTEYLNRPDATAAAFTEDGFFRTG
	DMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENA
	LLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPP
	ALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKI
	MKRALNRD

Malonate	MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT	18
transporter (matC)	LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT
Streptomyces	TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV
coelicolor	SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG
Accession:	GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA
NP_626686.1	AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS
	GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG
	AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
	AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY
	VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
	SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV
	SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY
	GGIVVAAVPALAWLVLVVPGFG

Malonate CoA-	MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARA	19
transferase (MdcA)	IELLETVIASGDRVCLEGNNQKQADFLSKCLSQCNPD
Acinetobacter	AVNDLHIVQSVLALPSHIDVFEKGIASKVDFSFAGPQS
calcoaceticus	LRLAQLVQQQKISIGSIHTYLELYGRYFIDLTPNICLITA
Accession:	HAADREGNLYTGPNTEDTPAIVEATAFKSGIVIAQVNE
AAB97627.1	IVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQI
	TEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIEL
	LLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDS
	VHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQ
	TAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAP
	NMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVV
	QTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMI
	YGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTP
	VGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLA
	AKSVKDLVKWSGGLYSPPSRFRNW

Pantothenate kinase	MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVE	20
(CoaX)	QLTSQQALPVRACRLVSVRSEQETSQLVARLEQLFPV
Pseudomonas	SALVASSGKQLAGVRNGYLDYQRLGLDRWLALVAA
aeruginosa	HHLAKKACLVIDLGTAVTSDLVAADGVHLGGYICPG
Accession:	MTLMRSQLRTHTRRIRYDDAEARRALASLQPGQATA
Q9HWCL1	EAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGG
	DAELVRDELAGARIMPDLVFVGLALACPIE

glutamyl-tRNA	MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSL	21
reductase (hemA^m)	LAQPMVQGGVVLSTCNRTELYLSVEEQDNLQEALIR
Salmonella	WLCDYHNLNEDDLRNSLYWHQDNDAVSHLMRVASG
typhimurium	LDSLVLGEPQILGQVKKAFADSQKGHLNASALRRMF
Accession:	QKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL
AAA88610.1	STVTVLLVGAGETIELVARHLREHKVQKMIIANRTRE
	RAQALADEVGAEVISLSDIDARLQDADIIISSTASPLPII
	GKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLA
	NAYLYSVDDLQSIISHNLAQRQAAAVEAETIVEQEASE
	FMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQ
	QGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDGD
	DERLNILRDSLGLE

5-aminolevulinic	MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQ	22
acid synthase	WNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHE
(ALAS)	ALEAVGAGSGGTRNISGTTAYHRRLEAEIADLHGKEA
Rhodobacter	ALVFSSAYIANDATLSTLRLLFPGLIIYSDSLNHASMIE
capsulatus	GIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFE
Accession:	SVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMY
CAA37857	GPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIA
	ASAKMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLK
	TAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHI
	VPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRG
	TERLRFTPSPVHDLKQIDGLVHAMDLLWARCA

Tyrosine ammonia-	MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTP	23
lyase (TAL)	ALRERCARAHARLEHAIAEQRHIYGITTGFGPLANRLI
Rhodobacter	GADQGAELQQNLIYHLATGVGPKLSWAEARALMLAR
capsulatus SB 1003	LNSILQGASGASPETIDRIVAVLNAGFAPEVPAQGTVG
Accession:	ASGDLTPLAHMVLALQGRGRMIDPSGRVQEAGAVM
ADE84832.1	DRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEA
	ARAIDAALRHSAVLMEVLSGHAEAWHPAFAELRPHP
	GQLRATERLAQALDGAGRVCRTLTAARRLTAADLRP
	EDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCE
	LNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDAL
	NAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQA
	GLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNG
	ANQDVVSMGTIAARRARAQLLPLSQIQAILALALAQA
	MDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRP
	LAGHIEAVAQGLRHPSAAADPPA

Tyrosine ammonia-	MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQ	24
lyase (TAL)	ATKTVVIDGHNMKVGDVVAVARHGAKVELAASVAG
Trichosporon	PVQASVDFKESKKHTSIYGVTTGFGGSADTRTSDTEA
cutaneum	LQISLLEHQLCGYLPTDPTYEGMLLAAMPIPIVRGAM
Accession:	AVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVPLR
XP_018276715	GTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLT
	APEAIAKYKLKTVRLASKEGLGLVNGTAVSAAAGAL
	ALYDAECLAMMSQTNTALTVEALDGHVGSFAPFIQEI
	RPHVGQIEAAKNIRHMLSNSKLAVHEEPELLADQDAG
	ILRQDRYALRTSAQWIGPQLEMLGLARQQIETELNSTT
	DNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQN
	LGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCK
	GLDIHCGAYCAELGFLANPMSNHVQSTEMHNQSVNS
	MAFASARKTMEANEVLSLLLGSQMYCATQALDLRV
	MEVKFKMAIVKLLNDTLTKHFSTFLTPEQLAKLNTTA
	AITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDALM
	VNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADG
	RNDLEPAEYLGQTRAVYEAIRSDLGVKVRRGDVAEG
	KSGKSIGSNVARIVEAMRDGRLMGAVSKMFF

Tyrosine ammonia-	MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNF	25
lyase (TAL)	LKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLI
Flavobacterium	RSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSV
johnsoniae	INLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLI
Accession:	GEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALING
WP_012023194	TSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAY
	DDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKR
	EDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETI
	NNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDY
	ISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFV
	NLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSI
	PNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIV
	QAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYP
	FVQKVKDHLINN

Tyrosine ammonia-	MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILA	26
lyase (TAL)	QIEASCAYINQAVKEHQPVYGVTTGFGGMANVIISPEE
Herpetosiphon	AAELQNNAIWYHKTGAGKLLPFTDVRAAMLLRANSH
aurantiacus DSM	MRGASGIRLEIIQRMVTFLNANVTPHVREFGSIGASGD
785	LVPLISITGALLGTDQAFMVDFNGETLDCISALERLGL
Accession:	PRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLA
ABX04526.1	LALEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWA
	ADHMLELLQGSQLSRNELDGSHDYRDGDLIQDRYSL
	RCLPQFLGPIIDGMAFISHHLRVEINSANDNPLIDTASA
	ASYHGGNFLGQYIGVGMDQLRYYMGLMAKHLDVQI
	ALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTANS
	IMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLA
	RQTIQTLQQYIAITLMFGVQAVDLRTHKLAGHYNAAE
	LLSPLTAKIYHAVRSIVKHPPSPERPYIWNDDEQVLEA
	HISALAHDIANDGSLVSAVEQTLSGLRSIILFR

Phenylalanine	MHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAME	27
ammonia-lyase	CSHLEEIKRMVDTYQNATQVMIEGATLTVPQVAAIAR
(PAL)	RPEVHVVLDAANARSRVDESSNWVLDRIMGGGDIYG
Physcomitrella	VTTGFGATSHRRTQQGVELQRELIRFLNAGVLSKGNS
patens	LPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKLL
Accession:	NAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKA
XP_001758374.1	VTEDGREVSALEALRIAGVEKPFELAPKEGLALVNGT
	AVGSALASTVCYDANIMVLLAEVLSALFCEVMQGKP
	EFADPLTHKLKHHPGQMEAAAVMEWVLDGSSFMKA
	AAKFNETDPLRKPKQDRYALRTSPQWLGPQVEVIRNA
	THAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSM
	DNMRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSG
	GPNPSLDYGMKGAEIAMASYLSEINYLANPVTTHVQS
	AEQHNQDVNSLGLVSARKTEEAMEILKLMSATFLVG
	LCQAIDLRHVEETMQSAVKQVVTQVAKKTLFMGSDG
	SLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLMEK
	LRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSR
	VVPAVREAYDTKGLSLVPNRIQDCRTYPLYKLVRGDL
	KTQLLSGQRTVSPGQEIEKVFNAISAGQLVAPLLECVQ
	GWTGTPGPFSARASC

Phenylalanine	MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNEL	28
ammonia-lyase	LEFLQKGRDQLENKLKENVAIYGINTGFGGNGDLIIPF
(PAL)	DKLDYHQSNLLDFLTCGTGDFFNDQYVRGIQFIIIIALS
Dictyostelium	RGWSGVRPMVIQTLAKHLNKGIIPQVPMHGSVGASG
discoideum AX4	DLVPLSYIANVLCGKGMVKYNEKLMNASDALKITSIE
Accession:	PLVLKSKEGLALVNGTRVMSSVSCISINKFETIFKAAIG
XP_644510.1	SIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNK
	YFNTSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRC
	APQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVL
	SGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMH
	SEFSKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQE
	AAPACIHSLTTEQFNQDIVSLGLHSANGAASMLIKLCD
	IVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIKIIPI
	LENDRRTDIDVREITNAILQDKLDFFNLNL

Phenylalanine	MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK	29
ammonia-lyase	GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV
(PAL)	TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD
Brevibacillus	EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
laterosporus LMG	ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE
15441	REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC
Accession:	LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ
WP_003337219.1	KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD
	SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK
	WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA
	MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
	RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS
	VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH
	LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD
	RALDGDIEKVVQLIRSGNLKKEIHDQNVND

Cinnamate-4-	MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGP	30
hydroxylase (C4H)	IPVPVFGNWLQVGDDLNHRNLTEMAKKFGEVFMLR
Rubus sp. SSL-2007	MGQRNLVWSSPDLAKEVLHTQGVEFGSRTRNVVFDI
Accession:	FTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQ
ABX74781.1	QYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQ
	LMMYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSR
	LAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLF
	KDYFVDERKKLSSTQATTNEGLKCAIDHILDAQQKGE
	INEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQ
	KKLRDELDTVLGRGVQITEPEIQKLPYLQAVVKETLR
	LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
	ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
	GVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQTQL
	DTTEKGGQFSLHILKHSPIVMKPRT

Cinnamate-4-	MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPV	31
hydroxylase (C4H)	PVFGNWLQVGDDLNHRNLTDMAKKFGDVFMLRMG
Fragaria vesca	QRNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFT
Accession:	GKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQY
XP_004294725.1	RHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQLM
	MYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLA
	QSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKD
	YFVDERKKLASTQVTTNEGLKCAIDHILDAQQKGEIN
	EDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQK
	KLRDELDTVLGHGVQVTEPELHKLPYLQAVVKETLR
	LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
	ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
	GVGRRSCPGIILALPILGVTLGRLVQNFEMLPPPGQTQ
	LDTTEKGGQFSLHILKHSTIVMKPRA

Cinnamate-4-	MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVP	32
hydroxylase (C4H)	VFGNWLQVGDDLNHRNLTEYAKKFGDVFLLRMGQR
Solanum tuberosum	NLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGK
Accession:	GQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRG
ABC69046.1	GWESEAASVVEDVKKNPESATNGIVLRKRLQLMMYN
	NMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFE
	YNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVD
	ERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNV
	LYIVENFNVAAIETTLWSIEWGIAELVNHPHIQKKLRD
	EIDTVLGPGMQVTEPDMPKLPYLQAVIKETLRLRMAI
	PLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNP
	AHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGR
	RSCPGIILALPILGITLGRLVQNFEMLPPPGQSKLDTSE
	KGGQFSLHILKHSTIVMKPRSF

4-coumarate-CoA	MGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKV	33
ligase (4CL)	SDRACLINGATGETFSYAQVELISRRVASGLNKLGIHQ
Daucus carota	GDTMMILLPNTPEYFFAFLGASYRGAVSTMANPFFTS
Accession:	PEVIKQLKASQAKLIITQACYVEKVKEYAAENNITVVC
AIT52344.1	IDEAPRDCLHFTTLMEADEAEMPEVAIDSDDVVALPY
	SSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIHS
	EDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIV
	PFLELIQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVR
	TVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEA
	GPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPE
	THASLPRNQSGEICIRGDQIMKGYLNDPESTKTTIDEE
	GWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAPAEI
	EALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGS
	TTTEEEIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKIL
	RKELRARIASGDLPK

4-coumarate-CoA	MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGS	34
ligase (4CL)	RPCLINGSTGEILTYDQVELASRRVGSGLHRLGIRQGD
Striga asiatica	TIMLLLPNSPEFVLAFLGASHIGAVSTMANPFFTPAEV
Accession:	VKQAAASRAKLIVTQACHVDKVRDYAAEHGVKVVC
GER48539.1	VDGAPPEECLPFSEVASGDEAELPAVKISPDDVVALPY
	SSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHS
	DDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIV
	PFLELIQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRT
	VMSGAAPLGRELEDAVRLKFPNAKLGQGYGMTEAGP
	VLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDTETG
	ASLGRNQPGEICIRGDQIMKGYLNDPESTERTIDKEGW
	LHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEA
	LLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTI
	TEDEIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKD
	LRARLAAGVPN

4-coumarate-CoA	MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISE	35
ligase (4CL)	FSSRPCLINGANNQIYTYADVELNSRKVAAGLHKQFGI
Capsicum annuum	QQKDTIMILLPNSPEFVFAFLGASYLGAISTMANPLFTP
Accession:	AEVVKQVKASNAEIIVTQACHVNKVKDYALENDVKI
KAF3620179.1	VCIDSAPEGCVHFSELIQADEHDIPEVQIKPDDVVALP
	YSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYI
	HSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKF
	DIVPFLELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLS
	SVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMT
	EAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVD
	PDTGNSLHRNQSGEICIRGDQIMKGYLNDPEATAGTID
	KEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQVA
	PAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVR
	SNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPS
	GKILRKDLRAKLAAGFPN

4-coumarate-CoA	MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSS	36
ligase (4CL)	KPCLINGSTGKVYTYSDVELTSRKVAAGFHNLGIQQR
Camellia sinensis	DTIMLLLPNCPEFVFAFLGASYLGAIITMANPFFTPAET
Accession:	IKQAKASNSKLIITQSSYTSKVLDYSSENNVKIICIDSPP
ASU87409.1	DGCLHFSELIQSNETQLPEVEIDSNEVVALPYSSGTTGL
	PKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMM
	CVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKL
	IQRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMS
	GAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLA
	MCLAFAKKPFEIKSGACGTVVRNAEMKIVDPDAGFSL
	PRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLH
	TGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEAL
	LLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVT
	ENEIKKYISEQVVFYKINRVYFVDAIPKAPSGKILRK
	DLRARLAAGIPS

Chaicone synthase	MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTY	37
(CHS)	PDYYFRITNSEHKTELKEKFKRMCEKSMIKTRYMHLT
Capsicum annuum	EEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEA
Accession:	AQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLA
XP_016566084.1	KLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
	NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGD
	GAAAIIMGSDPIPGVERPLFQLVSAAQTLLPDSEGAID
	GHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISD
	WNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVL
	SNYGNMSSACVLFILDEMRKASTKEGLGTSGEGLEW
	GVLFGFGPGLTVETVVLHSVAI

Chalcone synthase	MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYP	38
(CHS)	DYYFRITKSEHKAELKEKFQRMCDKSMIKKRYMYLT
Rosa chinensis	EEILKENPSMCEYMAPSLDARQDMVVVEIPKLGKEAA
Accession:	TKAIKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKL
AEC13058.1	LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
	GARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGA
	AAIIVGSDPLPEVEKPLFELVSAAQTILPDSDGAIDGHL
	REVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWN
	SLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEY
	GNMSSACVLFILDEVRRKSAANGHKTTGEGLEWGVL
	FGFGPGLTVETVVLHSVAA

Cha:cone synthase	MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQAD	39
(CHS)	YPDYYFRITNSDHMTDLKDKFKRMCEKSMITKRHMY
Morus alba var.	LTEEILKENPKMCEYMAPSLDARQDIVVVEVPKLGKE
multicaulis	AAAKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLT
Accession:	KLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENN
AHL83549.1	KGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDG
	AAAVILGADPDTSVERPIFELVSAAQTILPDSEGAIDGH
	LREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWN
	SIFWIAHPGGPAILDQVEAKLGLKQEKLSATRHVLSEY
	GNMSSACVLFILDEVRKKSVEEGKATTGEGLEWGVLF
	GFGPGLTVETIVLHSLPAV

Chalcone synthase	MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQAD	40
(CHS)	YPDYYFRITKSEHLTELKEKFKQMCDKSMIRKRYMYL
Dendrobium	TEEILKENPNICAFMAPSLDARQDIVVTEVPKLAREAS
catenatum	ARAIKEWGQPKSRITHLIFCTTSGVDMPGADYQLTRL
Accession:	LGLRPSVNRIMLYQQGCFAGGTVLRLAKDLAENNAG
ALE71934.1	ARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAA
	AIIVGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLRE
	MGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSI
	FWIAHPGGPAILDQVEIKLGLKEEKLASSRNVLAEYG
	NMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLF
	GFGPGLTVETVVLRSVPIAGAV

Chalcone isomerase	MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTI	41
(CHI)	EGNFIKFTAIGVYLEDVAVASLATKWKGKSSEELLET
Trifolium pratense	LDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVTENCVA
Accession:	HLKSVGTYGDAEVEAMEKFVEAFKPINFPPGASVFYR
PNX83855.1	QSPDGILGVSISIHFFP

Chalcone isomerase	MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGV	42
(CHI)	RGLTIEGNFIKFTGIGVYLEDQAVASLATKWKGKSSEE
Abrus precatorius	LVESLDFFRDIISGPFEKLIRGSKIRQLSGPEYSKKVME
Accession:	NCVAHMKSVGTYGDAEAAGIEEFAQAFKPVNFPPGA
XP_027366189.1	SVFYRQSPDGVLGLSFSQDATIPEEEAAVIKNKPVSAA
	VLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN

Chalcone isomerase	MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGER	43
(CHI)	GLTIDGKFIKFTGIGVYLEDKAVPSLAGKWKDKSSQQ
Arachis duranensis	LLQTLHFYRDIISGPFEKLIRGSKILALSGVEYSRKVME
Accession:	NCVAHMKSVGTYGDAEAEAIQQFAEAFKNVNFKPGA
XP_015942246.1	SVFYRQSPLGHLGLSFSQDGNIPEKEAAVIENKPLSSA
	VLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQH
	N

Chalcone isomerase	MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGER	44
(CHI)	GLEIQGKFIKFTAIGVYLEGDAVASLAVKWKGKSKEE
Cephalotus	LTDSVEFFRDIVTGPFEKFTQVTTILPLTGQQYSEKVSE
follicularis	NCVAFWKSVGIYTDAEAKAIEKFIEVFKEETFPPGSSIL
Accession:	FTQSPNGALTIAFSKDGVIPEVGKAVIENKLLAEGLLE
GAV77263.1	SIIGKHGVSPVAKQCLATRLSELL

Flavanone 3-	MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV	45
hydroxylase (F3H)	RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD
Abrus precatorius	EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
Accession:	MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
XP_027329642.1	WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN
	NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI
	IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI
	HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL
	GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK

Flavanone 3-	MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLER	46
hydroxylase (F3H)	GYIVRATVRDPANLKKVKHLLDLPKADTNLTLWKAD
Camellia sinensis	LNEEGSFDEAIEGCSGVFHVATPMDFESKDPENEVIKP
Accession:	TINGVLSIIRSCTKAKTVKRLVFTSSAGTVNVQEHQQP
AAT66505.1	VFDENNWSDLHFINKKKMTGWMYFVSKTLAEKAAW
	EAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRN
	EGHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSS
	HDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSF
	SSKKLIGMGFEFKYSLEDMFRGAIDTCREKGLLPHSFA
	ENPVNGNKV

Flavanone 3-	MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYI	47
hydroxylase (F3H)	VRATVRDPANMKKVKHLQELEKADKNLTLWKADLT
Nyssa sinensis	EEGSFDEAIKGCSGVFHVATPMDFESKDPENEVIKPTI
Accession:	NGVLSIVRSCVKAKTVKRLVFTSSAGTVNLQEHQQLV
KAA8531902.1	YDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAWE
	ATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEA
	HYSIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSH
	DATIYDLAKMIREKWPEYNVPTELKGIEKDLQTVSFSS
	KKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHE
	TPANANANANANVKKNQNENTEI

Flavanone 3-	MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV	48
hydroxylase (F3H)	RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
Rosa chinensis	EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
Accession:	MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN
XP_024167119.1	WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN
	NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII
	KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH
	EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE
	TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
	VDESSVVGVKVTG

Flavonoid 3′	MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIG	49
hydroxylase (F3′H)	NLPHMGQMPHHSLAAMARTYGPLMHLRLGFVDVIV
Cephalotus	AASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDL
follicularis	VFAPYGPRWRMLRKISSVHLFSGKALDDYRHVRQEE
Accession:	VAVLIRALARAESKQAVNLGQLLNVCTANALGRVML
GAV84063.1	GRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIG
	DFIPALDWLDLQGVAAKMKNLHKRFDTFLTGLLEEH
	KKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDT
	EIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQV
	LKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHP
	STPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDP
	KEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAG
	RRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE
	DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY

Flavonoid 3′	MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPII	50
hydroxylase (F3′H)	GNLPHMGPVPHHSLAALAKVYGPLMHLRLGFVDVV
Theobroma cacao	VAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQ
Accession:	DLVFAPYGPRWRMLRKISSVHLFSGKALDDFRHVRQ
EOY22049.1	DEVGVLVRALADAKTKVNLGQLLNVCTVNALGRVM
	LGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNI
	GDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEH
	KIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKA
	LLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVRKEL
	DSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLS
	LPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWK
	DPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRIC
	AGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLN
	MEEAFGLTLQRAAPLMVHPRPRLSPRAY

Flavonoid 3′	MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPW	51
hydroxylase (F3′H)	PVVGNLPHLGTIPHHSLAAMAKKYGPLMHLRLGFVD
Gerbera hybrida	VVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYN
Accession:	YQDLVFAPYGPRWRMLRKICSVHLFSTKALDDFRHV
ABA64468.1	RQEEVAILARALVGAGKSPVKLGQLLNVCTTNALAR
	VMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIG
	DFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHK
	TGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEI
	KALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARK
	EMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPL
	SLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW
	ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRIC
	VGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM
	EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV

Flavonoid 3′	MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGP	52
hydroxylase (F3′H)	RGWPVLGNLPQLGPKPHHTLHALSRAHGPLFRLRLGS
Phoenix dactylifera	VDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIA
Accession:	YNYQDLVFAPYGPGWRARRKLLNVHLFSGKALEDLR
XP_008791304.2	PVREGELALLVRALRDRAGANELVDLGRAANKCATN
	ALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAG
	VFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDG
	LIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGE
	GAKLTDTDIKALLLNLFTAGTDTTSSTVEWALSELVK
	HPDILRKAQLELDSVVGGDRLVSESDLPNLPFMQAIIK
	ETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNV
	WAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGND
	FGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWD
	LPEGQMPEKLDMEEAYGLTLQRATPLMVHPVPRLAP
	TAYQS

Cytochrome P450	MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI	53
reductase (CPR)	IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE
Camellia sinensis	AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK
Accession:	AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
XP_028084858	DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG
	NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ
	CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
	AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG
	SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE
	SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV
	ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA
	KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
	KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
	WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA
	KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
	DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA
	NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI
	SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL
	LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD
	LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED
	YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL
	QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV
	CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI
	IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
	CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK
	EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR
	DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR
	DVW

Cytochrome P450	MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI	54
reductase (CPR)	IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE
Cephalotus	AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK
follicularis	AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
Accession:	DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG
GAV59576.1	NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ
	CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
	AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG
	SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE
	SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV
	ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA
	KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
	KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
	WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA
	KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
	DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA
	NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI
	SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL
	LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD
	LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED
	YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL
	QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV
	CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI
	IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
	CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK
	EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR
	DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR
	DVW

Cytochrome P450	MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAE	55
reductase (CPR)	LSSMLIENRQFAMIISTSIAVLIGCIVMLLWRRSGGSGS
Brassica napus	SKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGT
Accession:	AEGFAKALGEEARARYEKTRFKIVDLDDYAADDDEY
XP_013706600.1	EEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEG
	DDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDI
	LVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPEL
	DTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKN
	LANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCT
	HLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLL
	DMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTR
	YACLLSSPKKSALLALAAHASDPTEAERLKHLASPAG
	KDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAV
	APRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIH
	KGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSD
	SKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVL
	FFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREGP
	TKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKG
	MARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGR
	YLRDVW

Flavonoid 3′, 5′-	MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPG	56
hydroxylase	PKGWPFVGALPLLGTMPHVALAQMAKKYGPVMYLK
(F3′5′H)	MGTCGMVVASTPDAARAFLKTLDLNFSNRPPNAGAT
Cephalotus	HLAYNAQDMVFADYGPRWKLLRKLSNLHMLGGKAL
follicularis	EDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLTY
Accession:	AMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTS
GAV62131	AGYFNIGDFIPSIAWMDLQGIERGMKKLHKRFDALLT
	KMFEEHMATAHERKGNPDLLDIVMANRDNSEGERLT
	TTNIKALLLNLFSAGTDTSSSIIEWSLAEMLKNPSILKR
	AHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHP
	STPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP
	EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGR
	RICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMD
	EAFGLALQKAVPLAAIVSPRLAPTAYVV

Flavonoid 3′, 5′-	MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGA	57
hydroxylase	LPFIGPMPHSGLALLARRYGPIMFLKMGIRRVVVASSS
(F3′5′H)	TAARTFLKTFDSHFSDRPSGVISKEISYNGQNMVFADY
Dendrobium	GPKWKLLRKVSSLHLLGSKAMSRWAGVRRDEALSMI
moniliforme	QFLKKHSDSEKPVLLPNLLVCAMANVIGRIAMSKRVF
Accession:	HEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGWL
AEB96145	DPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQ
	GNPDLLDLVVASEVKGEDGEGLCEDNIKGFISDLFVA
	GTDTSAIVIEWAMAEMLKNPSILRRAQEETDRVIGRH
	RLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEP
	CEVEGYHIPGETWLLVNIWAIGRDPDVWENPLVFDPE
	RFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMV
	MVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPK
	AVPLAVMATPRLPAAAYGLL

Dihydroflavonol 4-	MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVR	58
reductase (DFR)	DPDNEKKVKHLVELPKAKTHLTLWKADLSDEGSFDE
Acer palmatum	AIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIM
Accession:	KACKKAKTVKRLVFTSSAGTVDVEEHKKPVYDENSW
AWN08247.1	SDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEENSI
	DFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAII
	KQGNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATI
	LELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKIL
	DLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHV
	AAKGLIPNNSDEIHVAAAEKTTATA

Dihydroflavonol 4-	MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV	59
reductase (DFR)	RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD
Abrus precatorius	EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
Accession:	MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
XP_027329642.1	WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN
	NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI
	IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI
	HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL
	GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK

Dihydroflavonol 4-	MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRA	60
reductase (DFR)	TVRDPTNLKKVKPLLDLPRSNELLSIWKADLDGIEGSF
Dendrobium	DEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGI
moniliforme	LRSCKNAGSVQRVIFTSSAGTVNVEEHQAAAYDETC
Accession:	WSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFVKD
AEB96144.1	NHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHY
	SILKQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDST
	IYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKL
	MELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVL
	AAEKFEEVKEQIAVK

Dihydroflavonol 4-	MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV	61
reductase (DFR)	RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
Rosa chinensis	EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
Accession:	MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN
XP_024167119.1	WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN
	NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII
	KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH
	EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE
	TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
	VDESSVVGVKVTG

Leucoanthocyanidin	MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTT	62
reductase (LAR)	FLLVRSLDKGAIPSKSKTINSLHDKGAILIHGVIEDQEF
Camellia sinensis	VEGILKDHKIDIVISAVGGANILNQLTIVKAIKAVGTIK
Accession:	RFLPSEFGHDVDRANPVEPGLAMYKEKRMVRRLIEES
XP_028127206.1	GVPYTYICCNSIASWPYYDNTHPSEVIPPLDRFQIYGD
	GTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHFRPSC
	NFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAE
	NIIPQSIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPD
	ETFRTMDECFDDFVMKMDRWN

Leucoanthocyanidin	MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGK	63
reductase (LAR)	STYILSRAAARCPSKARAIKALEDQGAISIHGSVNDQE
Coffea arabica	FMEKTLKEHEIDIVISAVGGGNLLEQVILIRAMKAVGT
Accession:	IKRFLPSEFGHDVDRAEPVEPGLTMYNEKRRVRRLIEE
XP_027097479.1	SGVPYTYICCNSIASWPYYDNTHPSEVSPPLDQFQIYG
	DGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHFRPS
	CNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEE
	NIIPQSVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYP
	DEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRL
	VDVLTAPKLCA

Leucoanthocyanidin	MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEA	64
reductase (LAR)	SLDSGRPTYILARSSSNSPSKASTIKFLQDRGATVIYGSI
Theobroma cacao	TDKEFMEKVLKEHKIEVVISAVGGGSILDQFNLIEAIR
Accession:	NVDTVKRFLPSEFGHDTDRADPVEPGLTMYEQKRQIR
ADD51357.1	RQIEKSGIPYTYICCNSIAAWPYHDNTHPADVLPPLDR
	FKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKTVH
	FQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQM
	AKEMRIPQSVVAALTHDIFINGCQINFSLDKPTDVEVC
	SLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAI
	FVPTAKPGALPITAICT

Leucoanthocyanidin	MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLP	65
reductase (LAR)	TYVLVRPGPSRPSKSDTIKSLKDRGAIILHGVMSDKPL
Fragaria x	MEKLLKEHEIEIVISAVGGATILDQITLVEAITSVGTVK
ananassa	RFLPSEFGHDVDRADPVEPGLTMYLEKRKVRRAIEKS
Accession:	GVPYTYICCNSIASWPYYDNKHPSEVVPPLDQFQIYGD
ABH07785.2	GTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFRPSS
	NLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAEN
	RIPESIVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEE
	SFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVE
	PMAITATCA

Anthocyanin	MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRL	66
dioxygenase (ANS)	NEELTSMGNVFEEEKKEEGSQVPTIDIKDIASEDPEVR
Chenopodium	GKAIQELKRAAMEWGVMHLVNHGISDELIDRVKVAG
quinoa	QTFFELPVEEKEKYANDQASGNVQGYGSKLANSASG
Accession:	RLEWEDYYFHLSYPEDKRDLSIWPETPADYIPAVSEYS
XP_021735950.1	KELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQ
	FKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPG
	LQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSIL
	HRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEE
	PARYPPRTFAQHVQYKLFRKTQGPQTTITKN

Anthocyanin	MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDD	67
dioxygenase (ANS)	SLGDALEEIKKTEEGPQIPIVDLRGFDSGDEKERLHCM
Iris sanguinea	EEVKEAAVEWGVMHIVNHGIAPELIERVRAAGKGFFD
Accession:	LPVEAKERYANNQSEGKIQGYGSKLANNASGQLEWE
QCI56004.1	DYFFHLIFPSDKVDLSIWPKEPADYTEVMMEFAKQLR
	VVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKI
	NYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQV
	FHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVL
	HRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKR
	SPAKYPPRTFAQHIQHKLFKKAQEQLAGGVHIPEAIQN

Anthocyanin	MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGD	68
dioxygenase (ANS)	VFEEEKKLEGPQIPVVDLMGLEWENEEVFKKVEEDM
Magnolia sprengeri	KKAASEWGVMHIFNHGISMELMDRVRIAGKAFFDLPI
Accession:	EEKEMYANDQASGKIAGYGSKLANNASGQLEWEDYF
AHU88620.1	FHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQLRGLV
	TKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYY
	PKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFD
	DKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLV
	NKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFT
	PRTFSQHVRQKLFKKQQDALENLKSE

Anthocyanin	MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIG	69
dioxygenase (ANS)	NVFEEEKKEGPEVPTIDLTEIESEDEVVRARCHETLKK
Prosopis alba	AAQEWGVMNLVNHGIPEELLNQLRKAGETFFSLPIEE
Accession:	KEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFH
XP_028787846.1	LVFPEDKCDLSIWPRTPSDYIEVTSEYARQLRGLATKI
	LGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPIC
	PQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQ
	WITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNK
	EKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRT
	FAQHIQHKLFRKCQEGLSK

Anthocyanidin-3-	MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPN	70
O-glycotransferase	THFSFLNTSQSNNSIFSSDVYNRQPNLKAHNVWDGVP
(3GT)	EGYVFVGKPQESIELFVKAAPETFRKGVEAAVAETGR
Cephalotus	KVSCLVTDAFFWFAAEIAGELGVPWVPFWTAGPCSLS
follicularis	THVYTDLIRKTIGVGGIEGREDESLEFIPGMSQVVIRDL
Accession:	QEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEEL
GAV66155.1	DLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLP
	WLDQQKVASVAYISFGTVMAPSPPELVALAEALEASK
	IPFIWSLGEKLKVHLPKGFLDKTRTHGIVVPWAPQSDV
	LENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQ
	RLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLILS
	QARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALL
	NLVRLSI

Anthocyanidin-3-	MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRR	71
O-glycotransferase	LAAASPNTLFSFFSTSQSNNSLFSNTITNLPRNIKVFDV
(3GT)	ADGVPDGYVFAGKPQEDIELFMKAAPHNFTTSLDTCV
Prunus cerasifera	AHTGKRLTCLITDAFLWFGAHLAHDLGVPWLPLWLS
Accession:	GLNSLSLHVHTDLLRHTIGTQSIAGRENELITKNVNIPG
AKV89253.1	MSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN
	AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAA
	SPPLPEAPTAADDVTGCLSWLDKQKAASSVVYVSFGS
	VARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLN
	ELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWN
	SLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTV
	EDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLL
	AQQPVEPKGSSAQNFKLLLDVISGSTKV

Anthocyanidin-3-	MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFS	72
O-glycotransferase	FFNSAVSNSTLFNNGVLDSYDNIRVYHVWDGTPQGQ
(3GT)	AFTGSHFEAVGLFLKASPGNFDKVIDEAEVETGLKISC
Scutellaria	LITDAFLWFGYDLAEKRGVPWLAFWTSAQCALSAHM
baicalensis	YTHEILKAVGSNGVGETAEEELIQSLIPGLEMAHLSDL
Accession:	PPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEIDP
A0A482AQV3	IITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLD
	SQTRPKSVVYISFGTVITPPENELAALSEALETCNYPFL
	WSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLA
	HRSVGVFVTHCGWNSILESICSGVPLICRPFFGDQKLN
	SRMVEDSWKIGVRLEGGVLSKTATVEALGRVMMSEE
	GEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEIINAP
	QSS

Anthocyanidin-3-	MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPH	73
O-glycotransferase	AVFSFFSTSQSNASIFHDSMHTMQCNIKSYDISDGVPE
(3GT)	GYVFAGRPQEDIELFTRAAPESFRQGMVMAVAETGRP
Vitis vinifera	VSCLVADAFIWFAADMAAEMGLAWLPFWTAGPNSLS
Accession:	THVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFRDL
P51094	QEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL
	DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQ
	WLKERKPTSVVYISFGTVTTPPPAEVVALSEALEASRV
	PFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAE
	VLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFG
	DQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQIL
	SQEKGKKLRENLRALRETADRAVGPKGSSTENFITLV
	DLVSKPKDV

Acetyl-CoA	MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHS	74
carboxylase (ACC)	VITKVLICNNGIAAVKEIRSIRKWAYETFGDERAIEFTV
Ustilago maydis	MATPEDLKVNADYIRMADQYVEVPGGSNNNNYANV
521	DLIVDVAERAGVHAVWAGWGHASENPRLPESLAASK
Accession:	HKIIFIGPPGSAMRSLGDKISSTIVAQHADVPCMPWSG
XP_011390921.1	TGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAE
	KIGYPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLG
	EVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGR
	DCSVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAK
	LVGYVSAGTVEWLYSPESGEFAFLELNPRLQVEHPTT
	EMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRG
	NEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDT
	GFKPGMGALTELNFRSSTSTWGYFSVGTSGALHEYAD
	SQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVE
	YLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADL
	AVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIK
	TVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRTLV
	QLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSK
	TCLIEQENDPTQIRSPSPGKLVRFLVDSGDHVKANQAI
	AEIEVMKMYLPLVAAEDGVVSFVKTAGVALSPGDIIG
	ILSLDDPSRVQHAKPFAGQLPDFGMPVIVGNKPHQRY
	TALVEVLNDILDGYDQSFRMQAVIKELIETLRNPELPY
	GQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEFPA
	ARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYA
	GGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELR
	LQADGDLDKVVALQTSRNGINRKNALLLTLLDKHIKG
	TSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVS
	LDADMPSLADRSAQMQAILRGSVTSSKYGGDDEYHA
	PSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALC
	TYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQL
	PRSASSLKERERQVSISDLSMMDNNRRARPIRELRTGA
	MTSCADVADIPELLPKVLKFFKSSAGASGAPINVLNV
	AVVDQTDFVDAEVRSQLALYTNACSKEFSAARVRRV
	TYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALA
	YQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADT
	RFFVRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEV
	ALGQPEYRTADASHIFMSFIYQLDVSLVDVQKAIAGFL
	ERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNET
	GLVVRYETYEETVADDGSVILRGIEPQGKDATLNAQS
	AHFPYTTKVALQSRRSRAHALQTTFVYDFIDVLGQAV
	RASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRA
	PGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQ
	AGSFGPVEDRFFAAASKLARELGVPRLYISANSGARIG
	LATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQA
	KAPNSVMTKPVQAADGSVHNIITDIIGKPQGGLGVEC
	LSGSGLIAGETSRAKDQIFTATIITGRSVGIGAYLARLG
	ERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQ
	IMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGP
	LPIMPTTDSWDRAVTYQPPRGPYDPRWLINGTKAEDG
	TKLTGLFDEGSFVETLGGWATSVVTGRARLGGIPVGV
	IAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNS
	AYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMYD
	EILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVV
	VDSAINDNGMIEMSADVNSARGGVLEASGLVEIKYRA
	DKQRATMERLDSVYAKLSKEAAEATDFTAQTTARKA
	LAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRS
	ALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLK
	HVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLA
	ASKQLKAQYILAQISTLDPELRAQLAASLK

Acetyl-CoA	MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHT	75
carboxylase (ACC)	VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF
Hesseltinella	TVMATPEDLKANAEYIRMADNYIEVPGGTNNNNYAN
vesiculosa	VELIVDVAERTGVHAVWAGWGHASENPRLPEMLAKS
Accession:	KNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTMGW
ORX57605.1	SGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLA
	SAEKIGFPVMIKASEGGGGKGIRKVEDPSTFKQAFAQ
	VQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLF
	GRDCSVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLG
	KLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPT
	TEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN
	SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPD
	AGFKPSGGVMQELNFRSSTNVWGYFSVVSSGAMHEY
	ADSQFGHIFAYGENRQQARKNMVIALKELSIRGDFRT
	TVEYIIRLLETPDFTDNTINTGWLDMLISKKLTAERPDT
	MLAVFCGAVTKAHLASVECWQQYKNSLERGQIPSKE
	SLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKT
	QVGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMID
	GKTCLLEKESDPTQLRSPSPGKLVSLLLENGDHIRTGQ
	AYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGD
	IIGILSLDDPSRVKHALPFTGQVPKYGLPHLTGDKPHQ
	RFTHLKQTLEYVLQGYDNQGLIQTIVKELSEVLNNPEL
	PYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKG
	FPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIA
	ASYSNGLKQHEHSVYVDLMEQYYNVEVLFNSNQSRD
	EEVILALRDQHKDDLEKVINIILSHAKVNIKNNLILMLL
	DIIYPATSSEALDRCFLPILKHLSEIDSRGTQKVTLKAR
	EYLILCQLPSLEERQSQMYNILKSSVTESVYGGGTEYR
	TPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALE
	VYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSA
	GGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTL
	EELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMEN
	ILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALR
	RITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALA
	YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
	RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE
	IVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDR
	HGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNV
	SGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQP
	LSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAI
	HNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA
	IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDI
	TFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSG
	ARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEY
	DALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGL
	GVENLRGSGLIAGATSRAYDDIFTITLVTCRSVGIGAY
	LVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNL
	QLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPE
	CRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQ
	ENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGI
	PMGVVAVETRTVENIVPADPANADSQEQVVMEAGGV
	WFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGG
	QRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGEL
	RGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGI
	VEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKE
	QADELIAQVEAREQALLPVYQQVAIQFADLHDRSGR
	MEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRK
	MDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVA
	WFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAV
	VEGFSKLMESLSVDQRKEVLHKLATRF

Acetyl-CoA	MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVF	76
carboxylase (ACC)	MHGLGSSTSFWEAPFSRSNLSSRFRLIRYDFDGHGLSP
Rhodotorula	VSLLDAADDGAMIPLVDLVEDLAAVMEWTGVDKVA
toruloides	GIVGHSMSGLVASTFAAKYPQKVEKLVLLGAMRSLN
NBRC10032	PTVQTNMLKRADTVLESGLSAIVAQVVSAALSDKSKQ
Accession:	DSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDY
GEM08739.1	STIKAKTLVVSGESDYLSNKETTEALVNDIPGAKEVQ
	MDGVGHWHAVEDPAGLAKILDGFFLQGKFSGEAKA
	VNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPPS
	NVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKW
	AYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVE
	VPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGH
	ASENPRLPESLAASKHKIVFIGPPGSAMRSLGDKISSTI
	VAQHAEVPCMDWSGQGVDQVTQSLEGYVTVADDVY
	QQACVHDADEGLARASRIGYPVMIKASEGGGGKGIR
	KVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLE
	VQVLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAK
	PDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDK
	FAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGV
	PLHRIRDIRTLYGKAPNGSSEIDFEFENPESAKTQRKPS
	PKGHVVAVRITAENPDAGFKPSMGTLQELNFRSSTNV
	WGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKN
	MVVALKELSIRGDFRTTVEYLIKLLETDAFEQNTITTA
	WLDSLISARLTAERPDTTLAIICGAVTKAHLASEANIA
	EYKRILEKGQSPPKELLATVVPLEFVLEDVKYRATASR
	SSPSSWSIYVNGSNVSVGIRPLADGGLLILLDGRSYTC
	YAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSPGKL
	VRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQ
	FIKQPGATLEAGDILGILSLDDPSRVHHAKPFDGQLPA
	LGLPSIIGTKPHQRFAYLKDVLSNILMGYDNQAIMQSS
	IKELISVLRNPELPYGEANAVLSTLSGRIPAKLEQTLRQ
	YIDSAHESGAEFPSAKCRKAIDTTLEQLRPAEAQTVRN
	FLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKP
	FSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKN
	SLVLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKA
	TTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPT
	VYGEPGHDRTPRGEVLKDVIDSRFTVFDVLPSFFQHQ
	DQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEP
	ATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLT
	FLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFP
	DSNKGKLQQQPAASSSQEQWNVINVALTVPASAQVD
	EDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQ
	YPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSN
	FHLEPCPVENRQVHIYYATAKGNSSDCRFFVRALVRP
	GRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRR
	AADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRF
	WRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYE
	AYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEW
	LQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGK
	TAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVG
	WIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFY
	AVTQLARQLGLPRVYLSANSGARLGIAEELVDLFSVA
	WADSSRPEKGFKYLYLTAEKLGELKNKGEKSVITKRI
	EDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAY
	DDIFTITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTG
	AGALNKVLGREVYSSNLQLGGTQIMYKNGVSHLTAA
	NDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSI
	DYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSF
	QETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIP
	ADPANPLSNEQKIMEAGQVWYPNSSFKTGQAIFDFNR
	EGLPLIIFANWRGFSGGQQDMFDEVLKRGSLIVDGLS
	AYKQPVFVYIVPNGELRGGAWVVLDPSINAEGMMEM
	YVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTY
	HALRVKSTDASLSPTDAAQAKTELAAREKQLMPIYQQ
	VALQFADSHDKAGRILSKGCAREALEWSNARRYFYA
	RLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQ
	GVDLNNDLAAAAAFEQGAAAITERVKLARATTVAST
	LAQLAQDDKEAFAASLQQVLGDKLTAADLARILA

Malonyl-CoA	MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVAR	77
synthase (matB)	SGRMANVLVARGVKPGDRVAAQAEKSVAALVLYLA
Rhodopseudomonas	TVRAGAVYLPLNTAYTLHELDYFIGDAEPKLVVCDPA
palustris	KREGIAALAQKVGAGVETLDAKGQGSLSEAAAQASV
Accession:	DFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNLAS
WP_011661926.1	NSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLF
	ARASMIFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQ
	SDGLTKEAARHMRLFISGSAPLLADTHREWASRTGHA
	VLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSL
	RVVDPETGAELSPGEIGMIEVKGPNVFQGYWRMPEKT
	KAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGG
	FNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAV
	VVRDKGASVDEAAVLGALQGQLAKFKMPKRVLFVD
	DLPRNTMGKVQKNVLREAYAKLYAK

Malonyl-CoA	MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCS	78
synthase (matB)	ARITHVLVKLGVSPGDRVAVQVEKSAQALMLYLGCL
Rhizobium	RAGAVYLPLNTAYTPAELEYFLGDATPKLVVVSPCAA
sp. BUS003	EQLEPLARRVGTRLLTLGVNGDGSLMDMASLEPVEF
Accession:	ADIERKADDLAAILYTSGTTGRSKGAMLTHDNLLSNA
NKF42351.1	QTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAG
	GAIYLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQD
	ERLNKANTRHMRLFISGSAPLLAETHRLFEEYTGHAIL
	ERYGMTETNMITSNPCDGARVPGTVGYALPGVSVRIT
	DPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKE
	EFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNI
	YPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVG
	QPKAHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNT
	MGKVQKNVLRDRYRDLYLK

Malonyl-CoA	MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMT	79
synthase (matB)	GTARYANVLVGLGVKPGDRVAVQVEKSAGAIFLYLA
Ochrobactrum sp.	CVRAGAVFLPLNTAYTLTEIEYFLGDAEPALVVCDPA
3-3	RRDGITEVAKKTGVPAVETLGKGQDGSLFDKAAAAP
Accession:	ETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDNLA
WP_114216069.1	SNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTI
	LVAGASMLFLPKFDADKVFELMPRATTMMGVPTFYV
	RLVQDARLTREATKHMRLFISGSAPLLAETHKLFREK
	TGVSILERYGMTETNMNTSNPYDGDRVAGTVGFPLPG
	VALRVADPETGAAIPQGEIGVIEVKGPNVFSGYWRMP
	EKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLV
	ISGGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGE
	GVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVI
	VVDDLPRNTMGKVQKNLLRDAYKGLYGG

Malonate	MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT	80
transporter (matC)	LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT
Rhizobiales	TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV
bacterium	SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG
Accession:	GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA
MBN8942514.1	AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS
	GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG
	AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
	AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY
	VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
	SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV
	SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY
	GGIVVAAVPALAWLVLVVPGFG

Malonate	MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMI	81
transporter (matC)	IGMKTNEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL
Rhizobium	VECAVRLVRGRIGLIPWVMFLVAAIITGFGALGPAAV
leguminosarum	AILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSPI
Accession:	SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVFF
AAC83457.1	VFGGARVMKHDPASLGPLPELHPEGVSASIRGHGGTP
	AKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIG
	ALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSW
	STVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLV
	ALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAI
	GVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVL
	RQLLIYSALIAIIGPIVAWLVFVVPGLV

Malonate	MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLII	82
transporter (matC)	GMKPADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWL
Agrobacterium vitis	VERSVRMVRGRVGWIPWVMFLVAAIITGFGALGPAA
Accession:	VAILAPVALSFAVQYRIHPVLMGLMVIHGAQAGGFSPI
WP_180575084.1	SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLIFFI
	FGGLSILKQRSSVKGPLPELHPEGISASIKGHGGTPAKP
	FREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL
	VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTV
	LLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALL
	LCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGV
	VAAIAISTTIVDTSPFSTNGALVVANAPDDQRDKVMR
	QMLIYSALIALIGPVIAWLVFVVPGII

Malonate	MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLI	83
transporter (matC)	IGMKTSDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL
Neorhizobium sp.	VECAVRMVRGHVAWIPWVMFVVAAITGFGALGPAA
Accession:	VAILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSP
WP_105370917.1	ISVYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVF
	FVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGT
	PAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGI
	GALVFKFNVGLVAITVAVALALISPKTQKAAVDKVS
	WSTVLLIAGIITYVGVLEKAGTVNYVANGISSLGMPLL
	VALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHIS
	AVGVVAAIAISTTIVDTSPFSTNGALVVANAPDETREQ
	VLRQLLIYSALIAIIGPVVAWLVFVVPGLV

Malonate CoA-	MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALL	84
transferase (MdcA)	ETVIDRGDRVCLEGNNQKQADFLSKSLSSCNPDIVNG
Moraxella	LHIVQSVLALPSHIDVFERGIASKVDFSFAGPQSLRLAQ
catarrhalis	LVQAQKITIGAIHTYLELYGRYFIDLTPNVALITAHAA
Accession:	DKRGNLYTGANTEDTPAIVEATTFKSGIVIAQVNEIVD
WPO64617969.1	ELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITEIQ
	ILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTY
	AESLGLKGEICTHWALNPHPTLIPAIESGFIHSVHSFGS
	EVGMENYVKARSDVFFTGADGSMRSNRAFSQTAGLY
	ACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSD
	PHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVE
	TYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDD
	VTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLG
	RDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKS
	VKDLVRWSDRLYEPPSRFRNW

Malonate CoA-	MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEI	85
transferase (MdcA)	PVDRIIDLLEAVIQPGDRVCLEGNNQKQADFLSESLAD
Dechloromonas	CDPARINHLSMVQSVLALPSHVDLFERGLATRLDFSFS
aromatica	GPQGARLAKLVQEQRIEIGAIHTYLELFGRYFMDLTPN
Accession:	VALIAAQAADAEGNLYLGPNTEDTPAIVEATAFKGGI
WP_011289741.1	VIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPL
	FTRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIG
	FDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPA
	IEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMR
	SNRAFSQTAGLYACDMFIGSTLQMDLAGNSSTATLGR
	ITGFGGAPNMGSDPHGRRHASPAWLKAGREAYGPQA
	IRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASM
	GSDLPPIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAI
	RGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDP
	RQASRDLLAARSIKDLVRCSGGLYAPPSRFRNW

Malonate CoA-	MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVV	86
transferase (MdcA)	ALLEAVIEPGDRVCLEGNNQKQADFLARCLTEVDPAR
Pseudomonas	VHDLHMVQSVLSLAAHLDVFERGIAKRLDFSFSGPQA
cissicola	ARLAGLVSEGRIEIGAIHTYLELFGRYFIDLTPRIALVT
Accession:	AQAADRHGNLYTGPNTEDTPVIVEATAFKGGIVIAQV
WP_078590875.1	NEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPA
	QISEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIE
	LLLPTYAQSLGLKGKICRHWALNPHPALIPAIESGFVQ
	SVHSFGSELGMENYIAARPDIFFTGADGSMRSNRALS
	QTAGLYACDMFIGSTLQIDLQGNSSTATRDRIAGFGG
	APNMGSDARGRRHASAAWLKAGREAATPGEMPRGR
	KLVVQMVETFREHMAPAFVDRLDAWELAERANMPL
	PPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGV
	SGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRD
	ATRDLLAARTVKDLVRWSGGLYDPPKRFRNW

Malonate CoA-	MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTE	87
transferase (MdcA)	KIVEALTALIFPGDRVVIEGNNQKQASFLSKALSQVNP
Geobacillus	EKVNGLHIIMSSVSRPEHLDLFEKGIARKIDFSYAGPQS
subterraneus	LRMSQMLEDGKLVIGEIHTYLELYGRLFIDLTPSVALV
Accession:	AADKADASGNLYTGPNTEETPTLVEATAFRDGIVIAQ
WP_184319829.1	VNELADELPRVDIPGSWIDFVVAADHPYELEPLFTRDP
	RLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIE
	LLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVE
	SIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQ
	VAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGA
	PNMGHDPRGRRHSSPAWLDMITSDHPAAKGRKLVVQ
	MVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGE
	DVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLE
	RDPRKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSI
	EELVEWSEGLYEPPARFRSW

Pantothenate kinase	MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADE	88
(CoaX)	LAVLLQGLMGTHPLLGMELGEGIDGIAICSTVPAVLH
Streptomyces sp.	ELREVSRRYYGDVPAILVEPGVKTGVPILMDNPKEVG
CLI2509	TDRIINAVAAQHLYGGPAIVVDFGTATTFDAVSARGE
Accession:	YTGGVIAPGIEISVEALGLRGAQLRKIELARPRSVIGKS
WP_095682415.1	TVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVT
	VIATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERN
	AGRR

Pantothenate kinase	MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQA	89
(CoaX)	PAELLKGLTFRLERLNPDFIGVSSVLGQAVNNHVAESL
Streptomyces	ERLQKPFEFAQVHAKHALMSSDYNPAQLGVDRWLQ
cinereus	MLGIIEPSKKQCVIGCGTAVTIDLVDQGHHLGGYIFPSI
Accession:	YLQRESLFSGTRQISIIDGTFDSIDSGTNTQDAVHHGIM
WP_188874884.1	LSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI
	RQDLVLAGLQRFFAAKNNTKNQN

Pantothenate kinase	MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTAD	90
(CoaX)	ELAVLMQGLMGRQPGGAGRERVDGLAICSSVPAVLH
Kitasatospora	ELREVTRRYYGDLPAVLVAPGVKTGVHVLMDNPKEV
kifunensis	GADRIVNALAANHLYGGPCIVVDFGTATTFDAINERG
Accession:	DYVGGAIAPGIEISVEALGVRGAQLRKIELAKPRNVIG
WP_184936930.1	KNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPE
	DVQVIATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYE
	RNTAS

glutamyl-tRNA	MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQA	91
reductase (hemA)	LPMVQGGVVLSTCNRTEIYLSVEEQDNLREALIRWLC
Citrobacter	EYHNLNEEDLRNSLYWHQDNDAVSHLMRVASGLDS
freundii	LVLGEPQILGQVKKAFADSQKGHQNASALERMFQKS
Accession:	FSVAKRVRTETDIGSSAVSVAFAACTLARQIFESLSTV
NTY05430.1	TVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQ
	VLADEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKG
	MVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAY
	LYSVDDLQSIISHNLAQRKAAAVEAETIVEQEASEFMA
	WLRAQGASDTIREYRSQSEQIRDELTAKALAALQQGG
	DAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDSER
	LNILRDSLGLE

glutamyl-tRNA	MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQP	92
reductase (hemA)	LVQGGVVLSTCNRTELYLSVEQQENLQEQLVKWLCD
Pseudomonas	YHHLSADEVRKSLYWHQDNAAVSHLMRVASGLDSL
reactans	VVGEPQILGQVKKAFAESQHGQAVSGELERLFQKSFS
Accession:	VAKRVRTETDIGASAVSVAFAACTLARQIFESLSDVSV
NWA43040.1	LLVGAGETIELVARHLREHKVRHMMIANRTRERAQV
	LASEVGAEVITLQDIDARLADADIIISSTASPLPIIGKGM
	VERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYL
	YSVDDLHSIIQNNMAQRKAAAVQAESIVEQESSNFMA
	WLRSQGAVEIIRDYRSRADLVRAEAEAKALAAIAQGA
	DVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDVERL
	QILRDSLGLDQQ

glutamyl-tRNA	MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQ	93
reductase (hemA)	QPAVRGGVVLSTCNRTELYLSMEDKENSHEQLIRWLC
Gamma-	QYHQIEPNELQSSIYWHQDNQAVSHLMRVASGLDSL
proteobacteria	VLGEPQILGQVKKAFADSQNYDSLSSELERLFQKSFSV
Accession:	AKRVRTETQIGANAVSVAFAACTLARQIFESLSSLTILL
WP_193016510.1	VGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE
	VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVER
	ALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLYSV
	DDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWL
	RAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADA
	EKVIQQLSHQLMNRLIHTPTKSLQQAASDGDIERLNLL
	RESLGITHN

5-aminolevulinic	MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITI	94
acid synthase	PPNATVEMCPHAKAARDAARIAEDLAAAAASKQQPA
(ALAS)	KKAGGCPFHAAQAQAQAKPAAAPKETVATADKKGK
Schizophyllum	SPRAAGGFDYEKFYEEELDKKHQDKSYRYFNNINRLA
commune H4-8	ARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLET
Accession:	MHRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLH
XP_003036856.1	RKEGALVFTSCYVANDATLSTLGSKMPGCVIFSDRMN
	HASMIQGIRHSGTKKVIFEHNDLADLEKKLAEYPKETP
	KIIAFESVYSMCGSIGPIKEICDLAEKYGAITFLDEVHA
	VGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMD
	RVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIF
	TTSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREV
	KRRFAELDIPVVPGPSHIVPVLVGDAALAKQASDKLL
	AEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDH
	LIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQD
	FVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAVV
	AARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQ
	SSGLKLPVEEMTMGQTIAVAA

5-aminolevulinic	MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTAL	95
acid synthase	TERATKCPVMGPALNVRSKEIVAGYASVAANSDVALI
(ALAS)	HKEKGVFPPPGATVEMCPHASAARAAARMADDLAA
Crassisporium	AAEKKKGHFTSAAPRDEAAQAAAAGCPFHVKAAAD
junariophilum	AAAARKAAAAPAPVKAKEDGGFNYESFYVNELDKK
Accession:	HQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCA
KAF8165006.1	NDYLGMGNNPVVLETMHRTLDKYGHGAGGTRNIAG
	NGAMHLSLEQELATLHRKPAALVFSSCYVANDATLST
	LGAKLPGCIFFSDTMNHASMIQGMRHSGAKRVLFKH
	NDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKE
	ICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDY
	DAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGG
	YIAGSDDFVDMIRSYAPGFIFTTSLPPATVAGARASVV
	YQKHYVGDRQLKQVNVREVKRRFAELDVPVVPGPSH
	IVPVLVGDAALAKAASDKLLAEHNIYVQSINYPTVAR
	GEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA
	DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLD
	GTSPRTLRNGEAAVVDAMAVGQARAVFDNLLGPISG
	KLQSERSVLASSTPAAANPARPAARKVVKMKTGGVP
	MSEDIPLPPPDVSASA

5-aminolevulinic	MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSIS	96
acid synthase	ILTERATKCPVMGPALNVRSKEITAGYASVAGSSEVD
(ALAS)	QIHKQQGVTVPVNATVEMCPHASAARAAARMADDL
Dendrothele	AAAAAQKKVGSGASSAKAAAAGCPFHKSVAAGASA
bispora CBS	STASKPSAPIHKASVPGGFDYDNFYNNELEKKHKDKS
962.96	YRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLG
Accession:	MGNNPVVLETMHRTLDKYGHGAGGTRNIAGNGALH
THV05492.1	LGLEQELAALHRKEAALVFSSCYVANDATLSTLGSKL
	PGCILFSDKMNHASMIQGMRHSGAKKVIFNHNDLEDL
	ENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEK
	YGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAA
	GKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSM
	DFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYL
	GDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGD
	AALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRIT
	VTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAG
	GRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQR
	FLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD
	DELKVPLPTLVAAAA

5-aminolevulinic	MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHAL	97
acid synthase	WHSPKGKRDVVIWCSNDYLGMGQHPKVVGAMVETA
(ALAS)	TRVGTGAGGTRNIAGTHHPLVQLEAELADLHGKEASL
Bradyrhizobium	LFTSGYVSNQTGIATIAKLIPNCLILSDELNHNSMIEGIR
japonicum	QSGCERVVFRHNDLADLEEKLKAAGPNRPKLIACESL
Accession:	YSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMY
A0A0A3YXD2	GPRGGGIAERDGVMHRIDILEGTLAKAFGCLGGYIAA
	NGQIIDAVRSYAPGFIFTTALPPAICSAATAAIRHLKTS
	NWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFI
	GDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRI
	TPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKSLAAE

Cytochrome b5	MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLE	98
Petunia x hybrida.	EHPGGEEVLIESAGKDATKEFQDIGHSKAAKNLLFKY
Accession:	QIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAY
AAD10774.1	VIKEDPKPKYLTFVEYLLPFLAAAFYLYYRYLTGALQ
	F

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
	DHL	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 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.

2. The engineered host cell of claim 1, wherein the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.

3. The engineered host cell of claim 1, wherein 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.

4. The engineered host cell of claim 1, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

5. The engineered host cell of claim 1, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

6. The engineered host cell of claim 1, wherein the one or more genetic modifications cause reduction of formation of byproducts.

7. The engineered host cell of claim 1, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

8. The engineered host cell of claim 1, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

9. The engineered host cell of claim 1, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (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.

10. The engineered host cell of claim 1, wherein the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

11. The engineered host cell of claim 1, wherein the engineered host cell is E. coli.

12. The engineered host cell of claim 1, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

13. The engineered host cell of claim 1, wherein 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.

14. The engineered host cell of claim 1, wherein the flavonoid is catechin.

15. 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.

16. The method of claim 15, wherein the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation.

17. The method of claim 15, wherein 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.

18. The method of claim 15, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

19. The method of claim 15, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

20. The method of claim 15, wherein the one or more genetic modifications cause reduction in the production of byproducts.

21. The method of claim 15, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification for expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

22. The method of claim 15, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

23. The method of claim 15, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (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.

24. The method of claim 15, wherein the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

25. The method of claim 15, wherein the engineered host cell is E. coli.

26. The method of claim 15, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

27. The method of claim 15, wherein 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.

28. The method of claim 15, wherein the flavonoid is catechin.

29. 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, by the engineered host cells.

30. The plurality of engineered host cells of claim 29, wherein the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation.

31. The plurality of engineered host cells of claim 29, wherein 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.

32. The plurality of engineered host cells of claim 29, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

33. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

34. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications cause a reduction in the formation of byproducts.

35. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

36. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

37. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (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.

38. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

39. The plurality of engineered host cells of claim 29, wherein at least one of the engineered host cell is E. coli.

40. The plurality of engineered host cells of claim 29, wherein the one or more genetic modifications decrease fatty acid biosynthesis.

41. The plurality of engineered host cells of claim 29, wherein at least one of 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.

42. The plurality of engineered host cells of claim 29, wherein the flavonoid is catechin.

43. 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 that resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, through multiple chemical intermediates, by the engineered host cell.

44. The method of claim 43, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors.

45. The method of claim 43, wherein 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.

46. The method of claim 43, wherein the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol, and (vi) any combination thereof.

47. The method of claim 43, wherein the one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors.

48. The method of claim 43, wherein the one or more genetic modifications cause reduction in the production of byproducts.

49. The method of claim 43, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.

50. The method of claim 43, wherein at least one of the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

51. The method of claim 43, wherein at least one of the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (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.

52. The method of claim 43, wherein at least one of the engineered host cell comprises at least one or more peptides selected from the group consisting of: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

53. The method of claim 43, wherein at least one of the engineered host cell is E. coli.

54. The method of claim 43, wherein the one or more genetic modifications decreases fatty acid biosynthesis.

55. The method of claim 43, wherein at least one of 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.

56. The method of claim 43, wherein the flavonoid is catechin.