FLAVONOID AND ANTHOCYANIN BIOPRODUCTION USING MICROORGANISM HOSTS

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
II. SEQUENCE LISTING

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


III. FIELD OF THE INVENTION

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


IV. BACKGROUND OF THE INVENTION

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


V. SUMMARY OF THE INVENTION

To move away from agriculture and chemically derived products, we have created engineered cells for the bioproduction of flavonoids and anthocyanins. This approach provides a feasible route for the rapid, safe, economical, and sustainable production of a wide variety of important flavonoids.


Herein, a range of flavonoids and anthocyanins including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, and cyaninidin-3-glucoside are biomanufactured using a modified microbial host. Herein, the engineered cells include one or more genetic modifications that increase(s) flavonoid and anthocyanin bioproduction by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.


Provided herein are cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts. As nonlimiting examples, a genetic modification can be a modification for over-expressing or under-expressing one or more endogenous genes in the engineered host cell or can be a modification for expressing one or more non-native genes in the engineered host cell. Engineered cells as provided herein can include multiple genetic modifications.


Also provided are cell cultures for producing one or more flavonoids or anthocyanins. The cell cultures include engineered cells as disclosed herein in a culture medium that includes a carbon source that can also be an energy source, such as glycerol, sugar, or an organic acid. In various embodiments, the culture medium can include at least one feed molecule such as but not limited to one or more organic acids or amino acids that can be converted into a flavonoid precursor (such as tyrosine, p-coumaroyl-CoA or malonyl-CoA). Examples of feed molecules include, but are not limited to, acetate, malonate, tyrosine, phenylalanine, pantothenate, coumarate, etc. In some embodiments, the feed molecules may be of reduced or low purity. For example, glycerol as a feed molecule may be crude glycerol, including a biomass comprising glycerol, for example, glycerol obtained as a byproduct of biodiesel processing. Alternatively, or in addition, the culture medium can include a supplemental compound that can be a cofactor or a precursor of a cofactor used by an enzyme that functions in a flavonoid pathway, such as, for examples, bicarbonate, biotin, thiamine, pantothenate, alpha-ketoglutarate, ascorbate, or 5-aminolevulinic acid.


Further provided are methods for producing flavonoids and anthocyanins that include culturing a cell engineered for the production of flavonoids or anthocyanins as provided herein under conditions in which the cell produces flavonoids or anthocyanins. In some examples, the methods include culturing the engineered cells in a culture medium that includes at least one feed molecule or supplement such as but not limited to: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid. The methods can further include recovering at least one of the flavonoids from culture medium, whole culture, or the cells.


In a first aspect, provided herein are cells engineered to produce one or more flavonoids or anthocyanins, wherein the cells include, in addition to nucleic acid sequences encoding either tyrosine ammonia lyase activity and/or phenylalanine ammonia lyase activity and cinnamate-4-hydroxylase activity, 4-coumarate-CoA ligase activity, chalcone synthase activity, chalcone isomerase activity, flavanone-3-hydroxylase activity, flavonoid 3′-hydroxylase activity or flavonoid 3′5′-hydroxylase activity, cytochrome P450 reductase activity, leucoanthocyanidin reductase activity, and dihydroflavonol-4-reductase activity, one or more genetic modifications for improving production of the flavonoids or anthocyanins. As set forth herein, a cell that is engineered to produce one or more of the flavonoids is engineered to include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase activity that can form 4-coumaric acid using tyrosine as substrate (e.g., tyrosine ammonia lyase TAL, EC: 4.3.1.25) or, alternatively or in addition, an exogenous nucleic acid sequence encoding phenylalanine ammonia lyase activity that can convert phenylalanine to trans-cinnamic acid and an exogenous nucleic acid sequence encoding cinnamate-4-hydroxylase activity that forms 4-coumaric acid from trans-cinnamic acid, an exogenous nucleic acid sequence encoding CoA ligase activity that forms p-coumaroyl-CoA from coumaric acid (e.g., 4-coumarate-CoA ligase, 4CL, EC:6.2.1.12), an exogenous nucleic acid sequence encoding polyketide synthase activity that forms naringenin chalcone using malonyl-CoA and p-coumaroyl-CoA as substrates (e.g., chalcone synthase, CHS, EC:2.3.1.74), an exogenous nucleic acid sequence encoding chalcone isomerase activity that forms naringenin from naringenin chalcone via its cyclase activity (e.g., chalcone-flavonone isomerase, CHI, EC:5.5.1.6), an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase activity that forms dihydrokaempferol from naringenin or forms taxifolin from eriodictyol (e.g., naringenin 3-dioxygenase, F3H, EC: 1.14.11.9), an exogenous nucleic acid sequence encoding flavonoid 3′-hydroxylase or flavonoid 3′5′-hydroxylase activity coupled with an exogenous nucleic acid sequence encoding cytochrome P450 reductase activity to form taxifolin or dihydromyricetin from dihydrokaempferol or to form eriodictyol or pentahydroxyflavone from naringenin (e.g., flavonoid 3′-monooxygenase, F3′H, EC: 1.14.13.21, EC: 1.14.14.82; cytochrome P450/NADPH-P450 reductase, EC:1.14.14.1; F3′5′H, EC:1.14.14.81), an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase activity that forms leucocyanidin from taxifolin, leucodelphinidin from dihydromyricetin, or leucopelargonidin from dihydrokaempferol (e.g., dihydroflavonol 4-reductase, EC:1.1.1), and an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase activity that forms catechin from leucocyanidin (e.g., leucoanthocyanidin reductase, LAR, EC:1.17.1.3). Optionally, a cell that is engineered to produce anthocyanins is further engineered to include an exogenous nucleic acid sequence encoding anthocyanin synthase activity that forms cyanidin from catechin or leucocyanidin, forms delphinidin from leucodelphinidin, or forms pelargonidin from leucopelargonidin (e.g., anthocyanin synthase, ANS, EC:1.14.20.4) and to include an exogenous nucleic acid sequence encoding glucosyltransferase activity that forms cyanidin-3-O-beta-D-glucoside from cyanidin, delphinidin-3-O-beta-D-glucoside from delphinidin, or pelagonidin-3-O-beta-D-glucoside from pelagonidin (e.g., anthocyanidin 3-O-glucosyltransferase, 3GT, EC:2.4.1.115). The cells provided herein that are engineered to produce flavonoids or anthocyanins are further engineered to increase the production of flavonoids or anthocyanins product, for example by increasing metabolic flux to a flavonoid or anthocyanin pathway, or by decreasing byproduct formation.


A cell engineered to produce a flavonoid is further engineered to increase the supply of precursor malonyl-CoA. One strategy for increasing malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC) activity. In various embodiments, the ACC enzyme, which in most eukaryotes, including fungi, is a large single chain polypeptide, and in plant and bacteria such as E. coli is a multi-subunit enzyme, is overexpressed in the host strain. Examples of acetyl-CoA carboxylase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACC genes of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, Ustilago maydis, and orthologs of these ACCs in other species having at least 50% amino acid identity to these ACCs.


Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). In some embodiments, acetyl-CoA synthase (ACS) that converts acetate and CoA to acetyl-CoA is over-expressed in the host cells. Cultures of engineered host cells that include overexpressed nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium, orthologs of these ACSs in other species having at least 50% amino acid identity to these ACSs.


Also considered, in further embodiments, is an engineered host cell that overexpresses a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA. Further, in E. coli, a variant of the Lpd subunit of PDH can be expressed that includes a mutation (E354K) that reduces inhibition of PDH by NADH.


Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. In some embodiments, a cell engineered to produce a flavonoid, or an anthocyanin, is further engineered to increase the cell's supply of malonyl-CoA includes an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that generates malonyl-CoA from malonate. Examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, for example, of Streptomyces coelicolor, or a malonate transporter encoded by DctPQM of Sinorhizobium medicae.


In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. Examples of malonate CoA-transferase that can be expressed in an engineered cell as provided herein include, without limitation, the alpha subunit (mdcA) of malonate decarboxylase from Acinetobacter calcoaceticus, Geobacillus sp, or a transferase having at least 50% identity to any of these or other naturally occurring malonate CoA-transferases.


In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA supply include upregulating endogenous pantothenate kinase (PanK) (EC:2.7.1.33) that produces CoA from pantothenate. Alternatively, or in addition, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33). Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.


Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) can be disrupted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and acyl carrier protein (E. coli acpP).


Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption, or deletion of genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In an E. coli host cell, genes that are downregulated, disrupted, or deleted can include aldehyde-alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), and enzyme acetate kinase phosphate acetyltransferase (ackA-pta).


Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, actetyl-CoA, and/or p-coumaryol-CoA. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and ybgC can be downregulated, disrupted, or deleted.


Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for one or more of the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.


Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to coumaric acid is the first committed step of the pathway. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. Strategies to increase tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the Phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.


Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. Strategies to increase heme supply include overexpression of the genes that synthesize the precursor ALA. In an E. coli host, ALA is formed from the 5-carbon skeleton of glutamate (the C5 pathway). The three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (gltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include a mutated hemA (inserting two lysine residuals between Thr-2 and Leu-3 at N terminus of hemA gene from Salmonella typhimurium (EC:1.1.1.70). Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway (ALS is synthesized by the condensation of glycine and succinyl-CoA). Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Bradyrhizobium japonicum (EC: 2.3.1.37), ALAS of Rhodobacter capsulatus, or an ALAS having at least 50% sequence identity to a naturally occurring ALAS. Further, one or more of the downstream genes (e.g., in E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.


In another aspect, provided herein are cell cultures that include engineered cells as provided herein in a culture medium, where the culture medium includes a carbon source that is also an energy source for the cells, where the carbon source can be, for example, glycerol, a sugar, or an organic acid, as nonlimiting examples. The culture medium can further include a feed molecule that is used to produce flavonoids or anthocyanins. A feed molecule can be, for example, acetate, malonate, tyrosine, pantothenate, coumarate, biotin, alpha-ketoglutarate, ascorbate, 5-aminolevulinic acid, succinate, or glycine. In some embodiments, the culture comprises a culture medium that includes a carbon source and at least one supplement that is a cofactor of an enzyme or is a precursor of an enzyme cofactor.


In yet another aspect, methods for producing flavonoids and anthocyanins that include incubating a culture of engineered host cell as provided herein to produce flavonoids or anthocyanins. The methods can further include recovering at least one of the flavonoids from the cells, the culture medium, or the whole culture.


In yet another aspect, the invention provides an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.


In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing an engineered host cell that comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause increased metabolic flux to flavonoid precursors. In certain embodiments, one or more genetic modifications cause reduction in the formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in the biosynthesis pathway or cofactors. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. In certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. Coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.


In yet another aspect, the invention provides a plurality of engineered host cells, wherein each of the plurality of the engineered host cells comprises one or more genetic modifications resulting in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one the engineered host cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.


In yet another aspect, the invention provides a method of increasing the production of flavonoids or anthocyanins, the method comprising: providing a plurality of engineered host cells, wherein each of the plurality of the engineered host cell comprises one or more genetic modifications resulting production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, (v) a biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications cause reduction of formation of byproducts. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, wherein one or more of the selected molecules are the chemical intermediates, including molecules in biosynthesis pathway or cofactors. In certain embodiments, at least one of the engineered cells from the plurality of the engineered host cells comprise at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cell from the plurality of engineered host cells comprise at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one the engineered host cell is E. coli. In certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cell from the plurality of the engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof. In certain embodiments, the flavonoid is catechin.


In yet another aspect, the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the E. coli cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.


In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the E. coli cell further comprises genes from fungi. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.


In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.


In another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene. In certain embodiments, one or more genetic modifications are selected from a group consisting of: (i) expression or overexpression of (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of shikimate kinase (aroK); (v) deletion of tyrR; (vi) expression or overexpression of A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.


In another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.


In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.


In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), delphinidin or gallocatechin to delphindin-3-glucoside (De3G), or afzelechin or pelargonidin to pelargonidin-3-glucoside (Pe3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.


In another aspect, the invention provides a method of increasing the transformation of cyanidin to cyanidin-3-glucoside (Cy3G), delphindin to delphindin-3-glucoside (De3G), or pelargonidin to pelagonidin-3-glucoside (Pe3G), comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.


In another aspect, the invention provides an engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.


In another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the


N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98. In certain embodiments, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.





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


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



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



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



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





VII. DETAILED DESCRIPTION OF THE INVENTION

The present application provides engineered cells for producing one or more flavonoids, cultures that include the engineered cells, and methods of producing one or more flavonoids, or at least one anthocyanin. The terms “flavonoid”, “flavonoid product”, or “flavonoid compound” are used herein to refer to a member of a diverse group of phytonutrients found in almost all fruits and vegetables. As used herein, the terms “flavonoid”, “flavonoid product”, or “flavonoid compound” are used interchangeably to refer a molecule containing the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring. Flavonoids may include, but are not limited to, isoflavone type (e.g., genistein), flavone type (e.g., apigenin), flavonol type (e.g., kaempferol), flavanone type (e.g., naringenin), chalcone type (e.g., phloretin), anthocyanidin type (e.g., cyanidin), catechins, flavanones, and flavanonols. Flavonoid compounds of interest include, without limitation, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin, pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, and kaempferol. Anthocyanins are in the forms of anthocyanidin glycosides and acylated anthocyanins. Anthocyanin compounds of interest include, without limitation, cyanidin glycoside, delphinidin glycoside, pelargonidin glycoside, peonidin glycoside, and petunidin glycoside.


The terms ‘precursor’ or ‘flavonoid precursor’ as used herein may refer to any intermediate present in the biosynthetic pathway that leads to the production of catechins or anthocyanins. flavonoid precursors may include, but are not limited to tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.


Cells engineered for the production of a flavonoid or an anthocyanin can have one or multiple modifications, including, without limitation, the downregulation, disruption, or deletion of endogenous genes, the upregulation of an endogenous gene, and the introduction of exogenous genes.


The term “non-naturally occurring”, when used in reference to an enzyme is intended to mean that nucleic acids or polypeptides include at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids, and polypeptides can be referred to as “wild-type” or “original”. A host cell, organism, or microorganism that includes at least one genetic modification generated by human intervention can also be referred to as “non-naturally occurring”, “engineered”, “genetically engineered,” or “recombinant”.


A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to under-express or to have reduced expression of a gene, nucleic acid sequence, or to under-express an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Specifically included are gene disruptions, which include any insertions, deletions, or sequence mutations into or of the gene or a portion of the gene that affect its expression or the activity of the encoded polypeptide. Gene disruptions include “knockout” mutations that eliminate expression of the gene. Modifications to under-express a gene also include modifications to regulatory regions of the gene that can reduce its expression.


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


Genes or nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.


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


A homolog is a gene or genes that have the same or identical functions in different organisms. Genes that are orthologous can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Paralogs are genes related by duplication within a genome, and can evolve new functions, even if these are related to the original one.


An engineered cell for producing flavonoids include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase (TAL) activity (alternatively or in addition, an exogenous nucleic acid encoding phenylalanine ammonia-lyase (PAL) activity and an exogenous nucleic acid encoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding chalcone isomerase (CHI) activity. Optionally, the engineered cell can further include an exogenous nucleic acid sequence encoding an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding flavonoid 3′-hydroxlase (F3′H) activity or flavonoid 3′,5′-hydroxylase (F3′5′H), an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase (DFR) activity, and/or an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase (LAR) activity.


Tyrosine ammonia-lyase (TAL) can be, for example, a member of the aromatic amino acid deaminase family that catalyzes the elimination of ammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosine ammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). Also considered for use in the engineered cells provided herein are TALs with SEQ ID NOS: 23-26, TALs listed in Table 1, TAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:1 that have the activity of a tyrosine ammonia lyase that produces p-coumaric acid from tyrosine.









TABLE 1







Tyrosine ammonia-lyase










Organism
GenBank Accession Number







Rhodotorula glutini

AGZ04575.1




Flavobacterium johnsoniae

WP_012023194.1




Herpetosiphon aurantiacus

ABX02653.1




Rhodobacter capsulatus

ADE83766.1




Saccharothrix espanaensis

AKE50820.1




Trichosporon cutaneum

AKE50834.1









Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the non-oxidative deamination of L-phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is the Brevibacillus laterosporus phenylalanine ammonia-lyase (PAL; SEQ ID NO :2). Also considered for use in the engineered cells provided herein are PALs with SEQ ID NOS: 27-29, PAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 that have the activity of a phenylalanine ammonia lyase that produces trans-cinnamic acid from phenylalanine.


Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and catalyzes the formation of p-coumaric acid from trans-cinnamic acid. Considered for use in the engineered cells provided herein are C4H of Helianthus annuus L. (C4H; SEQ ID NO: 3), C4Hs with SEQ ID NOS: 30-32, and C4H homologs of other species, as well as variants of naturally occurring C4Hs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the SEQ ID NO: 3 (C4H, Helianthus annuus L.) that have the activity of a C4H.


4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester. Considered for use in the engineered cells provided herein are 4CLs of Petroselinum crispum (SEQ ID NO: 4), 4CLs in Table 2, 4CLs with SEQ ID NOS: 33-36, and 4CL homologs of other species, as well as variants of naturally occurring 4CLs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID No: 4 (4CL, Petroselinum crispum) that have the activity of a 4CL.









TABLE 2







4-coumarate-CoA ligases










Organism
GenBank Accession Number







Petroselinum crispum

CAA31697.1




Camellia sinensis

ASU87409.1




Capsicum annuum

KAF3620173.1




Castanea mollissima

KAF3954751.1




Daucus carota

AIT52344.1




Gynura bicolor

BAJ17664.1




Ipomoea purpurea

AHJ60263.1




Lonicera japonica

AGE10594.1




Lycium chinense

QDL52638.1




Nelumbo nucifera

XP_010265453.1




Nyssa sinensis

KAA8540582.1




Solanum lycopersicum

NP_001333770.1




Striga asiatica

GER48539.1









The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS, SEQ ID NO: 5). Also considered for use in the engineered cells provided herein are the genes listed in Table 3, CHSs with SEQ ID: 37-40, and CHS homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida) that have the activity of a chalcone synthase.









TABLE 3







Chalcone synthases










Organism
GenBank Accession Number







Petunia hybrida

AAF60297.1




Acer palmatum

AWN08245.1




Callistephus chinensis

CAA91930.1




Camellia japonica

BAI66465.1




Capsicum annuum

XP_016566084.1




Coffea arabica

XP_027118978.1




Curcuma alismatifolia

ADP08987.1




Dendrobium catenatum

ALE71934.1




Garcinia mangostana

ACM62742.1




Iochroma calycinum

AIY22758.1




Iris germanica

BAE53636.1




Lilium speciosum

BAE79201.1




Lonicera caerulea

ALU09326.1




Lycium ruthenicum

ATB56297.1




Magnolia liliiflora

AHJ60259.1




Matthiola incana

BBM96372.1




Morus alba var. multicaulis

AHL83549.1




Nelumbo nucifera

NP_001305084.1




Nyssa sinensis

KAA8548459.1




Paeonia lactiflora

AEK70334.1




Panax notoginseng

QKV26463.1




Ranunculus asiaticus

AYV99476.1




Rosa chinensis

AEC13058.1




Theobroma cacao

XP_007032052.2









Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ ID NO: 6), CHI of Table 4, CHIs with SEQ ID NOS: 41-44, and CHI homologs of other species, as well as variants of naturally occurring CHI having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 6 (CHI, Medicago sativa) that have the activity of a chalcone isomerase.









TABLE 4







Chalcone Isomerases










Organism
GenBank Accession Number







Medicago sativa

AGZ04578.1



Dendrobium hybrid cultivar
AGY46120.1




Abrus precatorius

XP_027366189.1




Antirrhinum majus

BA032070.1




Arachis duranensis

XP_015942246.1




Astragalus membranaceus

ATY39974.1




Camellia sinensis

XP_028119616.1




Castanea mollissima

KAF3958409.1




Cephalotus follicularis

GAV77263.1




Clarkia gracilis subsp.

QPF47150.1




sonomensis






Dianthus caryophyllus

CAA91931.1




Glycyrrhiza uralensis

AXO59749.1




Handroanthus impetiginosus

PIN05040.1




Lotus japonicus

CAD69022.1




Morus alba

AFM29131.1




Phaseolus vulgaris

XP_007142690.1




Punica granatum

ANB66204.1




Rhodamnia argentea

XP_030524476.1




Spatholobus suberectus

TKY50621.1




Trifolium subterraneum

GAU12132.1









A nucleic acid sequence encoding a CHI can in some embodiments be fused to a nucleic acid sequence encoding a CHS in an engineered cell as provided herein, such that the CHI activity is fused to the chalcone synthase activity, i.e., a fusion protein is produced in the engineered cell that has both condensing and cyclization activities.


Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substrates include (2S)-eriodictyol, (2S)-dihydrotricetin and (2S)-pinocembrin. Some F3H enzymes are bifunctional and also catalyzes as flavonol synthase (EC: 1.14.20.6). Considered for use in the engineered cells provided herein are F3H of Rubus occidentalis (SEQ ID NO: 7), F3Hs with SEQ ID NOS: 45-48, F3Hs listed in Table 5, and other F3H homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:7 (F3H, Rubus occidentalis) that have the activity of a F3H.









TABLE 5







Flavanone 3-hydroxylases










Organism
GenBank Accession Number







Rubus occidentalis

ACM17897.1




Abrus precatorius

XP_027347564.1




Nyssa sinensis

KAA8547483.1




Camellia sinensis

AAT68774.1




Morelia rubra

KAB1219056.1




Rosa chinensis

PRQ47414.1




Malus domestica

AAD26206.1




Vitis amurensis

ALB75302.1




Iochroma ellipticum

AMQ48669.1




Hibiscus sabdariffa

ALB35017




Cephalotus follicularis

GAV71832









Flavonoid 3′-hydroxylases (F3′H) belongs to the cytochrome P450 family with systematic name of flavonoid, NADPH:oxygen oxidoreductase (3′-hydroxylating). In the flavonoid biosynthetic pathway, F3′H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. Considered for use in the engineered cells provided herein are F3′H of Brassica napus (F3′H; SEQ ID NO: 8), F3′H with SEQ ID NOS: 49-52, those listed in Table 6, and homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these F3′H. F3′H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. Cytochrome P450 reductases are diflavin oxidoreductases that supply electrons to F3′Hs. The P450 reductase can be from the same species as F3′H or different species from F3′H. Considered for use in the engineered cells provided herein are CPR of Catharanthus roseus (SEQ ID NO: 9), additional CPRs listed in Table 7, CPRs with SEQ ID NOS: 53-55, CPR homologs of other species, and variants of naturally occurring CPRs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%,at least 98%, or at least 99% amino acid identity to these CPRs that have the activity of a CPR. In various embodiments, the N-terminal nucleic acid sequences in the genes of F3′H and/or CPR originated from eukaryotic cells can encode targeting leader peptides, which can be removed before introduction into prokaryotic host cells, if desired. In some embodiments, the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin).









TABLE 6







Flavonoid 3′-hydroxylases










Organism
GenBank Accession Number







Brassica napus

ABC58722.1



Gerbera hybrid cultivar D1
ABA64468.1




Cephalotus follicularis

GAV84063.1




Theobroma cacao

XP_007037548.1




Phoenix dactylifera

XP_008791304.2
















TABLE 7







Cytochrome P450 reductases










Organism
GenBank Accession Number







Catharanthus roseus

CAA49446.1




Brassica napus

XP_013706600.1




Cephalotus follicularis

GAV59576.1




Camellia sinensis

XP_028084858.1









A nucleic acid sequence encoding a F3′H can in some embodiments be fused to a nucleic acid sequence encoding a CPR in an engineered cell as provided herein, such that the F3′H activity is fused to the CPR activity.


In the cells engineered to produce dihydomyricetin, flavonoid 3′, 5′-hydroxylase (F3′5′H) can be used to convert dihydrokaempferol to dihydromyricetin or naringenin to pentahydroxyflavone, which is further converted to dihydromyricetin by a F3H. F3′5′H has the systematic name flavanone, NADPH:oxygen oxidoreductase and catalyzes the formation of 3′,5′-dihydroxyflavanone from flavanone. An exemplary F3′5′H is the Delphinium grandiflorum F3′5′H (SEQ ID NO: 10), Also considered for use in the engineered cells provided herein include F3′5′H with SEQ ID NOS:56-57, F3′5′H homologs of other species, and variants of naturally occurring F3′5′H having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NOS:10 that have the activity of a F3′5′H.


Dihydroflavonol 4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (Taxifolin, DHQ), or dihydromyricein (DHM) to reduce those compounds to the corresponding cis-flavan-3,4-diol (DHK to leucopelargonidin; Taxifolin to leucocyanidin; DHM to leucodelphinidin). An exemplary DFR is the Anthurium andraeanum DFR (SEQ ID NO: 11). Also considered for use in the engineered cells provided herein include DFRs in Table 8, DFRs with SEQ ID NOS: 58-61, and DFR homologs of other species, as well as variants of naturally occurring DFR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 11.









TABLE 8







Dihydroflavonol 4-reductases










Organism
GenBank Accession Number







Eustoma grandiflorum

BAD34461.1




Anthurium andraeanum

AAP20866.1




Camellia sinensis

AAT66505.1




Morelia rubra

KAB1203810.1




Dendrobium moniliforme

AEB96144.1




Fragaria × ananassa

AHL46451.1




Rosa chinensis

XP_024167119.1




Acer palmatum

AWN08247.1




Nyssa sinensis

KAA8531902.1




Vitis amurensis

I82380.1




Abrus precatorius

XP_027329642.1




Angelonia angustifolia

AHM27144.1




Pyrus pyrifolia

Q84KP0.1




Theobroma cacao

XP_017985307




Theobroma cacao

XP_007051597.2




Brassica oleracea var. capitata

QKO29328.1




Rubus idaeus

AXK92786.1




Citrus sinensis

AAY87035.1




Gerbera hybrida

P51105.1




Cephalotus follicularis

GAV76940.1




Ginkgo biloba

AGR34043.1




Dryopteris erythrosora

QFQ61498.1




Dryopteris erythrosora

QFQ61499.1




Cephalotus follicularis

GAV76942.1









Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin. Considered for use in the engineered cells provided herein are LAR of Desmodium uncinatum (SEQ ID NO: 12), LARs with SEQ ID NOS: 62-65, and LAR homologs of other species, as well as variants of naturally occurring LAR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 12 (LAR, Desmodium uncinatum) that have the activity of a LAR.


Optionally, the cells are further engineered to include an anthocyanin synthase (ANS) which catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin, or leucodelphinidin to delphinidin. Considered for use in the engineered cells provided herein are ANS of Carica papaya (SEQ ID NO: 13), ANS with SEQ ID NOS: 66-69, and ANS homologs of other species, as well as variants of naturally occurring ANS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:13 (ANS, Carica papaya) that have the activity of a ANS.


Optionally, the cells are further engineered to include a flavonoid-3-glucosyl transferase (3GT) to generate anthocyanins by transfer of a sugar moiety such as, without limitation, UDP-α-D-glucose to anthocyanidins to form glycosylated anthocyanins. Considered for use in the engineered cells provided herein are 3GT of Vitis labrusca (SEQ ID NO:14), 3GT with SEQ ID NOS: 70-73, and 3GT homologs of other species, as well as variants of naturally occurring 3GT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 14 (3GT, Vitis labrusca) that have the activity of a 3GT.


In various aspects, host cells may be engineered for enhanced production of flavonoids or anthocyanins by introducing additional exogenous pathways and/or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of flavonoids or anthocyanins can be increasing yield, titer, or rate of production.


Thus, a host cell engineered for the production of a flavonoid or anthocyanin can be engineered to include any or any combination of: overexpression of an acetyl-CoA carboxylase (ACC) or an ACC variant; expression or overexpression of at least one enzyme for increasing cell's malonyl-CoA supply that does not rely on the ACC step; expression or overexpression of at least one enzyme to increase tyrosine supply; expression or overexpression of at least one enzyme to increase CoA availability for synthesizing precursors malonyl-CoA or p-coumaryol-CoA; expression or overexpression at least one enzyme to increase heme biosynthesis; deletion or downregulation of at least one fatty acid synthesis enzyme; at least one alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, phosphate acetyl transferase, or acetate kinase; at least one enzyme of a fatty acid degradation pathway, at least one thioesterase, or at least one TCA gene. The foregoing list of modifications is nonlimiting.


Malonyl-CoA is the direct precursor for chalcone synthase to perform sequential condensations with p-coumaryol-CoA. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation of acetyl-CoA in a multistep reaction. First, the biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as a CO2 donor. In the second reaction, the carboxyl-group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes, including fungi, both reactions are catalyzed by a large single chain protein, but in E. coli and other bacteria, the activity is catalyzed by a multi-subunit enzyme. Host cells can be engineered for example to express an exogenous acetyl-CoA carboxylase or a variant ACC to increase malonyl-CoA synthesis from acetyl-CoA. For example, Mucor circinelloides (SEQ ID NO: 15) acetyl-CoA carboxylase can be introduced into the host cells. Additional examples of ACC genes that may be used in the engineered cells provided herein include, without limitation, the genes listed in Table 9, genes with SEQ ID NOS: 74-76, naturally occurring orthologs of these ACCs, or variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to referenced genes. Further, naturally occurring acetyl-CoA carboxylase genes can be further engineered to introduce single or multiple amino acid mutations to increase catalytic activity and/or remove feedback inhibition.









TABLE 9







Acetyl-CoA carboxylases










Organism
GenBank Accession Number







Lipomyces starkeyi

AJT60321.1




Rhodotorula toruloides

GEM08739.1




Ustilago maydis

XP_011390921.1




Mucor circinelloides

EPB82652.1




Kalaharituber pfeilii

KAF8466702.1




Aspergillus fumigatus

KEY77072.1




Rhodotorula diobovata

TNY18634.1




Leucosporidium creatinivorum

ORY74050.1




Microbotryum intermedium

SCV70467.1




Mixia osmundae

GAA98306.1




Puccinia graminis

KAA1079218.1




Suillus occidentalis

KAG1764021.1




Gymnopilus junonius

KAF8909366.1









Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). Acetyl-CoA can be synthesized from acetate by an acyl-CoA ligase in an ATP-dependent reaction. Acetyl-CoA synthetase (ACS) or acetate-CoA ligase (EC 6.2.1.1.) catalyzes the formation of a new chemical bond between acetate and CoA coenzyme A (CoA). ACSs with native activity on acetate will provide the function of increasing acetyl-CoA supply when cells are either supplied with acetate as a co-feed, or where acetate is produced as a by-product. Other acyl-CoA ligases, having their main activity on other acid substrates, may also have substantial activity on acetate, and are viable candidates for providing acetate-CoA ligase activity in the engineered cells provided herein. The ACSs expressed in the host cells can be prokaryotic or eukaryotic. Cultures of engineered host cells that overexpress a nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium (SEQ ID NO:16), and orthologs of these ACSs in other species having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these ACSs.


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


Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. For example, a cell engineered to produce a flavonoid or an anthocyanin as provided herein can include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from malonate. Acyl-CoA synthetase catalyzes the conversion of a carboxylic acid to its acyl-CoA thioester through an ATP-dependent two-step reaction. In the first step, the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate. In the second step, the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA product. Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced sequences. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. In Rhizobium trifolii, the math gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, for example of Streptomyces coelicolor (SEQ ID NO:18), of Rhizobiales bacterium (SEQ ID NO:80), of Rhizobium leguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ ID NO: 82), of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a naturally-occurring malonate transporter. Cell cultures of a host cell engineered to express a malonyl-CoA synthetase and a malonate transporter can include a culture medium that includes malonate.


In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase (EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase) that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity. The mdcA gene product, the α subunit, is malonate CoA-transferase, and mdcD gene product, the β subunit, is a malonyl-CoA decarboxylase. The mdcE gene product, the γ subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the δ subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. When the α subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an a subunit to exchange the acetyl group for a malonyl group. As the thioester transfer should be thermodynamically favorable, the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply. Examples of mdcAs that can be expressed in an engineered cell as provided herein include, without limitation, mdcA of Acinetobacter calcoaceticus (SEQ ID NO: 19), mdcAs of Table 10, mdcAs with SEQ ID NOS: 84-87, or a transferase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally occurring malonate CoA-transferases.









TABLE 10







Malonate CoA-transferases (malonate decarboxylase subunit alpha)










Organism
GenBank Accession Number







Acinetobacter calcoaceticus

AAB97627.1




Geobacillus sp.

QNU36929.1




Acinetobacter johnsonii

WP_087014029.1




Acinetobacter marinus

WP_092618543.1




Acinetobacter rudis

WP_016655668.1




Psychrobacter sp. G

WP_020444454.1




Moraxella catarrhalis

WP_064617969.1




Zoogloea sp.

MBL0283742.1




Dechloromonas sp.

KAB2923906.1




Stenotrophomonas rhizophila

WP_123729366.1




Xanthomonas cucurbitae

WP_159407614.1









In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA supply include expressing or overexpressing at least one enzyme of a CoA biosynthesis pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4′-phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-limiting step in the biosynthesis of CoA. Three distinct types of PanK have been identified—PanK-I (found in bacteria), PanK-II (mainly found in eukaryotes, but also in the Staphylococci) and PanK-III, also known as CoaX (found in bacteria). In E. coli, pantothenate kinase is competitively inhibited by CoA itself, as well as by some CoA esters. The type III enzymes CoaX are not subject to feedback inhibition by CoA. In some embodiments, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, without limitation, CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33, SEQ ID NO: 20), CoaX of Streptomyces sp. CL12509 (SEQ ID NO: 88), CoaX of Streptomyces cinereus (SEQ ID: 89), or CoaX of Kitasatospora kifunensis (SEQ ID NO: 90) Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.


Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavonoid biosynthesis for the precursor malonyl-CoA and thus limits flavonoid formation. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) can be disrupted, attenuated or deleted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and/or acyl carrier protein (E. coli acpP).


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


Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, acetyl-CoA, and/or p-coumaryol-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and/or ybgC can be downregulated, disrupted, or deleted.


In further embodiments, a cell engineered for the production of flavonoids or anthocyanins can have one or more of fatty acid degradation genes downregulated, disrupted, or deleted to improve precursor supply to the flavonoid pathway. In E. coli, for example, the acyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and/or gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted, or deleted.


Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.


Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from feedstock such as glucose or glycerol is necessary to make biological production economically viable. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The shikimate pathway is the central metabolic route leading to formation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), this pathway exclusively exists in plants and microorganisms. It starts with the condensation of intermediates of glycolysis and pentosephosphate-pathway, phosphoenolpyruvate (PEP), and erythrose-4-phosphate (E4P), respectively, which enter the pathway through a series of condensation and redox reactions via 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the central branch point metabolite chorismate is obtained via shikimate-3-phosphate under ATP hydrolysis and introduction of a second PEP. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. In E. coli three DAHP synthase isozymes exist (aroF, aroG, aroH), which are each feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), in contrast the two DAHP synthases of plants are not subject to feedback-inhibition. In plants and bacteria, the subsequent five steps are catalyzed by single enzymes. From the central intermediate chorismate the pathway branches off to anthranilate and prephenate leading to aromatic amino acid, para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA) synthesis, the latter being a precursor for folate metabolism. Strategies to increase L-tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the ppsA, aroG, and/or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.


Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There exist two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA synthase (ALAS). The C4 pathway is restricted to mammals, fungi and purple nonsulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions, including assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chains, and incorporation of reduced iron into the molecule, are required to convert ALA to heme. In an E. coli host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (G1tX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemA with SEQ ID NOS: 91-93. Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway. Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Rhodobacter capsulatus (SEQ ID:22), ALAS with SEQ ID NOS: 94-97, or an ALAS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally-occurring ALAS. Further, one or more of the downstream genes (E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.


Engineered cells that produce a flavonoid can be engineered to include multiple pathways to enhance flavonoid production. Those skilled in the art will recognize that the embodiments described herein can be combined in multiple ways. Examples of engineered cells having multiple genetic modifications are exemplary only and do not limit the scope of the invention.


Enzymes to be expressed or overexpressed in engineered cells according to the invention are set forth in Table 11.


Host Cells

A host cell as provided herein can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanobacteria) cells.


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


Nonlimiting examples of suitable microbial hosts for the bio-production of a flavonoid include, but are not limited to, any gram-negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coli, any gram-positive microorganism, for example Bacillus subtilis, Lactobacillus sp. or Lactococcus sp.; a yeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.


Culture Medium

In yet another aspect, methods for producing a flavonoid or an anthocyanin that include incubating a culture of an engineered host cell as provided herein to produce a flavonoid or an anthocyanin. The methods can further include recovering the flavonoid or anthocyanin from the culture medium, whole culture, or cells.


The culture comprises cells engineered for the production of flavonoids or anthocyanins in a culture medium. In various embodiments the engineered cells can be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source that is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. Such carbon sources may be purified or crude, including a biomass comprising glycerol, for example, crude glycerol produced as a byproduct of biodiesel production from corn waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply such as, without limitation, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, alpha-ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.


Culture conditions can include aerobic, microaerobic or any combination alternating aerobic/microaerobic growth conditions. Further, culture conditions can include shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving a flavonoid product include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing flavonoid product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation protocol, the cells are grown in a bioreactor that is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds. The desired temperature can be from, for example, 20-37° C., depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH of the bioreactor can be controlled to range from 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for examples, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. To purify the flavonoids and/or anthocyanins to homogeneity the solution containing the flavonoids and/or anthocyanins was concentrated and the product purified via ion exchange or silica-based chromatography. The resulting solution was either lyophilized to yield the products in a solid form or was concentrated into a liquid solution.


In some embodiments, a method of producing a flavonoid or an anthocyanin comprises culturing an engineered cell disclosed herein in a culture medium to produce a flavonoid or an anthocyanin. In some embodiments, glycerol is used as a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In some embodiments, the method comprises isolating naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside. In some embodiments the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to about 99%, e.g., from about 50% to about 95% (for example from: about 50%, 55%, 60%, 65%, 70%, 75%, 80% to about: 85%, 90%, 95%, 97.5%, 99% or 99.9%). In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to: about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 55% to: about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 60% to: about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 65% to: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 70% to: about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 75% to: about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80% to about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 85% to: about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 90% to about 95%, or about 99%, or from about 95% to about 99% or greater.


VIII. EXAMPLES
Using The Modified Cell To Create Products
Example 1
Production of Naringenin in E. coli

An E. coli cell derived from MG1655 was engineered to overexpress ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) to produce naringenin when substrates tyrosine and coumaric acid were supplied in culture medium. ACC was expressed on a medium-copy plasmid (15-20 copies) while TAL, 4CL, CHS, and CHI were expressed on the chromosome. Cells of an OD 2.5 were cultured in a 48-well plate at 30 degree for 24 hours with a shaking speed of 600 RPM in minimal medium supplied with trace element, vitamins, 1 mM tyrosine,1 mM coumaric acid, and 2% glycerol. Cell cultures were extracted with DMSO at 1:1 ratio and centrifuged for 15 mins. The supernatant was analyzed for naringenin with HPLC. The cells produced 232 μM naringenin.


Variants of the foregoing host cell may be prepared using one or more of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) with one or more homologs of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), or CHI (SEQ ID NO: 6), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.


Example 2
Production of Dihydrokaempferol in E. coli

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


Variants of the foregoing host cell may be prepared using a homolog of F3H (SEQ ID NO: 7), wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzyme.


Example 3
Production of Taxifolin in E. coli

An E. coli strain derived from MG1655 was engineered to overexpress F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) to produce taxifolin when the substrate naringenin was supplied in culture medium. F3H was overexpressed on the chromosome while F3′H and CPR were overexpressed on a medium-copy plasmid. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glucose, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with 50% DMSO and centrifuged for 15 minutes. The supernatant was analyzed for taxifolin with HPLC. The cells produced 500 μM taxifolin.


Variants of the foregoing host cell may be prepared using one or more of F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) along with one or more homologs of F3H (SEQ ID NO: 7), F3′H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.


Example 4
Production of Anthocyanidins and Anthocyanins

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


Variants of the foregoing host cell may be prepared using one or both of ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) along with a homolog of ANS (SEQ ID NO: 13), 3GT (SEQ ID NO: 14), or both, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.


Analytical Methods
Example 5
Flavonoid Precursors and Flavonoids

For sampling naringenin, eriodictyol, dihydrokaempferol and taxifolin, extraction of total flavonoids from E. coli were performed on whole cell broth. 500 μL of whole cell broth was vortexed for 30 seconds with 500 μL of DMSO (dimethyl sulfoxide) and centrifuged for 15 minutes. For HPLC analysis, 50 μL of supernatant was transferred to an HPLC vial.


The HPLC method was as follows: An Agilent 1200 HPLC was fitted with an Ascentis C18 Column 150 mm×4.6 mm, 3μm, equipped with a R-18 (3 μm) guard column. The column was heated to 30 ° C. with the sample block being maintained at 25 ° C. For each sample, 5 μL was injected and the product was eluted at a flow rate of 1.5 mL/min using 0.1% phosphoric acid in water (solvent A), acetonitrile (solvent B), and methanol (solvent C) with the following gradient:

















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




















0
85
10
5



2.5
85
10
5



7.5
70
25
5



12.5
50
45
5



15
85
10
5









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


Example 6
Anthocyanidins and Anthocyanins

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


Example 7
Flavonoid Production

The example provides a combination of modifications to the E. coli host genome including deletions and overexpression of enzymes from other organisms to recapitulate the bioproduction pathway described in FIG. 4. Accordingly, the invention provides an engineered host cell that comprises one or more genetic modifications (as shown in FIG. 4 and described in this Example 7 and herein above in this application) that result in production of flavonoid or anthocyanin from a carbon source that can also be an energy source, through multiple chemical intermediates, by the engineered host cell. In certain embodiments, the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation. In certain embodiments, the carbon source is selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, and (v) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from a group consisting of: (i) glycerol, (ii) a sugar, (iii) an organic acid, (iv) an amino acid, and (v) any combination thereof. As shown in FIG. 4, in certain embodiments, one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors. As shown in FIG. 4, in certain embodiments, one or more of the genetic modifications cause reduction of formation of byproducts. As shown in FIG. 4, in certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for over-expressing one or more endogenous genes in the engineered host cells; (ii) one or more modifications for under-expressing one or more endogenous genes in the engineered host cells; (iii) one or more genetic modification is expressing one or more non-native genes in the engineered host cells; and (iv) a combination thereof.


As shown in FIG. 4, in certain embodiments, the engineered host cell is cultured in a medium comprising molecules selected from: tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.


As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) a nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprises a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.


As shown in FIG. 4, in certain embodiments, one or more genetic modifications decreases fatty acid biosynthesis. As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combinations thereof.


As shown in FIG. 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from: (i) nucleic acid sequences encoding tyrosine ammonia lyase activity; (ii) nucleic acid sequences encoding phenylalanine ammonia lyase activity; (iii) nucleic acid sequences encoding cinnamate 4-hydroxylase activity; (iv) nucleic acid sequences encoding 4-courmarate-CoA ligase (4CL) activity; and (v) any combination thereof.


As shown in FIG. 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) nucleic acid sequence encoding cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) nucleic acid sequence encoding 4-courmarate-CoA ligase activity, wherein 4-courmarate-CoA ligase forms p-coumaroyl-CoA from coumaric acid (v) nucleic acid sequence encoding chalcone synthase activity, wherein chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) nucleic acid sequence encoding chalcone isomerase activity, wherein chalcone isomerase forms naringenin from naringenin chalcone; (vii) nucleic acid sequence encoding flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combinations thereof.


The compositions as described above, can be used in methods described herein for increasing the production of flavonoids or anthocyanins. Such methods involve providing any of the compositions described above to result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (such as shown in part or in whole in FIG. 4).


In yet another aspect, it is envisioned that the pathway illustrated in FIG. 4 can be carried out using a plurality of engineered host cells, as opposed to a single host cell as described above. In such embodiments, the plurality of the engineered host cells have one or more genetic modifications that result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (as shown in FIG. 4).


Aspects of the invention are now described with reference herein to FIG. 4.


Step 1: conversion of pyruvate to acetate. poxB is deleted to reduce carbon loss and eliminate the byproducts.


Step 2: conversion of pyruvate to lactate. ldhA is deleted to reduce carbon loss and eliminate the byproducts.


Step 3: conversion of Acetyl-CoA to acetate. ackA-pta is deleted to reduce carbon loss and eliminate the byproducts.


Step 4: conversion of Acetyl-CoA to ethanol (EtOH). adhE is deleted to reduce carbon loss and eliminate the byproducts.


Step 5: conversion of acetyl-CoA to a substrate for the tricarboxylic acid cycle (TCA).


Step 6: conversion of acetyl-CoA to mal-CoA. Heterologous ACC is expressed to increase the concentration of available mal-CoA. Heterologous ACC may be obtained from fungal species. Accordingly, embodiments of the invention provide an engineered host cell that comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.


In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof.


Step 7: conversion of mal-CoA to malonyl-ACP (acyl carrier protein). malonyl-coA-ACP transacylase (fabD) is downregulated to increase carbon flux.


Step 8: conversion of malonyl-ACP to 3-ketyoacyl-ACP. beta-ketoacyl-ACP synthase II (fabF) is downregulated to increase carbon flux.


Step 9: conversion to mal-CoA to naringenin chalcone; conversion of coumaryl-CoA to naringenin chalcone. A heterologous CHS is overexpressed.


Step 10: conversion to naringenin chalcone to naringenin. A heterologous CHI is overexpressed.


Steps 11, 12, and 13: conversion of naringenin to dihydrokaempferol (DHK); conversion of naringenin to eriodictyol (EDL); conversion of eriodictyol (EDL) to dihydroquercetin (DHQ); conversion of (DHK) to dihydroquercetin (DHQ); conversion of dihydrokaempferol (DHK) to dihydromyricetin (DHM); conversion of pentahydroxyflayaone (PHF) to dihydromyricein (DHM). Heterologous F3′5′H, F3H, F3H, and/or CPR are overexpressed. Accordingly, as shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.


As shown in FIG. 4, in another aspect, the invention provides method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3′-hydroxylase (F3′H) or flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises peptides selected from a group consisting of: (i) flavonoid 3′-hydroxylase (F3′H); (ii) cytochrome P450 reductase (CPR); and (iii) any combination thereof. In certain embodiments, the engineered host cell produces eriodictyol or taxifolin. In certain embodiments, the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7. In certain embodiments, flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9. In certain embodiments, flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57. In certain embodiments, the engineered host cell further comprises cytochrome b5. In certain embodiments, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.


Step 14: conversion of dihydroquercetin (DHQ) to leucocyanidin (LC); conversion of dihydrokaempferol (DHK) to leucopelargonidin (LP); and conversion of dihydromyricetin (DHM) to leucodelphinidin (LD). Heterologous DFR is overexpressed.


Step 15: conversion of leucocyanidin (LC) to catechin; conversion of leucodelphinidin (LD) to gallocatechin; and conversion of leucopelargonidin (LP) to afzelechin. Heterologous LAR is overexpressed.


Step 16: conversion of catechin to cyanidin; conversion of leucocyanidin (LC) to catechin; conversion to leucodelphinidin (LD) to delphinidin; conversion of gallocatechin to delphinidin; conversion of leucopelargonidin (LP) to pelargonidin; or conversion of afzelechin to pelargonidin. Heterologous ANS is overexpressed. Step 16 could be carried in vivo or in a cell-free medium. Accordingly, as shown in FIG. 4, in another aspect, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.


In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.


In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof.


In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof.


Step 17: conversion of pelargonidin to callistephin; conversion of delphinidin to myrtillin (De3G); conversion of cyanidin to Cy3G. Heterologous 3GT was overexpressed in E. coli. Step 17 could be carried in vivo or as a cell-free reaction.


Step 18: conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine.


Step 19: conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA is overexpressed to upregulate tyrosine.


Step 20: conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). aroG variant is overexpressed to upregulate tyrosine.


Step 21: conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) to dehydroquinate (DHQ).


Step 22: conversion of dehydroquinate (DHQ) to 3-dehydroshikimate (DHS).


Step 23: conversion of 3-dehydroshikimate (DHS) to shikimic acid (SHK). aroE is overexpressed to upregulate tyrosine.


Step 24: conversion of shikimic acid (SHK) to shikimate-3-phosphate (S3P).


Step 25: conversion of shikimate-3-phosphate (S3P) to 5-enolpyruvylshikimate-3-phosphate (EPSP).


Step 26: conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismic acid (CHA).


Step 27: conversion of chorismic acid (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variant is overexpressed.


Step 28: conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine; conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Accordingly, as shown in FIG. 4, embodiments of the invention provide an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene.


As shown in FIG. 4, in another aspect, the invention provides a method of increasing endogenous biosynthesis of tyrosine comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combinations thereof. In certain embodiments, one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprises expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprises disruption of tyrR gene.


Step 29: conversion of tyrosine to coumaric acid. A heterologous TAL is overexpressed.


Step 30: conversion of courmaric acid to coumaryl-CoA. A heterologous 4CL is overexpressed.


Step 31: conversion of glutamate (Glut) to glutamyl-tRNA.


Step 32: conversion of glutamyl-tRNA to glutamate semialdehyde (GSA). hemA is overexpressed to upregulate ALA.


Step 33: conversion of glutamate semialdehyde (GSA) to δ amino levulinic acid (ALA). hemL is overexpressed to upregulate ALA.


Step 34: conversion of δ amino levulinic acid (ALA) to porphobilinogen (PBG).


Step 35: conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB).


Step 36: conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III (UPPIII).


Step 37: conversion of uroporphyrinogen III (UPPIII) to coproporphyrinogen III (CPPIII).


Step 38: conversion of coproporphyrinogen III (CPPIII) to protoporphyrinogen IX (PPPIX).


Step 39: conversion of protoporphyrinogen IX (PPPIX) to protoporphyrin IX, which is subsequently covered to heme.


Step 40: conversion of prephenate (PPA) to phenylpyruvate (POPP).


Step 41: conversion of phenylalanine (Phe) to cinnamate. Heterologous PAL and/or TAL are overexpressed.


Step 42: conversion of cinnamate to coumaric acid. Heterologous C4H/CPR are overexpressed.









TABLE 11







Enzyme Sequences:









Enzyme:
Sequence:
SEQ ID:





Tyrosine ammonia-
MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTDEEIVRMGAS
 1


lyase (TAL)
ARTIEEYLKSDKPIYGLTQGFGPLVLFDADSELEQGGSLISHLGT




Saccharothrix

GQGAPLAPEVSRLILWLRIQNMRKGYSAVSPVFWQKLADLWNKGF




espanaensis

TPAIPRHGTVSASGDLQPLAHAALAFTGVGEAWTRDADGRWSTVP



Accession:
AVDALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHRSALRLV



ABC88669.1
RACAVLSARLATLLGANPEHYDVGHGVARGQVGQLTAAEWIRQGL




PRGMVRDGSRPLQEPYSLRCAPQVLGAVLDQLDGAGDVLAREVDG




CQDNPITYEGELLHGGNFHAMPVGFASDQIGLAMHMAAYLAERQL




GLLVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRIRQLVF




PASLTTLPTNGWNQDHVPMALNGANSVFEALELGWLTVGSLAVGV




AQLAAMTGHAAEGVWAELAGICPPLDADRPLGAEVRAARDLLSAH




ADQLLVDEADGKDFG






Phenylalanine
MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH
 2


ammonia-lyase
ISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK



(PAL)
AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL




Brevibacillus

EVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK




laterosporus LMG

VLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL



15441
AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA



Accession:
SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS



WP_003337219.1
IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNG




GNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPN




LIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRS




TEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRD




SKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK




KEIHDQNVND






Cinnamate-4-
MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLPVPIFGN
 3


hydroxylase (C4H)
WLQVGDDLNHRNLTDLAKKFGEIFLLRMGQRNLVVVSSPDLAKEV




Helianthus annuus

LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV



L.
PFFTNKVVQQYRYGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQL



Accession:
MMYNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQSFEYNYG



QJC72299.1
DFIPILRPFLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV




DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAIETTLWSI




EWGIAELVNHPEIQAKLRNELDTKLGPGVQVTEPDLHKLPYLQAV




IKETLRLRMAIPLLVPHMNLHDAKLGGYDIPAESKILVNAWWLAN




NPEQWKKPEEFRPERFFEEESKVEANGNDFRYLPFGVGRRSCPGI




ILALPILGITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKH




STIVAKPRAL






4-coumarate-CoA
MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKVGDKSCL
 4


ligase (4CL)
INGATGETFTYSQVELLSRKVASGLNKLGIQQGDTIMLLLPNSPE




Petroselinum

YFFAFLGASYRGAISTMANPFFTSAEVIKQLKASQAKLIITQACY




crispum

VDKVKDYAAEKNIQIICIDDAPQDCLHFSKLMEADESEMPEVVIN



Accession:
SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYMH



P14912.1
SEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKFDIVPFLEL




IQKYKVTIGPFVPPIVLAIAKSPVVDKYDLSSVRTVMSGAAPLGK




ELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGA




CGTVVRNAEMKIVDPETNASLPRNQRGEICIRGDQIMKGYLNDPE




STRTTIDEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAP




AELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRTNGFTTTEE




EIKQFVSKQVVFYKRIFRVFFVDAIPKSPSGKILRKDLRARIASG




DLPK






Chaicone synthase
MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTYPDYYFRITN
 5


(CHS)
SEHKTDLKEKFKRMCEKSMIKKRYMHLTEEILKENPSMCEYMAPS




Petunia x hybrida

LDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV



Accession:
DMPGCDYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN



AAF60297.1
NKGARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGAGAIIIG




SDPIPGVERPLFELVSAAQTLLPDSHGAIDGHLREVGLTFHLLKD




VPGLISKNIEKSLEEAFRPLSISDWNSLFWIAHPGGPAILDQVEI




KLGLKPEKLKATRNVLSNYGNMSSACVLFILDEMRKASAKEGLGT




TGEGLEWGVLFGFGPGLTVETVVLHSVAT







Chalcone isomerase

MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERGLTIEGNFI
 6


(CHI)
KFTAIGVYLEDIAVASLAAKWKGKSSEELLETLDFYRDIISGPFE




Medicago sativa

KLIRGSKIRELSGPEYSRKVMENCVAHLKSVGTYGDAEAEAMQKF



Accession:
AEAFKPVNFPPGASVFYRQSPDGILGLSFSPDTSIPEKEAALIEN



P28012.1
KAVSSAVLETMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN






Flavanone 3-
MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFSNEIPIISL
 7


hydroxylase (F3H)
SGIDEVEGRRAEICNKIVEACEDWGVFQIVDHGVDAKLISEMTRL




Rubus occidentalis

ARDFFALPPEEKLRFDMSGGKKGGFIVSSHLQGEAVQDWREIVTY



Accession:
FSYPVRHRDYSRWPDKPEGWRAVTQQYSDELMGLACKLLEVLSEA



ACM17897.1
MGLEKEALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDPGTI




TLLLQDQVGGLQATRDGGKTWITVQPVEGAFVVNLGDHGHFLSNG




RFKNADHQAVVNSNHSRLSIATFQNPAQEAIVYPLKVREGEKPIL




EEPITYTEMYKKKMSKDLELARLKKLAKEQQPEDSEKAKLEVKQV




DDIFA






Flavonoid 3′
MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIGNLPHM
 8


hydroxylase (F3′H)
GPKPHQTLAAMVTTYGPILHLRLGFADVVVAASKSVAEQFLKVHD




Brassica napus

ANFASRPPNSGAKHMAYNYQDLVFAPYGQRWRMLRKISSVHLFSA



Accession:
KALEDFKHVRQEEVGTLMRELARANTKPVNLGQLVNMCVLNALGR



ABC58723.1
EMIGRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFVPALDCL




DLQGVAGKMKRLHKRFDAFLSSILEEHEAMKNGQDQKHTDMLSTL




ISLKGTDFDGEGGTLTDTEIKALLLNMFTAGTDTSASTVDWAIAE




LIRHPEIMRKAQEELDSVVGRGRPINESDLSQLPYLQAVIKENFR




LHPPTPLSLPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWS




DPLTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAGLSLGL




RTIQLLTATLVHGFEWELAGGVTPEKLNMEETYGITLQRAVPLVV




HPKLRLDMSAYGLGSA






Cytochrome P450
MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPAVMAMLLE
 9


reductase (CPR)
NKELVMILTTSVAVLIGCVVVLIWRRSSGSGKKVVEPPKLIVPKS




Catharanthus

VVEPEEIDEGKKKFTIFFGTQTGTAEGFAKALAEEAKARYEKAVI




roseus

KVIDIDDYAADDEEYEEKFRKETLAFFILATYGDGEPTDNAARFY



Accession:
KWFVEGNDRGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVAEQ



Q05001
GGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDNLLRDEDDTTVS




TTYTAAIPEYRVVFPDKSDSLISEANGHANGYANGNTVYDAQHPC




RSNVAVRKELHTPASDRSCTHLDFDIAGTGLSYGTGDHVGVYCDN




LSETVEEAERLLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCT




LRTALTRYADLLNTPKKSALLALAAYASDPNEADRLKYLASPAGK




DEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFAAIAPRLQPRFYS




ISSSPRMAPSRIHVTCALVYEKTPGGRIHKGVCSTWMKNAIPLEE




SRDCSWAPIFVRQSNFKLPADPKVPVIMIGPGTGLAPFRGFLQER




LALKEEGAELGTAVFFFGCRNRKMDYIYEDELNHFLEIGALSELL




VAFSREGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDAKGMAR




DVHRTLHTIAQEQGSMDSTQAEGFVKNLQMTGRYLRDVW






Flavonoid 3′, 5′-
MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKGWPVVGA
10


hydroxylase
LPMLGNMPHAALADLSRRYGPIVYLKLGSRGMVVASTPDSARAFL



(F3′5′H)
KTQDLNFSNRPTDAGATHIAYNSQDMVFADYGPRWKLLRKLSSLH




Delphinium

MLGGKAVEDWAVVRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAM




grandiflorum

ANMLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFNVGDFIP



Accession:
SIAWMDLQGIVRGMKRLHKKFDALLDKILREHTATRRERKEKPDL



BAO66642
VDVLMDNRDNKSEQERLTDTNIKALLLNLFSAGTDTSSSTIEWAL




TEMIKNPSIFGRAHAEMDQVIGRNRRLEESDIPKLPYLQAICKET




FRKHPSTPLNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNV




WENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRICAGTRMG




IVLVEYILGSLVHAFEWKLRDGETLNMEETFGIALQKAVPLAAVV




TPRLPPSAYVV






Dihydroflavonol 4-
MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATVRDPSNMKKVK
11


reductase (DFR)
HLLDLPGAANRLTLWKADLVDEGSFDEPIQGCTGVFHVATPMDFE




Anthurium

SKDPESEMIKPTIEGMLNVLRSCARASSTVRRVVFTSSAGTVSIH




andraeanum

EGRRHLYDETSWSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK



Accession:
NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPHYSILNP



AAP20866.1
VQFVHLDDLCNAHIFLFECPDAKGRYICSSHDVTIAGLAQILRQR




YPEFDVPTEFGEMEVFDIISYSSKKLTDLGFEFKYSLEDMFDGAI




QSCREKGLLPPATKEPSYATEQLIATGQDNGH






Leucoanthocyanidin
MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYPTFLLVRP
12


reductase (LAR)
GPVSPSKAVIIKTFQDKGAKVIYGVINDKECMEKILKEYEIDVVI




Desmodium

SLVGGARLLDQLTLLEAIKSVKTIKRFLPSEFGHDVDRTDPVEPG




uncinatum

LTMYKEKRLVRRAVEEYGIPFTNICCNSIASWPYYDNCHPSQVPP



Accession:
PMDQFQIYGDGNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRP



Q84V83.1
SSNCYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENIIPESI




VSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKFRSLDDCYED




FVPMVHDKIHAGKSGEIKIKDGKPLVQTGTIEEINKDIKTLVETQ




PNEEIKKDMKALVEAVPISAMG






Anthocyanin
MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFDEEKKD
13


dioxygenase (ANS)
EGPQVPTIDLRDIDSDDQQVRQRCRDELKKAAVDWGVMHLVNHGI




Carica papaya

PDHLIDRVKKAGQAFFELPVEVKEKYANDQASGNIQGYGSKLANN



Accession:
ASGQLEWEDYYFHLIFPEEKRDLAIWPNNPADYIEVTSEYARQLR



XP_021901846.1
RLVSKILGVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCPQ




PELALGVEAHTDISALTFILHNMVPGLQLFYEGKWVTAKCVPNSI




VMHVGDTIEILSNGKYKSILHRGLVNKEKVRISWAVFCEPPKEKI




ILKPLPETVSENEPPLFPPRTFAQHIQHKLFRKNQENLEAK






Anthocy anidin-3-
MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPHAVFSFFST
14


O-glycotransferase
SESNASIFHDSMHTMQCNIKSYDVSDGVPEGYVFTGRPQEGIDLF



(3GT)
MRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGVA




Vitis labrusca

WLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS



Accession:
KVRFRDLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFEEL



ABR24135
DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKP




TSVVYISFGTVTTPPPAELVALAEALEASRVPFIWSLRDKARMHL




PEGFLEKTRGHGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESV




AGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSC




FDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTLVDLV




SKPKDV






Acetyl-CoA
MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHTVISKVLIAN
15


carboxylase (ACC)
NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIR




Mucor

MADNFVEVPGGSNNNNYANVELIVDVAERTAVHAVWAGWGHASEN




circinelloides

PRLPEMLAKSKHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTM



1006PhL
GWSGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKAAEKIGF



Accession:
PVMIKASEGGGGKGIRMVKDGSNFAQLFAQVQGEIPGSPIFIMKL



EPB82652.1
AGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIA




KPDVFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDDQFYFLELNP




RLQVEHPTTEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPN




SASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAGFKPSS




GIMQELNFRSSTNVWGYFSVVSAGGLHEYADSQFGHIFAYGENRQ




QARKNMVIALKELSIRADFRSTVEYIIRLLETPDFEENTINTGWL




DMLISKKLTAERPDTMLAVFCGAVTKAHMASLDCFQQYKQSLEKG




QVPSKGSLKTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQ




VGIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGKTCLLEKE




SDPTQLRSPSPGKLVNLLVENGDHLNAGDAYAEIEVMKMYMPLIA




TEDGHVQFIKQAGATLEAGDIIGILSLDDPSRVKHALPFNGTVPA




FGAPHITGDKPVQRFNATKLTLQHILQGYDNQALVQTVVKDFADI




LNNPDLPYSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP




AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFARYRNGLTE




HAYSNYVELMEAYYDVEILFNQQREEEVILSLRDQHKDDLDKVLA




VTLSHAKVNIKNNVILMLLDLINPVSTGSALDKYFTPILKRLSEI




ESRATQKVTLKARELLILCQLPSYEERQAQMYQILKNSVTESVYG




GGSEYRTPSYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEV




YCRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANGIDSNKRIA




SYSDLTFLLNKTEEEPIRTGAMTACNSLADLQAELPRILTAFEEE




PLPPMLQRNAAPKEERMENILNIAVRADEDMDDTAFRTKICEMIT




ANADVFRQAHLRRLSVVVCRDNQWPDYYTFRERENYQEDETIRHI




EPAMAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDCRFF




IRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHEYKNS




DCNHLFINFIPTFAIEADDVEHALKDFVDRHGKRLWKLRVTGAEI




RFNVQSKKPDAPIIPMRFTVDNVSGFILKVEVYQEVKTEKSGWIL




KSVNKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPE




LFRQSVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVLQEIDR




APGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDITFKIGSFGVAE




DQVFYKASELARALGIPRIYLSANSGARIGLADELISQFRAAWKD




ASNPTAGFKYLYLTPAEYDVLAQQGDAKSVLVEEIQDEGETRLRI




TDVIGHTDGLGVENLKGSGLIAGATSRAYDDIFTITLVTCRSVGI




GAYLVRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQLGGT




QIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVRNAPVSMRLGAD




PIDRDIEYTPPKGPSDPRFFLAGKSENGKWLSGFFDQDSFVETLS




GWARTVVVGRARLGGIPMGVVSVETRTVENIVPADPANSDSTEQV




FMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQR




DMYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGAWVVVDPT




INKDMMEMYADNNARGGVLEPEGIVEIKYRKPALLATMERLDATY




ASLKKQLAEEGKTDEEKAALKVQVEAREQELLPVYQQISIQFADL




HDRAGRMKAKGVIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVT




ATSAAPMPREQMLDLVKQWFTNDNETVNFEDADELVSEWFEKRAS




VIDQRISKLKSDATKEQIVSLGNADQEAVIEGFSQLIENLSEDAR




AEILRKLNSRF






Acetyl-CoA
MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTFWGEQGKI
16


synthase (ACS)
LDWITPYQKVKNTSFAPGNVSIKWYEDGTLNLAANCLDRHLQENG




Salmonella

DRTAIIWEGDDTSQSKHISYRELHRDVCRFANTLLDLGIKKGDVV




typhimurium

AIYMPMVPEAAVAMLACARIGAVHSVIFGGFSPEAVAGRIIDSSS



Accession:
RLVITADEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRTGS



NP_463140.1
DIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPLFILYTSGSTGK




PKGVLHTTGGYLVYAATTFKYVFDYHPGDIYWCTADVGWVTGHSY




LLYGPLACGATTLMFEGVPNWPTPARMCQVVDKHQVNILYTAPTA




IRALMAEGDKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE




KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFFGVQPALVD




NEGHPQEGATEGNLVITDSWPGQARTLFGDHERFEQTYFSTFKNM




YFSGDGARRDEDGYYWITGRVDDVLNVSGHRLGTAEIESALVAHP




KIAEAAVVGIPHAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRK




EIGPLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNLGDTS




TLADPGVVEKLLEEKQAIAMPS






Malonyl-CoA
MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAAAGATAGRI
17


synthase (matB)
GGAGRVAVWATPAMETGVAVVAALLAGVAAVPLNPKSGDKELAHI




Streptomyces

LSDSAPSLVLAPPDAELPPALGALERVDVDVRARGAVPEDGADDG




coelicolor

DPALVVYTSGTTGPPKGAVIPRRALATTLDALADAWQWTGEDVLV



Accession:
QGLPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAARELNDGAT



WP_011028356
MLFGVPTMYHRIAETLPADPELAKALAGARLLVSGSAALPVHDHE




RIAAATGRRVIERYGMTETLMNTSVRADGEPRAGTVGVPLPGVEL




RLVEEDGTPIAALDGESVGEIQVRGPNLFTEYLNRPDATAAAFTE




DGFFRTGDMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENAL




LEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPPALGTLADHV




AARLAPHKRPRVVRYLDAVPRNDMGKIMKRALNRD






Malonate
MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA
18


transporter (matC)
DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR




Streptomyces

VALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL




coelicolor

LMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS



Accession:
LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG



NP_626686.1
SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTP




ARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRA




VGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAA




VLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAAL




AVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA




VPALAWLVLVVPGFG






Malonate CoA-
MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARAIELLETVIAS
19


transferase (MdcA)
GDRVCLEGNNQKQADFLSKCLSQCNPDAVNDLHIVQSVLALPSHI




Acinetobacter

DVFEKGIASKVDFSFAGPQSLRLAQLVQQQKISIGSIHTYLELYG




calcoaceticus

RYFIDLTPNICLITAHAADREGNLYTGPNTEDTPAIVEATAFKSG



Accession:
IVIAQVNEIVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQ



AAB97627.1
ITEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIELLLPTYAA




SLGLKGQICTNWALNPHPTLIPAIESGFVDSVHSFGSEVGMEDYI




KERPDVFFTGSDGSMRSNRAFSQTAGLYACDSFIGSTLQIELQGN




SSTATVDRISGFGGAPNMGSDPHGRRHASYAYTKAGREATDGKLI




KGRKLVVQTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMIYG




EDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTPVGLKRDAAK




VEELRQRGIIQRPEDLGIDPTQVSRDLLAAKSVKDLVKWSGGLYS




PPSRFRNW






Pantothenate kinase
MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVEQLTSQQAL
20


(CoaX)
PVRACRLVSVRSEQETSQLVARLEQLFPVSALVASSGKQLAGVRN




Pseudomonas

GYLDYQRLGLDRWLALVAAHHLAKKACLVIDLGTAVTSDLVAADG




aeruginosa

VHLGGYICPGMTLMRSQLRTHTRRIRYDDAEARRALASLQPGQAT



Accession:
AEAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGGDAELVRD



Q9HWCL1
ELAGARIMPDLVFVGLALACPIE






glutamyl-tRNA
MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSLLAQPMVQG
21


reductase (hemAm)
GVVLSTCNRTELYLSVEEQDNLQEALIRWLCDYHNLNEDDLRNSL




Salmonella

YWHQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHL




typhimurium

NASALRRMFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFE



Accession:
SLSTVTVLLVGAGETIELVARHLREHKVQKMIIANRTRERAQALA



AAA88610.1
DEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKS




RRNQPMLLVDIAVPRDVEPEVGKLANAYLYSVDDLQSIISHNLAQ




RQAAAVEAETIVEQEASEFMAWLRAQGASETIREYRSQSEQIRDE




LTTKALSALQQGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDG




DDERLNILRDSLGLE






5-aminolevulinic
MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQWNRPDGGK
22


acid synthase
QDITVWCGNDYLGMGQHPVVLAAMHEALEAVGAGSGGTRNISGTT



(ALAS)
AYHRRLEAEIADLHGKEAALVFSSAYIANDATLSTLRLLFPGLII




Rhodobacter

YSDSLNHASMIEGIKRNAGPKRIFRHNDVAHLRELIAADDPAAPK




capsulatus

LIAFESVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMYGPR



Accession:
GAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIAASAKMVDAVRSY



CAA37857
APGFIFSTSLPPAIAAGAQASIAFLKTAEGQKLRDAQQMHAKVLK




MRLKALGMPIIDHGSHIVPVVIGDPVHTKAVSDMLLSDYGVYVQP




INFPTVPRGTERLRFTPSPVHDLKQIDGLVHAMDLLWARCA






Tyrosine ammonia-
MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCAR
23


lyase (TAL)
AHARLEHAIAEQRHIYGITTGFGPLANRLIGADQGAELQQNLIYH




Rhodobacter

LATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAVLN




capsulatus SB 1003

AGFAPEVPAQGTVGASGDLTPLAHMVLALQGRGRMIDPSGRVQEA



Accession:
GAVMDRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEAARAID



ADE84832.1
AALRHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALD




GAGRVCRTLTAARRLTAADLRPEDHPAQDAYSLRVVPQLVGAVWD




TLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALAS




DALNAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQAGLQSGF




MGAQVTATALLAEMRANATPVSVQSLSTNGANQDVVSMGTIAARR




ARAQLLPLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDR




IRAVSPGLRADRPLAGHIEAVAQGLRHPSAAADPPA






Tyrosine ammonia-
MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQATKTVVIDG
24


lyase (TAL)
HNMKVGDVVAVARHGAKVELAASVAGPVQASVDFKESKKHTSIYG




Trichosporon

VTTGFGGSADTRTSDTEALQISLLEHQLCGYLPTDPTYEGMLLAA




cutaneum

MPIPIVRGAMAVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVP



Accession:
LRGTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLTAPEA



XP_018276715
IAKYKLKTVRLASKEGLGLVNGTAVSAAAGALALYDAECLAMMSQ




TNTALTVEALDGHVGSFAPFIQEIRPHVGQIEAAKNIRHMLSNSK




LAVHEEPELLADQDAGILRQDRYALRTSAQWIGPQLEMLGLARQQ




IETELNSTTDNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQN




LGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCKGLDIHCGA




YCAELGFLANPMSNHVQSTEMHNQSVNSMAFASARKTMEANEVLS




LLLGSQMYCATQALDLRVMEVKFKMAIVKLLNDTLTKHFSTFLTP




EQLAKLNTTAAITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDAL




MVNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADGRNDLEPA




EYLGQTRAVYEAIRSDLGVKVRRGDVAEGKSGKSIGSNVARIVEA




MRDGRLMGAVSKMFF






Tyrosine ammonia-
MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNFLKEFSG
25


lyase (TAL)
NKVIYGVNTGFGPMAQYRIKESDQIQLQYNLIRSHSSGTGKPLSP




Flavobacterium

VCAKAAILARLNTLSLGNSGVHPSVINLMSELINKDITPLIFEHG




johnsoniae

GVGASGDLVQLSHLALVLIGEGEVFYKGERRPTPEVFEIEGLKPI



Accession:
QVEIREGLALINGTSVMTGIGVVNVYHAKKLLDWSLKSSCAINEL



WP_012023194
VQAYDDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKREDHL




YSGENTEEIFKEKVQEYYSLRCVPQILGPVLETINNVASILEDEF




NSANDNPIIDVKNQHVYHGGNFHGDYISLEMDKLKIVITKLTMLA




ERQLNYLLNSKINELLPPFVNLGTLGFNFGMQGVQFTATSTTAES




QMLSNPMYVHSIPNNNDNQDIVSMGTNSAVITSKVIENAFEVLAI




EMITIVQAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYPF




VQKVKDHLINN






Tyrosine ammonia-
MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILAQIEASCAY
26


lyase (TAL)
INQAVKEHQPVYGVTTGFGGMANVIISPEEAAELQNNAIWYHKTG




Herpetosiphon

AGKLLPFTDVRAAMLLRANSHMRGASGIRLEIIQRMVTFLNANVT




aurantiacus

PHVREFGSIGASGDLVPLISITGALLGTDQAFMVDFNGETLDCIS



DSM 785
ALERLGLPRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLALA



Accession:
LEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWAADHMLELLQGS



ABX04526.1
QLSRNELDGSHDYRDGDLIQDRYSLRCLPQFLGPIIDGMAFISHH




LRVEINSANDNPLIDTASAASYHGGNFLGQYIGVGMDQLRYYMGL




MAKHLDVQIALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTAN




SIMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLARQTIQT




LQQYIAITLMFGVQAVDLRTHKLAGHYNAAELLSPLTAKIYHAVR




SIVKHPPSPERPYIWNDDEQVLEAHISALAHDIANDGSLVSAVEQ




TLSGLRSIILFR






Phenylalanine
MHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAMECSHLEEIKRM
27


ammonia-lyase
VDTYQNATQVMIEGATLTVPQVAAIARRPEVHVVLDAANARSRVD



(PAL)
ESSNWVLDRIMGGGDIYGVTTGFGATSHRRTQQGVELQRELIRFL




Physcomitrella

NAGVLSKGNSLPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKL




patens

LNAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKAVTEDGR



Accession:
EVSALEALRIAGVEKPFELAPKEGLALVNGTAVGSALASTVCYDA



XP_001758374.1
NIMVLLAEVLSALFCEVMQGKPEFADPLTHKLKHHPGQMEAAAVM




EWVLDGSSFMKAAAKFNETDPLRKPKQDRYALRTSPQWLGPQVEV




IRNATHAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSMDN




MRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSGGPNPSLDYGMK




GAEIAMASYLSEINYLANPVTTHVQSAEQHNQDVNSLGLVSARKT




EEAMEILKLMSATFLVGLCQAIDLRHVEETMQSAVKQVVTQVAKK




TLFMGSDGSLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLME




KLRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSRVVPAVR




EAYDTKGLSLVPNRIQDCRTYPLYKLVRGDLKTQLLSGQRTVSPG




QEIEKVFNAISAGQLVAPLLECVQGWTGTPGPFSARASC






Phenylalanine
MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNELLEFLQ
28


ammonia-lyase
KGRDQLENKLKENVAIYGINTGFGGNGDLIIPFDKLDYHQSNLLD



(PAL)
FLTCGTGDFFNDQYVRGIQFIIIIALSRGWSGVRPMVIQTLAKHL




Dictyostelium

NKGIIPQVPMHGSVGASGDLVPLSYIANVLCGKGMVKYNEKLMNA




discoideum AX4

SDALKITSIEPLVLKSKEGLALVNGTRVMSSVSCISINKFETIFK



Accession:
AAIGSIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNKYFN



XP_644510.1
TSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRCAPQILGIISEN




ISNAKIVIKREILSVNDNPLIDPYYGDVLSGGNFMGNHIARIMDG




IKLDISLVANHLHSLVALMMHSEFSKGLPNSLSPNPGIYQGYKGM




QISQTSLVVWLRQEAAPACIHSLTTEQFNQDIVSLGLHSANGAAS




MLIKLCDIVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIK




IIPILENDRRTDIDVREITNAILQDKLDFFNLNL






Phenylalanine
MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAKGTFEAFTFH
29


ammonia-lyase
ISEEANKRIEECNELKHEIMNQHNPIYGVTTGFGDSVHRQISGEK



(PAL)
AWDLQRNLIRFLSCGVGPVADEAVARATMLIRTNCLVKGNSAVRL




Brevibacillus

EVIHQLIAYMERGITPIIPERGSVGASGDLVPLSYLASILVGEGK




laterosporus

VLYKGEEREVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFACL



LMG 15441
AYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQKPHLGQMA



Accession:
SAKNIYTLLEGSQLSKEYSQIVGNNEKLDSKAYLELTQSIQDRYS



WP_003337219.1
IRCAPHVTGVLYDTLDWVKKWLEVEINSTNDNPIFDVETRDVYNG




GNFYGGHVVQAMDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPN




LIPRFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVSVFSRS




TEAHNQDKVSMGTISSRDARTIVELTQHVAAIHLIALCQALDLRD




SKKMSPQTTKIYNMIRKQVPFVERDRALDGDIEKVVQLIRSGNLK




KEIHDQNVND






Cinnamate-4-
MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGPIPVPVFGN
30


hydroxylase (C4H)
WLQVGDDLNHRNLTEMAKKFGEVFMLRMGQRNLVWSSPDLAKEVL




Rubus sp. SSL-2007

HTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVP



Accession:
FFTNKVVQQYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQLM



ABX74781.1
MYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSRLAQSFEYNYGD




FIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLSSTQATT




NEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSIE




WGIAELVNHPEIQKKLRDELDTVLGRGVQITEPEIQKLPYLQAVV




KETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLANN




PAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGII




LALPILGITLGRLVQNFELLPPPGQTQLDTTEKGGQFSLHILKHS




PIVMKPRT






Cinnamate-4-
MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPVPVFGN
31


hydroxylase (C4H)
WLQVGDDLNHRNLTDMAKKFGDVFMLRMGQRNLVVVSSPDLAKEV




Fragaria vesca

LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV



Accession:
PFFTNKVVQQYRHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQL



XP_004294725.1
MMYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLAQSFEYNYG




DFIPVLRPFLRGYLKICKEVKEKRIQLFKDYFVDERKKLASTQVT




TNEGLKCAIDHILDAQQKGEINEDNVLYIVENINVAAIETTLWSI




EWGIAELVNHPEIQKKLRDELDTVLGHGVQVTEPELHKLPYLQAV




VKETLRLRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWLAN




NPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPFGVGRRSCPGI




ILALPILGVTLGRLVQNFEMLPPPGQTQLDTTEKGGQFSLHILKH




STIVMKPRA






Cinnamate-4-
MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVPVFGN
32


hydroxylase (C4H)
WLQVGDDLNHRNLTEYAKKFGDVFLLRMGQRNLVVVSSPELAKEV




Solanum tuberosum

LHTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTV



Accession:
PFFTNKVVQQYRGGWESEAASVVEDVKKNPESATNGIVLRKRLQL



ABC69046.1
MMYNNMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFEYNYG




DFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVDERKKLANTKSM




DSNALKCAIDHILEAQQKGEINEDNVLYIVENFNVAAIETTLWSI




EWGIAELVNHPHIQKKLRDEIDTVLGPGMQVTEPDMPKLPYLQAV




IKETLRLRMAIPLLVPHMNLHDAKLAGYDIPAESKILVNAWWLAN




NPAHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGRRSCPGI




ILALPILGITLGRLVQNFEMLPPPGQSKLDTSEKGGQFSLHILKH




STIVMKPRSF






4-coumarate-CoA
MGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKVSDRACL
33


ligase (4CL)
INGATGETFSYAQVELISRRVASGLNKLGIHQGDTMMILLPNTPE




Daucus carota

YFFAFLGASYRGAVSTMANPFFTSPEVIKQLKASQAKLIITQACY



Accession:
VEKVKEYAAENNITVVCIDEAPRDCLHFTTLMEADEAEMPEVAID



AIT52344.1
SDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIH




SEDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIVPFLEL




IQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVRTVMSGAAPLGK




ELEDAVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPYEIKSGA




CGTVVRNAEMKIVDPETHASLPRNQSGEICIRGDQIMKGYLNDPE




STKTTIDEEGWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAP




AEIEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGSTTTEE




EIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKILRKELRARIASG




DLPK






4-coumarate-CoA
MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGSRPCLI
34


ligase (4CL)
NGSTGEILTYDQVELASRRVGSGLHRLGIRQGDTIMLLLPNSPEF




Striga asiatica

VLAFLGASHIGAVSTMANPFFTPAEVVKQAAASRAKLIVTQACHV



Accession:
DKVRDYAAEHGVKVVCVDGAPPEECLPFSEVASGDEAELPAVKIS



GER48539.1
PDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIH




SDDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIVPFLEL




IQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRTVMSGAAPLGR




ELEDAVRLKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKSGA




CGTVVRNAEMKIVDTETGASLGRNQPGEICIRGDQIMKGYLNDPE




STERTIDKEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAP




AELEALLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTITED




EIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKDLRARLAAG




VPN






4-coumarate-CoA
MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISEFSSRPC
35


ligase (4CL)
LINGANNQIYTYADVELNSRKVAAGLHKQFGIQQKDTIMILLPNS




Capsicum annuum

PEFVFAFLGASYLGAISTMANPLFTPAEVVKQVKASNAEIIVTQA



Accession:
CHVNKVKDYALENDVKIVCIDSAPEGCVHFSELIQADEHDIPEVQ



KAF3620179.1
IKPDDVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLY




IHSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFDIVPFL




ELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLSSVRTVMSGAAPL




GKELEDTVRAKFPNAKLGQGYGMTEAGPVLAMCLAFAKEPFEIKS




GACGTVVRNAEMKIVDPDTGNSLHRNQSGEICIRGDQIMKGYLND




PEATAGTIDKEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQV




APAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVRSNGSTIT




EDEVKEFISKQVIFYKRIKRVFFVDAVPKSPSGKILRKDLRAKLA




AGFPN






4-coumarate-CoA
MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSSKPCLI
36


ligase (4CL)
NGSTGKVYTYSDVELTSRKVAAGFHNLGIQQRDTIMLLLPNCPEF




Camellia sinensis

VFAFLGASYLGAIITMANPFFTPAETIKQAKASNSKLIITQSSYT



Accession:
SKVLDYSSENNVKIICIDSPPDGCLHFSELIQSNETQLPEVEIDS



ASU87409.1
NEVVALPYSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHS




EDMMMCVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKLI




QRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMSGAAPLGKE




LEDAVRAKFPHAKLGQGYGMTEAGPVLAMCLAFAKKPFEIKSGAC




GTVVRNAEMKIVDPDAGFSLPRNQPGEICIRGDQIMKGYLNDPEA




TERTIDKQGWLHTGDIGYIDDDDELFIVDRLKELIKYKGFQVAPA




ELEALLLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVTENE




IKKYISEQVVFYKKINRVYFVDAIPKAPSGKILRKDLRARLAAGI




PS






Chaicone synthase
MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTYPDYYFRITN
37


(CHS)
SEHKTELKEKFKRMCEKSMIKTRYMHLTEEILKENPNMCAYMAPS




Capsicum annuum

LDARQDIVVVEVPKLGKEAAQKAIKEWGQPKSKITHLVFCTTSGV



Accession:
DMPGCDYQLAKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN



XP_016566084.1
NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAIIMG




SDPIPGVERPLFQLVSAAQTLLPDSEGAIDGHLREVGLTFHLLKD




VPGLISKNIEKSLVEAFQPLGISDWNSLFWIAHPGGPAILDQVEL




KLGLKPEKLKATREVLSNYGNMSSACVLFILDEMRKASTKEGLGT




SGEGLEWGVLFGFGPGLTVETVVLHSVAI






Chaicone synthase
MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYPDYYFRITK
38


(CHS)
SEHKAELKEKFQRMCDKSMIKKRYMYLTEEILKENPSMCEYMAPS




Rosa chinensis

LDARQDMVVVEIPKLGKEAATKAIKEWGQPKSKITHLVFCTTSGV



Accession:
DMPGADYQLTKLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN



AEC13058.1
NKGARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGAAAIIVG




SDPLPEVEKPLFELVSAAQTILPDSDGAIDGHLREVGLTFHLLKD




VPGLISKNIEKSLNEAFKPLNITDWNSLFWIAHPGGPAILDQVEA




KLGLKPEKLEATRHILSEYGNMSSACVLFILDEVRRKSAANGHKT




TGEGLEWGVLFGFGPGLTVETVVLHSVAA






Chaicone synthase
MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQADYPDYYFR
39


(CHS)
ITNSDHMTDLKDKFKRMCEKSMITKRHMYLTEEILKENPKMCEYM




Morus alba var.

APSLDARQDIVVVEVPKLGKEAAAKAIKEWGQPKSKITHLIFCTT




multicaulis

SGVDMPGADYQLTKLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDL



Accession:
AENNKGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDGAAAV



AHL83549.1
ILGADPDTSVERPIFELVSAAQTILPDSEGAIDGHLREVGLTFHL




LKDVPGLISKNIEKSLVEAFTPIGISDWNSIFWIAHPGGPAILDQ




VEAKLGLKQEKLSATRHVLSEYGNMSSACVLFILDEVRKKSVEEG




KATTGEGLEWGVLFGFGPGLTVETIVLHSLPAV






Chaicone synthase
MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQADYPDYYFRI
40


(CHS)
TKSEHLTELKEKFKQMCDKSMIRKRYMYLTEEILKENPNICAFMA




Dendrobium

PSLDARQDIVVTEVPKLAREASARAIKEWGQPKSRITHLIFCTTS




catenatum

GVDMPGADYQLTRLLGLRPSVNRIMLYQQGCFAGGTVLRLAKDLA



Accession:
ENNAGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAAAII



ALE71934.1
VGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLREMGLTFHLL




KDVPGLISKNIQKSLVETFKPLGIHDWNSIFWIAHPGGPAILDQV




EIKLGLKEEKLASSRNVLAEYGNMSSACVLFILDEMRRRSAEAGQ




ATTGEGLEWGVLFGFGPGLTVETVVLRSVPIAGAV






Chaicone isomerase
MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTIEGNFIK
41


(CHI)
FTAIGVYLEDVAVASLATKWKGKSSEELLETLDFYRDIISGPFEK




Trifolium pratense

LIRGSKIRELSGPEYSRKVTENCVAHLKSVGTYGDAEVEAMEKFV



Accession:
EAFKPINFPPGASVFYRQSPDGILGVSISIHFFP



PNX83855.1







Chaicone isomerase
MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGVRGLTIEGNF
42


(CHI)
IKFTGIGVYLEDQAVASLATKWKGKSSEELVESLDFFRDIISGPF




Abrus precatorius

EKLIRGSKIRQLSGPEYSKKVMENCVAHMKSVGTYGDAEAAGIEE



Accession:
FAQAFKPVNFPPGASVFYRQSPDGVLGLSFSQDATIPEEEAAVIK



XP_027366189.1
NKPVSAAVLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN






Chaicone isomerase
MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGERGLTIDGK
43


(CHI)
FIKFTGIGVYLEDKAVPSLAGKWKDKSSQQLLQTLHFYRDIISGP




Arachis duranensis

FEKLIRGSKILALSGVEYSRKVMENCVAHMKSVGTYGDAEAEAIQ



Accession:
QFAEAFKNVNFKPGASVFYRQSPLGHLGLSFSQDGNIPEKEAAVI



XP_015942246.1
ENKPLSSAVLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQ




HN



Chaicone isomerase
MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGERGLEIQGK
44


(CHI)
FIKFTAIGVYLEGDAVASLAVKWKGKSKEELTDSVEFFRDIVTGP




Cephalotus

FEKFTQVTTILPLTGQQYSEKVSENCVAFWKSVGIYTDAEAKAIE




follicularis

KFIEVFKEETFPPGSSILFTQSPNGALTIAFSKDGVIPEVGKAVI



Accession:
ENKLLAEGLLESIIGKHGVSPVAKQCLATRLSELL



GAV77263.1







Flavanone 3-
MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV
45


hydroxylase (F3H)
KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF




Abrus precatorius

ESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT



Accession:
EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE



XP_027329642.1
NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQ




GQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQK




YPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA




VETCKEKRLLSETAEISGTTQK






Flavanone 3-
MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLERGYIVRATVR
46


hydroxylase (F3H)
DPANLKKVKHLLDLPKADTNLTLWKADLNEEGSFDEAIEGCSGVF




Camellia sinensis

HVATPMDFESKDPENEVIKPTINGVLSIIRSCTKAKTVKRLVFTS



Accession:
SAGTVNVQEHQQPVFDENNWSDLHFINKKKMTGWMYFVSKTLAEK



AAT66505.1
AAWEAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRNE




GHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSSHDATIHD




LAKLMREKWPEYNVPTEFKGIDKDLPVVSFSSKKLIGMGFEFKYS




LEDMFRGAIDTCREKGLLPHSFAENPVNGNKV






Flavanone 3-
MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYIVRATVRDPA
47


hydroxylase (F3H)
NMKKVKHLQELEKADKNLTLWKADLTEEGSFDEAIKGCSGVFHVA




Nyssa sinensis

TPMDFESKDPENEVIKPTINGVLSIVRSCVKAKTVKRLVFTSSAG



Accession:
TVNLQEHQQLVYDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAW



KAA8531902.1
EATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEAHY




SIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSHDATIYDLAK




MIREKWPEYNVPTELKGIEKDLQTVSFSSKKLIGMGFEFKYSLED




MYKGAIDTCREKGLLPYSTHETPANANANANANVKKNQNENTEI






Flavanone 3-
MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV
48


hydroxylase (F3H)
KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF




Rosa chinensis

ESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE



Accession:
ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE



XP_024167119.1
NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQ




GQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREK




YPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGA




VDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG






Flavonoid 3′
MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIGNLPHM
49


hydroxylase (F3′H)
GQMPHHSLAAMARTYGPLMHLRLGFVDVIVAASASVASQLLKTHD




Cephalotus

ANFSSRPHNSGAKYIAYNYQDLVFAPYGPRWRMLRKISSVHLFSG




follicularis

KALDDYRHVRQEEVAVLIRALARAESKQAVNLGQLLNVCTANALG



Accession:
RVMLGRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIGDFIPA



GAV84063.1
LDWLDLQGVAAKMKNLHKRFDTFLTGLLEEHKKMLVGDGGSEKHK




DLLSTLISLKDSADDEGLKLTDTEIKALLLNMFTAGTDTSSSTVE




WAIAELIRHPKILAQVLKELDTVVGRDRLVTDLDLPQLTYLQAVI




KETFRLHPSTPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARD




PKEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAGRRICAG




MSLGLRMVQLLTATLVHAFDWDLTSGLMPEDLSMEEAYGLTLQRA




EPLMVHPRPRLSPNVY






Flavonoid 3′
MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPIIGNLPH
50


hydroxylase (F3′H)
MGPVPHHSLAALAKVYGPLMHLRLGFVDVVVAASASVAAQFLKVH




Theobroma cacao

DANFSSRPPNSGAKYVAYNYQDLVFAPYGPRWRMLRKISSVHLFS



Accession:
GKALDDFRHVRQDEVGVLVRALADAKTKVNLGQLLNVCTVNALGR



EOY22049.1
VMLGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNIGDFIPAL




EWLDLQGVQAKMKKLHKRFDRFLSAILEEHKIKARDGSGQHKDLL




STFISLEDADGEGGKLTDTEIKALLLNMFTAGTDTSSSTVEWAIA




ELIRHPKILAQVRKELDSVVGRDRLVSDLDLPNLTYFQAVIKETF




RLHPSTPLSLPRMASESCEINGYHIPKGATLLVNVWAIARDPDEW




KDPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRICAGMSLG




LRMVQLLAATLVHAFDWELADGLMPEKLNMEEAFGLTLQRAAPLM




VHPRPRLSPRAY






Flavonoid 3′
MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPWPVVGNLP
51


hydroxylase (F3′H)
HLGTIPHHSLAAMAKKYGPLMHLRLGFVDVVVAASASVAAQFLKT




Gerbera hybrida

HDANFADRPPNSGAKHIAYNYQDLVFAPYGPRWRMLRKICSVHLF



Accession:
STKALDDFRHVRQEEVAILARALVGAGKSPVKLGQLLNVCTTNAL



ABA64468.1
ARVMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIGDFIPVLDW




LDLQGVTKKMKKLHAKFDSFLNTILEEHKTGAGDGVASGKVDLLS




TLISLKDDADGEGGKLSDIEIKALLLNLFTAGTDTSSSTIEWAIA




ELIRNPQLLNQARKEMDTIVGQDRLVTESDLGQLTFLQAIIKETF




RLHPSTPLSLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW




ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRICVGMSLG




LRMVQLLTATLIHAFDWELADGLNPKKLNMEEAYGLTLQRAAPLV




VHPRPRLAPHVYETTKV






Flavonoid 3′
MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGPRGWPVLG
52


hydroxylase (F3′H)
NLPQLGPKPHHTLHALSRAHGPLFRLRLGSVDVVVAASAAVAAQF




Phoenix dactylifera

LRAHDANFSNRPPNSGAEHIAYNYQDLVFAPYGPGWRARRKLLNV



Accession:
HLFSGKALEDLRPVREGELALLVRALRDRAGANELVDLGRAANKC



XP_008791304.2
ATNALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAGVFNVGDF




VPGIGWLDLQGVVRRMKELHRRYDGFLDGLIAAHRRAAEGGGGGG




KDLLSVLLGLKDEDLDFDGEGAKLTDTDIKALLLNLFTAGTDTTS




STVEWALSELVKHPDILRKAQLELDSVVGGDRLVSESDLPNLPFM




QAIIKETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNVWA




IARDPAVWRDPLEFRPARFLPDGGCEGMDVKGNDFGIIPFGAGRR




ICAGMSLGIRMVQFMTATLAHAFHWDLPEGQMPEKLDMEEAYGLT




LQRATPLMVHPVPRLAPTAYQS






Cytochrome P450
MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF
53


reductase (CPR)
LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFY




Camellia sinensis

GTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQK



Accession:
LKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY



XP_028084858
GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIE




DDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDP




LSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKT




MASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF




LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGT




QTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK




KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGV




FSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD




FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTT




TSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHL




EFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSV




HADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA




LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFP




SAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGP




SPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDP




SIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR




RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAA




QVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE




VIVKKLQMEGRYLRDVW






Cytochrome P450
MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVIIGLLFF
54


reductase (CPR)
LLKRSSDRSKESKPVVISKPLLVEEEEEEDEVEAGSGKTKVTMFY




Cephalotus

GTQTGTAEGFAKSLAKEIKARYEKAIVKVVDLDDYAADDDQYEQK




follicularis

LKKETLVFFMLATYGDGEPTDDAARFYKWFTEENERGAWLQQLTY



Accession:
GVFSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIE



GAV59576.1
DDFAAWRETLWPELDQLLRDEDDANTVSTPYAAAIPEYRVVIHDP




LSGRGEAPSFSIDSHLTICEIWSTSREGSNQQISEYFWTSNSLKT




MASNSNLIRSIESALGVSFGSESVSDTAIVVVTTSVAVIIGLLFF




LLKRSSDRSKESKPVVISKPLLVEEEEDEVEAGSGKTKVTLFYGT




QTGTAEGFAKSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK




KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGAWLQQLTYGV




FSLGNRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQCIEDD




FAAWRETLWPELDQLLRDEDDANTVSTPYTAAIPEYRVVIHDPTT




TSYEDKNLNMANGNASYDIHHPCRVNVAVQRELHKPESDRSCIHL




EFDISGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLELLFSV




HADKDDGTSLGGSLPPPFPGPCTLRDALAHYADLLNPPRKAALSA




LAAHAVEPSEAERLKFLSSPQGKEDYSQWVVASQRSLLEIMAEFP




SAKPPLGVFFAAVAPRLQPRYYSISSSPRFVPNRVHVTCALVYGP




SPTGRIHKGVCSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDP




SIPIIMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFGCRNR




RMDFIYEDELNNFVDQGAVSELVVAFSREGPEKEYVQHKLNAKAA




QVWGLISQGGYLYVCGDAKGMARDVHRMLHTIVEQQENVDSRKAE




VIVKKLQMEGRYLRDVW






Cytochrome P450
MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAELSSMLIE
55


reductase (CPR)
NRQFAMIISTSIAVLIGCIVMLLWRRSGGSGSSKRAETLKPLVLK




Brassica napus

PPREDEVDDGRKKVTIFFGTQTGTAEGFAKALGEEARARYEKTRF



Accession:
KIVDLDDYAADDDEYEEKLKKEDVAFFFLATYGDGEPTDNAARFY



XP_013706600.1
KWFTEGDDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDILVEQ




GAQRLVHVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVT




PYTAAVLEYRVSIHNSADALNEKNLANGNGHAVFDAQHPYRANVA




VRRELHTPESDRSCTHLEFDIAGSGLTYETGDHVGVLSDNLNETV




EEALRLLDMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALT




RYACLLSSPKKSALLALAAHASDPTEAERLKHLASPAGKDEYSKW




VVESQRSLLEVMAEFPSAKPPLGVFFAAVAPRLQPRFYSISSSPK




IAETRIHVTCALVYEKMPTGRIHKGVCSTWMKSAVPYEKSENCCS




APIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVES




GVELGPSVLFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREF




GPTKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKGMARDVHRSL




HTIAQEQGSMDSTKAESFVKNLQMSGRYLRDVW






Flavonoid 3′, 5′-
MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPGPKGWPF
56


hydroxylase
VGALPLLGTMPHVALAQMAKKYGPVMYLKMGTCGMVVASTPDAAR



(F3′5′H)
AFLKTLDLNFSNRPPNAGATHLAYNAQDMVFADYGPRWKLLRKLS




Cephalotus

NLHMLGGKALEDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLT




follicularis

YAMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTSAGYFNIGD



Accession:
FIPSIAWMDLQGIERGMKKLHKRFDALLTKMFEEHMATAHERKGN



GAV62131
PDLLDIVMANRDNSEGERLTTTNIKALLLNLFSAGTDTSSSIIEW




SLAEMLKNPSILKRAHEEMDQVIGRNRRLEESDIKKLPYLQAICK




ESFRKHPSTPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP




EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGRRICAGTR




MGIVLVEYILGTLVHSFDWSLPHGVKLNMDEAFGLALQKAVPLAA




IVSPRLAPTAYVV






Flavonoid 3′, 5′-
MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGALPFI
57


hydroxylase
GPMPHSGLALLARRYGPIMFLKMGIRRVVVASSSTAARTFLKTFD



(F3′5′H)
SHFSDRPSGVISKEISYNGQNMVFADYGPKWKLLRKVSSLHLLGS




Dendrobium

KAMSRWAGVRRDEALSMIQFLKKHSDSEKPVLLPNLLVCAMANVI




moniliforme

GRIAMSKRVFHEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGW



Accession:
LDPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQGNPDLLDLV



AEB96145
VASEVKGEDGEGLCEDNIKGFISDLFVAGTDTSAIVIEWAMAEML




KNPSILRRAQEETDRVIGRHRLLDESDIPNLPYLQAICKEALRKH




PPTPLSIPHYASEPCEVEGYHIPGETWLLVNIWAIGRDPDVWENP




LVFDPERFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMVMV




QYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPKAVPLAVMATPR




LPAAAYGLL






Dihydroflavonol 4-
MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVRDPDNEKKV
58


reductase (DFR)
KHLVELPKAKTHLTLWKADLSDEGSFDEAIHGCTGVFHVATPMDF




Acer palmatum

ESKDPENEVIKPTINGVLGIMKACKKAKTVKRLVFTSSAGTVDVE



Accession:
EHKKPVYDENSWSDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEE



AWN08247.1
NSIDFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAIIKQ




GNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATILELSKFLRER




YPEYDLPTEYKGVDDSLENVVFCSKKILDLGFQFKYSLEDMFTGA




VETCREKGLIPLTNIDKKHVAAKGLIPNNSDEIHVAAAEKTTATA






Dihydroflavonol 4-
MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATVRDPANMKKV
59


reductase (DFR)
KHLLELPNAKTNLSLWKADLAEEGSFDEAIKGCTGVFHVATPMDF




Abrus precatorius

ESKDPENEVIKPTINGLIDIMKACMKAKTVRRLVFTSSAGTVDVT



Accession:
EHPKPLFDESCWSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKE



XP_027329642.1
NNIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAIIKQ




GQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATIHDIASLLNQK




YPEFNVPTKFKNIPDQLEIIRFSSKKITDLGFKFKYSLEDMFTGA




VETCKEKRLLSETAEISGTTQK






Dihydroflavonol 4-
MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRATVRDPTNLKK
60


reductase (DFR)
VKPLLDLPRSNELLSIWKADLDGIEGSFDEVIRGSIGVFHVATPM




Dendrobium

NFQSKDPENEVIQPAINGLLGILRSCKNAGSVQRVIFTSSAGTVN




moniliforme

VEEHQAAAYDETCWSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFV



Accession:
KDNHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHYSIL



AEB96144.1
KQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDSTIYGLAEMLK




NRYPTYAIPHKFKEIDPDIKCVSFSSKKLMELGFKYKYTMEEMFD




DAIKTCREKKLIPLNTEEIVLAAEKFEEVKEQIAVK






Dihydroflavonol 4-
MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATVRDPANKKKV
61


reductase (DFR)
KHLLDLPKAATHLTLWKADLAEEGSFDEAIKGCTGVFHVATPMDF




Rosa chinensis

ESKDPENEVIKPTINGVLDIMKACLKAKTVRRLVFTASAGSVNVE



Accession:
ETQKPVYDESNWSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKE



XP_024167119.1
NNIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSIIKQ




GQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIHEIAKLLREK




YPEYNVPTTFKGIEENLPKVHFSSKKLLETGFEFKYSLEDMFVGA




VDACKAKGLLPPPTERVEKQEVDESSVVGVKVTG






Leucoanthocyanidin
MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTTFLLVRS
62


reductase (LAR)
LDKGAIPSKSKTINSLHDKGAILIHGVIEDQEFVEGILKDHKIDI




Camellia sinensis

VISAVGGANILNQLTIVKAIKAVGTIKRFLPSEFGHDVDRANPVE



Accession:
PGLAMYKEKRMVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV



XP_028127206.1
IPPLDRFQIYGDGTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHF




RPSCNFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAENIIPQ




SIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPDETFRTMDECF




DDFVMKMDRWN






Leucoanthocyanidin
MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGKSTYILS
63


reductase (LAR)
RAAARCPSKARAIKALEDQGAISIHGSVNDQEFMEKTLKEHEIDI




Coffea arabica

VISAVGGGNLLEQVILIRAMKAVGTIKRFLPSEFGHDVDRAEPVE



Accession:
PGLTMYNEKRRVRRLIEESGVPYTYICCNSIASWPYYDNTHPSEV



XP_027097479.1
SPPLDQFQIYGDGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHF




RPSCNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEENIIPQ




SVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYPDEPFQTMDECF




NEFAGKIEEDKKHVVGTKGKNIAHRLVDVLTAPKLCA






Leucoanthocyanidin
MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEASLDSGRPTY
64


reductase (LAR)
ILARSSSNSPSKASTIKFLQDRGATVIYGSITDKEFMEKVLKEHK




Theobroma cacao

IEVVISAVGGGSILDQFNLIEAIRNVDTVKRFLPSEFGHDTDRAD



Accession:
PVEPGLTMYEQKRQIRRQIEKSGIPYTYICCNSIAAWPYHDNTHP



ADD51357.1
ADVLPPLDRFKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKT




VHFQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQMAKEMR




IPQSVVAALTHDIFINGCQINFSLDKPTDVEVCSLYPDTPFRTIN




ECFEDFAKKIIDNAKAVSKPAASNNAIFVPTAKPGALPITAICT






Leucoanthocyanidin
MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLPTYVLVR
65


reductase (LAR)
PGPSRPSKSDTIKSLKDRGAIILHGVMSDKPLMEKLLKEHEIEIV




Fragaria x

ISAVGGATILDQITLVEAITSVGTVKRFLPSEFGHDVDRADPVEP




ananassa

GLTMYLEKRKVRRAIEKSGVPYTYICCNSIASWPYYDNKHPSEVV



Accession:
PPLDQFQIYGDGTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFR



ABH07785.2
PSSNLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAENRIPES




IVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEESFRTLDECFN




DFLVKVGGKLETDKLAAKNKAAVGVEPMAITATCA






Anthocyanin
MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRLNEELTSM
66


dioxygenase (ANS)
GNVFEEEKKEEGSQVPTIDIKDIASEDPEVRGKAIQELKRAAMEW




Chenopodium

GVMHLVNHGISDELIDRVKVAGQTFFELPVEEKEKYANDQASGNV




quinoa

QGYGSKLANSASGRLEWEDYYFHLSYPEDKRDLSIWPETPADYIP



Accession:
AVSEYSKELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQF



XP_021735950.1
KINYYPKCPQPELALGVEAHTDVSALTFILHNMVPGLQLFYEGKW




VTAKCVPNSIIMHIGDTIEILSNGKYKSILHRGLVNKEKVRISWA




VFCEPPKEKIILKPLPDLVSDEEPARYPPRTFAQHVQYKLFRKTQ




GPQTTITKN






Anthocyanin
MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDDSLGDALEE
67


dioxygenase (ANS)
IKKTEEGPQIPIVDLRGFDSGDEKERLHCMEEVKEAAVEWGVMHI




Iris sanguinea

VNHGIAPELIERVRAAGKGFFDLPVEAKERYANNQSEGKIQGYGS



Accession:
KLANNASGQLEWEDYFFHLIFPSDKVDLSIWPKEPADYTEVMMEF



QCI56004.1
AKQLRVVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKINYY




PKCPQPELALGVEAHTDVSSLSFILHNGVPGLQVFHGGRWVNARL




VPGSLVVHVGDTLEILSNGRYKSVLHRGLVNKEKVRISWAVFCEP




PKEKIVLEPLAELVDKRSPAKYPPRTFAQHIQHKLFKKAQEQLAG




GVHIPEAIQN






Anthocyanin
MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGDVFEEEKK
68


dioxygenase (ANS)
LEGPQIPVVDLMGLEWENEEVFKKVEEDMKKAASEWGVMHIFNHG




Magnolia sprengeri

ISMELMDRVRIAGKAFFDLPIEEKEMYANDQASGKIAGYGSKLAN



Accession:
NASGQLEWEDYFFHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQL



AHU88620.1
RGLVTKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYYPKCP




QPDLALGVEAHTDVSALTFILHNMVPGLQVFFDDKWVTAKCIPGA




LVVHIGDSLEILSNGKYRSILHRGLVNKEKVRISWAIFCEPPKEK




VVLQPLPELVSEAEPARFTPRTFSQHVRQKLFKKQQDALENLKSE






Anthocyanin
MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIGNVFEEEK
69


dioxygenase (ANS)
KEGPEVPTIDLTEIESEDEVVRARCHETLKKAAQEWGVMNLVNHG




Prosopis alba

IPEELLNQLRKAGETFFSLPIEEKEKYANDQASGKIQGYGSKLAN



Accession:
NASGQLEWEDYFFHLVFPEDKCDLSIWPRTPSDYIEVTSEYARQL



XP_028787846.1
RGLATKILGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPICP




QPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQWITAKCVPNS




IFMHIGDTVEILSNGRYKSILHRGLVNKEKVRISWAVFCEPPKEK




IILKPLPELVTDDEPARFPPRTFAQHIQHKLFRKCQEGLSK






Anthocy anidin-3-
MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPNTHFSFLN
70


O-glycotransferase
TSQSNNSIFSSDVYNRQPNLKAHNVWDGVPEGYVFVGKPQESIEL



(3GT)
FVKAAPETFRKGVEAAVAETGRKVSCLVTDAFFWFAAEIAGELGV




Cephalotus

PWVPFWTAGPCSLSTHVYTDLIRKTIGVGGIEGREDESLEFIPGM




follicularis

SQVVIRDLQEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEE



Accession:
LDLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLPWLDQQK



GAV66155.1
VASVAYISFGTVMAPSPPELVALAEALEASKIPFIWSLGEKLKVH




LPKGFLDKTRTHGIVVPWAPQSDVLENGAVGVFITHCGWNSLLES




IAGGVPMICRPFFGDQRLNGRMVQDVWEIGVTATGGPFTTEGVMG




DLDLILSQARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALLNL




VRLSI






Anthocy anidin-3-
MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRRLAAASPNT
71


O-glycotransferase
LFSFFSTSQSNNSLFSNTITNLPRNIKVFDVADGVPDGYVFAGKP



(3GT)
QEDIELFMKAAPHNFTTSLDTCVAHTGKRLTCLITDAFLWFGAHL




Prunus cerasifera

AHDLGVPWLPLWLSGLNSLSLHVHTDLLRHTIGTQSIAGRENELI



Accession:
TKNVNIPGMSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN



AKV89253.1
AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAASPPLPEAP




TAADDVTGCLSWLDKQKAASSVVYVSFGSVARPPEKELLAMAQAL




EASGVPFLWSLKDSFKTPLLNELLIKASNGMVVPWAPQPRVLAHA




SVGAFVTHCGWNSLLETIAGGVPMICRPFFGDQRVNARLVEDVLE




IGVTVEDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLLAQQP




VEPKGSSAQNFKLLLDVISGSTKV






Anthocy anidin-3-
MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFSFFNSAVS
72


O-glycotransferase
NSTLFNNGVLDSYDNIRVYHVWDGTPQGQAFTGSHFEAVGLFLKA



(3 GT)
SPGNFDKVIDEAEVETGLKISCLITDAFLWFGYDLAEKRGVPWLA




Scutellaria

FWTSAQCALSAHMYTHEILKAVGSNGVGETAEEELIQSLIPGLEM




baicalensis

AHLSDLPPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEID



Accession:
PIITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLDSQTR



A0A482AQV3
PKSVVYISFGTVITPPENELAALSEALETCNYPFLWSLNDRAKKS




LPTGFLDRTKELGMIVPWAPQPRVLAHRSVGVFVTHCGWNSILES




ICSGVPLICRPFFGDQKLNSRMVEDSWKIGVRLEGGVLSKTATVE




ALGRVMMSEEGEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEI




INAPQSS






Anthocy anidin-3-
MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPHAVFSFFST
73


O-glycotransferase
SQSNASIFHDSMHTMQCNIKSYDISDGVPEGYVFAGRPQEDIELF



(3GT)
TRAAPESFRQGMVMAVAETGRPVSCLVADAFIWFAADMAAEMGLA




Vitis vinifera

WLPFWTAGPNSLSTHVYIDEIREKIGVSGIQGREDELLNFIPGMS



Accession:
KVRFRDLQEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL



P51094
DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQWLKERKP




TSVVYISFGTVTTPPPAEVVALSEALEASRVPFIWSLRDKARVHL




PEGFLEKTRGYGMVVPWAPQAEVLAHEAVGAFVTHCGWNSLWESV




AGGVPLICRPFFGDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSC




FDQILSQEKGKKLRENLRALRETADRAVGPKGSSTENFITLVDLV




SKPKDV






Acetyl-CoA
MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHSVITKVLIC
74


carboxylase (ACC)
NNGIAAVKEIRSIRKWAYETFGDERAIEFTVMATPEDLKVNADYI



Ustilago maydis
RMADQYVEVPGGSNNNNYANVDLIVDVAERAGVHAVWAGWGHASE



521
NPRLPESLAASKHKIIFIGPPGSAMRSLGDKISSTIVAQHADVPC



Accession:
MPWSGTGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAEKIG



XP_011390921.1
YPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLGEVPGSPVFVMK




LAGQARHLEVQLLADQYGNAISIFGRDCSVQRRHQKIIEEAPVTI




APEDARESMEKAAVRLAKLVGYVSAGTVEWLYSPESGEFAFLELN




PRLQVEHPTTEMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDP




RGNEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDTGFKPG




MGALTELNFRSSTSTWGYFSVGTSGALHEYADSQFGHIFAYGADR




SEARKQMVISLKELSIRGDFRTTVEYLIKLLETDAFESNKITTGW




LDGLIQDRLTAERPPADLAVICGAAVKAHLLARECEDEYKRILNR




GQVPPRDTIKTVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRT




LVQLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSKTCLIEQ




ENDPTQIRSPSPGKLVRFLVDSGDHVKANQAIAEIEVMKMYLPLV




AAEDGVVSFVKTAGVALSPGDIIGILSLDDPSRVQHAKPFAGQLP




DFGMPVIVGNKPHQRYTALVEVLNDILDGYDQSFRMQAVIKELIE




TLRNPELPYGQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEF




PAARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYAGGLKA




HEGNVLASFLQKYYEVESQFTGEADVVLELRLQADGDLDKVVALQ




TSRNGINRKNALLLTLLDKHIKGTSLVSRTSGATMIEALRKLASL




QGKSTAPIALKAREVSLDADMPSLADRSAQMQAILRGSVTSSKYG




GDDEYHAPSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALCT




YVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQLPRSASSLKER




ERQVSISDLSMMDNNRRARPIRELRTGAMTSCADVADIPELLPKV




LKFFKSSAGASGAPINVLNVAVVDQTDFVDAEVRSQLALYTNACS




KEFSAARVRRVTYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIE




PALAYQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADTRFF




VRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEVALGQPEYRTA




DASHIFMSFIYQLDVSLVDVQKAIAGFLERHGTRFFRLRITGAEI




RMILNGPNGEPRPIRAFVTNETGLVVRYETYEETVADDGSVILRG




IEPQGKDATLNAQSAHFPYTTKVALQSRRSRAHALQTTFVYDFID




VLGQAVRASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRAPG




MNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQAGSFGPVEDRF




FAAASKLARELGVPRLYISANSGARIGLATEALDLFKVKFVGDDP




AKGFEYIYLDDESLQAVQAKAPNSVMTKPVQAADGSVHNIITDII




GKPQGGLGVECLSGSGLIAGETSRAKDQIFTATIITGRSVGIGAY




LARLGERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQIM




YKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGPLPIMPTTDSWD




RAVTYQPPRGPYDPRWLINGTKAEDGTKLTGLFDEGSFVETLGGW




ATSVVTGRARLGGIPVGVIAVETRTLERVVPADPANPNSTEQRIM




EAGQVWYPNSAYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMY




DEILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVVVDSAIND




NGMIEMSADVNSARGGVLEASGLVEIKYRADKQRATMERLDSVYA




KLSKEAAEATDFTAQTTARKALAEREKQLAPIFTAIATEYADAHD




RAGRMLATGVLRSALPWENARRYFYWRLRRRLTEVAAERTVGEAN




PTLKHVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLAASKQL




KAQYILAQISTLDPELRAQLAASLK






Acetyl-CoA
MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHTVISKVLIAN
75


carboxylase (ACC)
NGMAAMKEIRSVRKWAYETFGNERAIEFTVMATPEDLKANAEYIR




Hesseltinella

MADNYIEVPGGTNNNNYANVELIVDVAERTGVHAVWAGWGHASEN




vesiculosa

PRLPEMLAKSKNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTM



Accession:
GWSGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLASAEKIGF



ORX57605.1
PVMIKASEGGGGKGIRKVEDPSTFKQAFAQVQGEIPGSPIFIMKL




AGNARHLEVQLLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIA




KPDIFEQMEKAAVRLGKLVGYVSAGTVEYLYSHHDEKFYFLELNP




RLQVEHPTTEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN




SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPDAGFKPSG




GVMQELNFRSSTNVWGYFSVVSSGAMHEYADSQFGHIFAYGENRQ




QARKNMVIALKELSIRGDFRTTVEYIIRLLETPDFTDNTINTGWL




DMLISKKLTAERPDTMLAVFCGAVTKAHLASVECWQQYKNSLERG




QIPSKESLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKTQ




VGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMIDGKTCLLEKE




SDPTQLRSPSPGKLVSLLLENGDHIRTGQAYAEIEVMKMYMPLVA




SEDGHVQFIKQVGATLEAGDIIGILSLDDPSRVKHALPFTGQVPK




YGLPHLTGDKPHQRFTHLKQTLEYVLQGYDNQGLIQTIVKELSEV




LNNPELPYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKGFP




AVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIAASYSNGLKQ




HEHSVYVDLMEQYYNVEVLFNSNQSRDEEVILALRDQHKDDLEKV




INIILSHAKVNIKNNLILMLLDIIYPATSSEALDRCFLPILKHLS




EIDSRGTQKVTLKAREYLILCQLPSLEERQSQMYNILKSSVTESV




YGGGTEYRTPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAAL




EVYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSAGGGFNNQR




IASYSDLTFLLNKTEEEPIRTGAMVALKTLEELEAELPRIMTAFE




EEPLPPMLMKQPPPDKTEERMENILNISIQGQDMEDDTLRKNMTT




LIQAHSDAFRKAALRRITLVVCRDNQTPDYYTFRERNGYEEDETI




RHIEPALAYQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC




RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLEIVSHDY




KNSDCNHLFINFIPTFAIEADEVETALKDFVDRHGKRLWKLRVTG




AEIRFNIQSKRPDAPVIPLRFTVDNVSGYILKVDVYQEVKTDKNG




WILKSVGKIPGAMHMQPLSTPYPTKEWLQPRRYKAHLMGTTYVYD




FPELFRQAIHNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA




IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDITFKIGSFG




VQEDLVFYKASEYARELGVPRVYLSANSGARIGLADELISRFHVA




WKDEDQPGSGFEYLYLLPEEYDALIQQGDAQSVLVQEVQDKGERR




FRITDIIGHTDGLGVENLRGSGLIAGATSRAYDDIFTITLVTCRS




VGIGAYLVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNLQL




GGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPECRGAPLPMRA




GADPIDREIEYLPPKGPSDPRFFLAGKQENGKWLSGFFDHGSFVE




TLSGWARTVVVGRARLGGIPMGVVAVETRTVENIVPADPANADSQ




EQVVMEAGGVWFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSG




GQRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGELRGGAWVVV




DSTINEDMMEMYADTQARGGVLEPEGIVEIKYRRPQLLATMERLD




PVYSDLKRRLAALDDSQKEQADELIAQVEAREQALLPVYQQVAIQ




FADLHDRSGRMEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIR




KMDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVAWFDAKNLLLD




QRIAKLKSEKLKDHVVQLASVDQDAVVEGFSKLMESLSVDQRKEV




LHKLATRF






Acetyl-CoA
MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVFMHGLGSST
76


carboxylase (ACC)
SFWEAPFSRSNLSSRFRLIRYDFDGHGLSPVSLLDAADDGAMIPL




Rhodotorula

VDLVEDLAAVMEWTGVDKVAGIVGHSMSGLVASTFAAKYPQKVEK




toruloides

LVLLGAMRSLNPTVQTNMLKRADTVLESGLSAIVAQVVSAALSDK



NBRC10032
SKQDSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDYSTIKAK



Accession:
TLVVSGESDYLSNKETTEALVNDIPGAKEVQMDGVGHWHAVEDPA



GEM08739.1
GLAKILDGFFLQGKFSGEAKAVNGSHAVDETPKKPKYDHGRVVKY




LGGNSLESAPPSNVADWVRERGGHTVITKILIANNGIAAVKEIRS




VRKWAYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVEVPGG




TNNNNYANVDVIVDVAERAGVHAVWAGWGHASENPRLPESLAASK




HKIVFIGPPGSAMRSLGDKISSTIVAQHAEVPCMDWSGQGVDQVT




QSLEGYVTVADDVYQQACVHDADEGLARASRIGYPVMIKASEGGG




GKGIRKVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLEVQV




LADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAKPDTFEQMEKS




AVRLAKLVGYVSAGTVEFLYSAADDKFAFLELNPRLQVEHPTTEM




VSGVNLPAAQLQVAMGVPLHRIRDIRTLYGKAPNGSSEIDFEFEN




PESAKTQRKPSPKGHVVAVRITAENPDAGFKPSMGTLQELNFRSS




TNVWGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKNMVVALK




ELSIRGDFRTTVEYLIKLLETDAFEQNTITTAWLDSLISARLTAE




RPDTTLAIICGAVTKAHLASEANIAEYKRILEKGQSPPKELLATV




VPLEFVLEDVKYRATASRSSPSSWSIYVNGSNVSVGIRPLADGGL




LILLDGRSYTCYAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSP




GKLVRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQFIKQ




PGATLEAGDILGILSLDDPSRVHHAKPFDGQLPALGLPSIIGTKP




HQRFAYLKDVLSNILMGYDNQAIMQSSIKELISVLRNPELPYGEA




NAVLSTLSGRIPAKLEQTLRQYIDSAHESGAEFPSAKCRKAIDTT




LEQLRPAEAQTVRNFLVAFDDIVYRYRSGLKHHEWSTLAGIFAAY




AETEKPFSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKNSL




VLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKATTKVALKARE




VLIHIQLPSLDERLGQLEQILKASVTPTVYGEPGHDRTPRGEVLK




DVIDSRFTVFDVLPSFFQHQDQWVSLAALDTYVRRAYRSYNLLNI




EHIEADAAEDEPATVAWSFRMRKAASESEPPTPTTGLTSQRTASY




SDLTFLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFPDSNK




GKLQQQPAASSSQEQWNVINVALTVPASAQVDEDALRADFAAHVN




AMSAEIDARGMRRLTLLICREGQYPSYYTVRKQDGTWKELETIRD




IEPALAFQLELGRLSNFHLEPCPVENRQVHIYYATAKGNSSDCRF




FVRALVRPGRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRRA




ADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRFWRLRVTGAE




IRIVLEDAQGNIQPIRAIIENVSGFVVKYEAYREVTTDKGQVILK




SIGPQGALHLQPVNFPYPTKEWLQPKRYKAHVVGTTYVYDFPDLF




RQAIRKQWKAVGKTAPAELLVAKELVLDEFGKPQEVARPPGTNNI




GMVGWIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFYAV




TQLARQLGLPRVYLSANSGARLGIAEELVDLFSVAWADSSRPEKG




FKYLYLTAEKLGELKNKGEKSVITKRIEDEGETRYQITDIIGLQE




GLGVESLKGSGLIAGETSRAYDDIFTITLVTARSVGIGAYLVRLG




QRAVQVEGQPIILTGAGALNKVLGREVYSSNLQLGGTQIMYKNGV




SHLTAANDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSID




YTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSFQETLSGWAQT




VVVGRARLGGIPMGAIAVETRTIERIIPADPANPLSNEQKIMEAG




QVWYPNSSFKTGQAIFDFNREGLPLIIFANWRGFSGGQQDMFDEV




LKRGSLIVDGLSAYKQPVFVYIVPNGELRGGAWVVLDPSINAEGM




MEMYVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTYHALRV




KSTDASLSPTDAAQAKTELAAREKQLMPIYQQVALQFADSHDKAG




RILSKGCAREALEWSNARRYFYARLRRRLAEEAAVKRLGEADPTL




SRDERLAIVHDAVGQGVDLNNDLAAAAAFEQGAAAITERVKLARA




TTVASTLAQLAQDDKEAFAASLQQVLGDKLTAADLARILA






Malonyl-CoA
MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVARSGRMANVL
77


synthase (matB)
VARGVKPGDRVAAQAEKSVAALVLYLATVRAGAVYLPLNTAYTLH




Rhodopseudomonas

ELDYFIGDAEPKLVVCDPAKREGIAALAQKVGAGVETLDAKGQGS




palustris

LSEAAAQASVDFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNL



Accession:
ASNSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLFARASM



WP_011661926.1
IFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQSDGLTKEAARHM




RLFISGSAPLLADTHREWASRTGHAVLERYGMTETNMNTSNPYDG




ARVPGAVGPALPGVSLRVVDPETGAELSPGEIGMIEVKGPNVFQG




YWRMPEKTKAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITG




GFNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAVVVRDKG




ASVDEAAVLGALQGQLAKFKMPKRVLFVDDLPRNTMGKVQKNVLR




EAYAKLYAK






Malonyl-CoA
MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCSARITHVL
78


synthase (matB)
VKLGVSPGDRVAVQVEKSAQALMLYLGCLRAGAVYLPLNTAYTPA




Rhizobium

ELEYFLGDATPKLVVVSPCAAEQLEPLARRVGTRLLTLGVNGDGS



sp. BUS003
LMDMASLEPVEFADIERKADDLAAILYTSGTTGRSKGAMLTHDNL



Accession:
LSNAQTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAGGAI



NKF42351.1
YLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQDERLNKANTRHM




RLFISGSAPLLAETHRLFEEYTGHAILERYGMTETNMITSNPCDG




ARVPGTVGYALPGVSVRITDPVSGEPLAAGEPGMIEVKGPNVFQG




YWNMPDKTKEEFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISG




GYNIYPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVGQPK




AHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNTMGKVQKNVLR




DRYRDLYLK






Malonyl-CoA
MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMTGTARYANVL
79


synthase (matB)
VGLGVKPGDRVAVQVEKSAGAIFLYLACVRAGAVFLPLNTAYTLT




Ochrobactrum sp.

EIEYFLGDAEPALVVCDPARRDGITEVAKKTGVPAVETLGKGQDG



3-3
SLFDKAAAAPETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDN



Accession:
LASNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTILVAGAS



WP_114216069.1
MLFLPKFDADKVFELMPRATTMMGVPTFYVRLVQDARLTREATKH




MRLFISGSAPLLAETHKLFREKTGVSILERYGMTETNMNTSNPYD




GDRVAGTVGFPLPGVALRVADPETGAAIPQGEIGVIEVKGPNVFS




GYWRMPEKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLVIS




GGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGEGVTAVVVAEK




GASLDEATIIKTLEQRLARYKLPKRVIVVDDLPRNTMGKVQKNLL




RDAYKGLYGG






Malonate
MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGTLVADLDA
80


transporter (matC)
DGIFAGFPGDLFVVLVGVTYLFAIARANGTTDWLVHAAVRLVRGR




Rhizobiales

VALIPWVMFALTGALTAIGAVSPAAVAIVAPVALSFATRYSISPL




bacterium

LMGTMVVHGAQAGGFSPISIYGSIVNGIVEREKLPGSEIGLFLAS



Accession:
LVANLLIAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGSG



MBN8942514.1
SDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGGAEGTGVRLTP




ARVATLVALVALVVAVLGFDLDAGLTAVTLAVVLSTAWPDDSRRA




VGEIAWSTVLLICGVLTYVGVLEEMGTITWAGEGVGGIGVPLLAA




VLLCYIGAIVSAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAAL




AVSATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVYGGIVVAA




VPALAWLVLVVPGFG






Malonate
MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMIIGMKT
81


transporter (matC)
NEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRLVRGR




Rhizobium

IGLIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV




leguminosarum

MMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS



Accession:
FFFNLAIAVLVFFVFGGARVMKHDPASLGPLPELHPEGVSASIRG



AAC83457.1
HGGTPAKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIGAL




VFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSWSTVLLIAGIIT




YVGVMEKAGTVDYVANGISSLGMPLLVALLLCFTGAIVSAFASST




ALLGAIIPLAVPFLLQGHISAIGVVAAIAISTTIVDTSPFSTNGA




LVVANAPDDSREQVLRQLLIYSALIAIIGPIVAWLVFVVPGLV






Malonate
MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLIIGMKP
82


transporter (matC)
ADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWLVERSVRMVRGR




Agrobacterium vitis

VGWIPWVMFLVAAIITGFGALGPAAVAILAPVALSFAVQYRIHPV



Accession:
LMGLMVIHGAQAGGFSPISIYGGITNQIVAKAGLPFAPTSLFLSS



WP_180575084.1
FFFNLAIAVLIFFIFGGLSILKQRSSVKGPLPELHPEGISASIKG




HGGTPAKPFREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL




VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTVLLISGIIT




YVGVMEKAGTIDYVAHGISSLGMPLLVALLLCFTGAIVSAFASST




ALLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGA




LVVANAPDDQRDKVMRQMLIYSALIALIGPVIAWLVFVVPGII






Malonate
MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLIIGMKT
83


transporter (matC)
SDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWLVECAVRMVRGH




Neorhizobium sp.

VAWIPWVMFVVAAITGFGALGPAAVAILAPVALSFAVQYRIHPVM



Accession:
MGLMVIHGAQAGGFSPISVYGGITNQIVAKAGLPFAPTSLFLSSF



WP_105370917.1
FFNLAIAVLVFFVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGH




GGTPAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGIGALV




FKFNVGLVAITVAVALALISPKTQKAAVDKVSWSTVLLIAGIITY




VGVLEKAGTVNYVANGISSLGMPLLVALLLCFTGAIVSAFASSTA




LLGAIIPLAVPFLLQGHISAVGVVAAIAISTTIVDTSPFSTNGAL




VVANAPDETREQVLRQLLIYSALIAIIGPVVAWLVFVVPGLV






Malonate CoA-
MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALLETVIDRGDR
84


transferase (MdcA)
VCLEGNNQKQADFLSKSLSSCNPDIVNGLHIVQSVLALPSHIDVF




Moraxella

ERGIASKVDFSFAGPQSLRLAQLVQAQKITIGAIHTYLELYGRYF




catarrhalis

IDLTPNVALITAHAADKRGNLYTGANTEDTPAIVEATTFKSGIVI



Accession:
AQVNEIVDELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITE



WPO64617969.1
IQILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTYAESLG




LKGEICTHWALNPHPTLIPAIESGFIHSVHSFGSEVGMENYVKAR




SDVFFTGADGSMRSNRAFSQTAGLYACDLFIGSTLQIDLQGNSST




ATADRIAGFGGAPNMGSDPHGRRHASYAYMKAGREAVDGSPIKGR




KLVVQMVETYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDDV




THIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLGRDDTMVAR




LRERKVIQRPEDLGINPMHATRDLLAAKSVKDLVRWSDRLYEPPS




RFRNW






Malonate CoA-
MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEIPVDRIIDLL
85


transferase (MdcA)
EAVIQPGDRVCLEGNNQKQADFLSESLADCDPARINHLSMVQSVL




Dechloromonas

ALPSHVDLFERGLATRLDFSFSGPQGARLAKLVQEQRIEIGAIHT




aromatica

YLELFGRYFMDLTPNVALIAAQAADAEGNLYLGPNTEDTPAIVEA



Accession:
TAFKGGIVIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPLF



WP_011289741.1
TRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIGFDTAAIELL




LPTYAADLGLKGKICTHWALNPHPTLIPAIEAGFVESVHCFGSEV




GMDDYISARSDIFFTGADGSMRSNRAFSQTAGLYACDMFIGSTLQ




MDLAGNSSTATLGRITGFGGAPNMGSDPHGRRHASPAWLKAGREA




YGPQAIRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASMGSDLP




PIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAIRGVAGFTPVGM




ARDKGTVENLRDRGIIRRPEDLGIDPRQASRDLLAARSIKDLVRC




SGGLYAPPSRFRNW






Malonate CoA-
MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVVALLEAVIEPG
86


transferase (MdcA)
DRVCLEGNNQKQADFLARCLTEVDPARVHDLHMVQSVLSLAAHLD




Pseudomonas

VFERGIAKRLDFSFSGPQAARLAGLVSEGRIEIGAIHTYLELFGR




cissicola

YFIDLTPRIALVTAQAADRHGNLYTGPNTEDTPVIVEATAFKGGI



Accession:
VIAQVNEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPAQI



WP_078590875.1
SEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIELLLPTYAQS




LGLKGKICRHWALNPHPALIPAIESGFVQSVHSFGSELGMENYIA




ARPDIFFTGADGSMRSNRALSQTAGLYACDMFIGSTLQIDLQGNS




STATRDRIAGFGGAPNMGSDARGRRHASAAWLKAGREAATPGEMP




RGRKLVVQMVETFREHMAPAFVDRLDAWELAERANMPLPPVMIYG




DDVSHVLTEEGIANLLLCRTPEEREQAIRGVSGYTAVGLGRDKRM




VENLRDRGVIKRPDDLGIRPRDATRDLLAARTVKDLVRWSGGLYD




PPKRFRNW






Malonate CoA-
MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTEKIVEALTA
87


transferase (MdcA)
LIFPGDRVVIEGNNQKQASFLSKALSQVNPEKVNGLHIIMSSVSR




Geobacillus

PEHLDLFEKGIARKIDFSYAGPQSLRMSQMLEDGKLVIGEIHTYL




subterraneus

ELYGRLFIDLTPSVALVAADKADASGNLYTGPNTEETPTLVEATA



Accession:
FRDGIVIAQVNELADELPRVDIPGSWIDFVVAADHPYELEPLFTR



WP_184319829.1
DPRLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIELLLP




TYGESLGLKGKICKHWALNPHPTLIPAIETGWVESIHCFGGEVGM




EKYIAARPDIFFTGKDGNLRSNRTLSQVAGQYAVDLFIGSTLQID




RDGNSSTVTNGRLAGFGGAPNMGHDPRGRRHSSPAWLDMITSDHP




AAKGRKLVVQMVETFQKGNRPVFVESLDAIEVGRSARLATTPIMI




YGEDVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLERDP




RKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSIEELVEWSEGL




YEPPARFRSW






Pantothenate kinase
MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADELAVLLQG
88


(CoaX)
LMGTHPLLGMELGEGIDGIAICSTVPAVLHELREVSRRYYGDVPA




Streptomyces sp.

ILVEPGVKTGVPILMDNPKEVGTDRIINAVAAQHLYGGPAIVVDF



CLI2509
GTATTFDAVSARGEYTGGVIAPGIEISVEALGLRGAQLRKIELAR



Accession:
PRSVIGKSTVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVTV



WP_095682415.1
IATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERNAGRR






Pantothenate kinase
MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQAPAELLKGLTF
89


(CoaX)
RLERLNPDFIGVSSVLGQAVNNHVAESLERLQKPFEFAQVHAKHA




Streptomyces

LMSSDYNPAQLGVDRWLQMLGIIEPSKKQCVIGCGTAVTIDLVDQ




cinereus

GHHLGGYIFPSIYLQRESLFSGTRQISIIDGTFDSIDSGTNTQDA



Accession:
VHHGIMLSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI



WP_188874884.1
RQDLVLAGLQRFFAAKNNTKNQN






Pantothenate kinase
MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTADELAVLMQG
90


(CoaX)
LMGRQPGGAGRERVDGLAICSSVPAVLHELREVTRRYYGDLPAVL




Kitasatospora

VAPGVKTGVHVLMDNPKEVGADRIVNALAANHLYGGPCIVVDFGT




kifunensis

ATTFDAINERGDYVGGAIAPGIEISVEALGVRGAQLRKIELAKPR



Accession:
NVIGKNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPEDVQVI



WP_184936930.1
ATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYERNTAS






glutamyl-tRNA
MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQALPMVQGGV
91


reductase (hemA)
VLSTCNRTEIYLSVEEQDNLREALIRWLCEYHNLNEEDLRNSLYW




Citrobacter

HQDNDAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQKGHQNA




freundii

SALERMFQKSFSVAKRVRTETDIGSSAVSVAFAACTLARQIFESL



Accession:
STVTVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQVLADE



NTY05430.1
VGAEVISLSDIDARLQDADIIISSTASPLPIIGKGMVERALKNRR




NQPMLLVDIAVPRDVEPEVGKLSNAYLYSVDDLQSIISHNLAQRK




AAAVEAETIVEQEASEFMAWLRAQGASDTIREYRSQSEQIRDELT




AKALAALQQGGDAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDS




ERLNILRDSLGLE






glutamyl-tRNA
MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQPLVQGGV
92


reductase (hemA)
VLSTCNRTELYLSVEQQENLQEQLVKWLCDYHHLSADEVRKSLYW




Pseudomonas

HQDNAAVSHLMRVASGLDSLVVGEPQILGQVKKAFAESQHGQAVS




reactans

GELERLFQKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL



Accession:
SDVSVLLVGAGETIELVARHLREHKVRHMMIANRTRERAQVLASE



NWA43040.1
VGAEVITLQDIDARLADADIIISSTASPLPIIGKGMVERALKARR




NQPMLMVDIAVPRDIEPEVGKLANAYLYSVDDLHSIIQNNMAQRK




AAAVQAESIVEQESSNFMAWLRSQGAVEIIRDYRSRADLVRAEAE




AKALAAIAQGADVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDV




ERLQILRDSLGLDQQ






glutamyl-tRNA
MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQQPAVRGGV
93


reductase (hemA)
VLSTCNRTELYLSMEDKENSHEQLIRWLCQYHQIEPNELQSSIYW




Gammaproteobacteria

HQDNQAVSHLMRVASGLDSLVLGEPQILGQVKKAFADSQNYDSLS



Accession:
SELERLFQKSFSVAKRVRTETQIGANAVSVAFAACTLARQIFESL



WP_193016510.1
SSLTILLVGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE




VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVERALKKRR




NQPMLLVDIAVPRDIEQDVEKLNNVYLYSVDDLEAIIQHNREQRQ




AAAVQAEHIVQQESGQFMDWLRAQGAVGAIREYRDSAETLRAEMT




EKAITLIQNGADAEKVIQQLSHQLMNRLIHTPTKSLQQAASDGDI




ERLNLLRESLGITHN






5-aminolevulinic
MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITIPPNATVEMC
94


acid synthase
PHAKAARDAARIAEDLAAAAASKQQPAKKAGGCPFHAAQAQAQAK



(ALAS)
PAAAPKETVATADKKGKSPRAAGGFDYEKFYEEELDKKHQDKSYR




Schizophyllum

YFNNINRLAARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLETM




commune H4-8

HRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLHRKEGALVFTS



Accession:
CYVANDATLSTLGSKMPGCVIFSDRMNHASMIQGIRHSGTKKVIF



XP_003036856.1
EHNDLADLEKKLAEYPKETPKIIAFESVYSMCGSIGPIKEICDLA




EKYGAITFLDEVHAVGLYGPRGAGVAEHLDYDLHKAAGDSPDAIP




GTVMDRVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIFT




TSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREVKRRFAELDIP




VVPGPSHIVPVLVGDAALAKQASDKLLAEHDIYVQAINYPTVARG




EERLRITVTQRHTLEQMDHLIGAVDQVFNELNINRVQDWKRLGGR




ASVGVPGGQDFVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAV




VAARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQSSGLKL




PVEEMTMGQTIAVAA






5-aminolevulinic
MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTALTERATK
95


acid synthase
CPVMGPALNVRSKEIVAGYASVAANSDVALIHKEKGVFPPPGATV



(ALAS)
EMCPHASAARAAARMADDLAAAAEKKKGHFTSAAPRDEAAQAAAA




Crassisporium

GCPFHVKAAADAAAARKAAAAPAPVKAKEDGGFNYESFYVNELDK




junariophilum

KHQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCANDYLGMGN



Accession:
NPVVLETMHRTLDKYGHGAGGTRNIAGNGAMHLSLEQELATLHRK



KAF8165006.1
PAALVFSSCYVANDATLSTLGAKLPGCIFFSDTMNHASMIQGMRH




SGAKRVLFKHNDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGP




IKEICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDYDAHVAA




GESPHPIKGSVMDRVDIITGTLGKAYGAVGGYIAGSDDFVDMIRS




YAPGFIFTTSLPPATVAGARASVVYQKHYVGDRQLKQVNVREVKR




RFAELDVPVVPGPSHIVPVLVGDAALAKAASDKLLAEHNIYVQSI




NYPTVARGEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA




DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLDGTSPRTLRNG




EAAVVDAMAVGQARAVFDNLLGPISGKLQSERSVLASSTPAAANP




ARPAARKVVKMKTGGVPMSEDIPLPPPDVSASA






5-aminolevulinic
MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSISILTER
96


acid synthase
ATKCPVMGPALNVRSKEITAGYASVAGSSEVDQIHKQQGVTVPVN



(ALAS)
ATVEMCPHASAARAAARMADDLAAAAAQKKVGSGASSAKAAAAGC




Dendrothele

PFHKSVAAGASASTASKPSAPIHKASVPGGFDYDNFYNNELEKKH



bispora CBS
KDKSYRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLGMGNNP



962.96
VVLETMHRTLDKYGHGAGGTRNIAGNGALHLGLEQELAALHRKEA



Accession:
ALVFSSCYVANDATLSTLGSKLPGCILFSDKMNHASMIQGMRHSG



THV05492.1
AKKVIFNHNDLEDLENKLKQYPKETPKIIAFESVYSMCGSIGPIK




EICDLAEKYGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAAGK




SPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSMDFVDTIRSYA




PGFIFTTSLPPATVSGAQASVAYQKEYLGDRQLKQVNVREVKRRF




AELDIPVIPGPSHILPVLVGDAALAKAASDKLLTDHDIYVQSINY




PTVAVGEERLRITVTPRHTLEQMDKLVRAVNQVFTELNINRISDW




KVAGGRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQRFLVD




AQGVTAARGRFDTLLGPMSGSLQANPTLPLVD




DELKVPLPTLVAAAA






5-aminolevulinic
MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHALWHSPKGKR
97


acid synthase
DVVIWCSNDYLGMGQHPKVVGAMVETATRVGTGAGGTRNIAGTHH



(ALAS)
PLVQLEAELADLHGKEASLLFTSGYVSNQTGIATIAKLIPNCLIL




Bradyrhizobium

SDELNHNSMIEGIRQSGCERVVFRHNDLADLEEKLKAAGPNRPKL




japonicum

IACESLYSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMYGPRG



Accession:
GGIAERDGVMHRIDILEGTLAKAFGCLGGYIAANGQIIDAVRSYA



AOAOA3YXD2
PGFIFTTALPPAICSAATAAIRHLKTSNWERERHQDRAARVKAIL




NAAGLPVMSSDTHIVPLFIGDAEKCKQASDLLLEQHGIYIQPINY




PTVAKGTERLRITPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKS




LAAE






Cytochrome b5
MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLEEHPGGEEV
98



Petunia x hybrida,

LIESAGKDATKEFQDIGHSKAAKNLLFKYQIGYLQGYKASDDSEL



Accession:
ELNLVTDSIKEPNKAKEMKAYVIKEDPKPKYLTFVEYLLPFLAAA



AAD10774.1
FYLYYRYLTGALQF
















TABLE 12







Glossary of abbreviations










Abbreviation
Full Name






3GT
anthocyanidin-3-O-glycotransferase



4CL
4-coumarate-CoA ligase



ACC
acetyl-CoA carboxylase



ACOT
acyl-CoA thioesterase



acpP
acyl carrier protein



ACS
acetyl-CoA synthase



adhE
aldehyde-alcohol dehydrogenase



ADP
adenosine diphosphate



ALA
5-aminolevulinic acid



ALAS
ALA synthase



ANS
anthocyanin dioxygenase



aroG
DAHP synthase



aroK
shikimate kinase



aroL
shikimate kinase



ATP
adenosine triphosphate



C3G
cyanidin-3-O-glycoside



C4H
cinnimate-4-hydroxylase



CHI
chalcone isomerase



CHS
chalcone synthase



CoA
coenzyme A



CPR
cytochrome P450 Reductase



DAD
diode array detector



DAHP
deoxy-d-arabino-heptulosonate-7-phosphate



DctPQM
a malonate transporter



DFR
dihydroflavonol 4-reductase



DHK
dihydrokaempferol



DHM
dihydromyricein



DHQ
dihydroquercetin



DMSO
dimethyl sulfoxide



E4P
erythrose-4-phosphate



F3′H
flavonoid 3′ hydroxylase



F3H
flavanone 3-hydroxylase



fabB
beta-ketoacyl-ACP synthase I



fabD
malonyl-coA-ACP transacylase



fabF
beta-ketoacyl-ACP synthase II



FadA
3-ketoacyl-CoA thiolase



FadB
fatty acid oxidation complex subunit alpha



FadE
acyl-CoA dehydrogenase



GltX
glutamyl-tRNA synthetase



hemA
glutamyl-tRNA reductase



hemL
glutamate-1-semialdehyde aminotransferase



HPLC
high performance liquid chromatography



ldhA
lactate dehydrogenase



LAR
leucoanthocyanidin reductase



matB
malonyl-CoA synthase



matC
malonate transporter



mdcA
malonate coA-transferase



mdcC
acyl-carrier protein, subunit of mdc



mdcD
malonyl-CoA decarboxylase, subunit of mdc



mdcE
co-decarboxylase, subunit of mdc



pABA
para-aminobenzoic acid



PAL
phenylalanine ammonia-lyase



PanK
pantothenase kinase



Pdh
pyruvate dehydrogenase



PEP
phosphoenolpyruvate



pHBA
para-hydroxybenzoic acid



PHE
phenylalanine



pheA
chorismate mutase/prephenate dehydrogenase



poxB
pyruvate dehydrogenase



ppsA
phosphoenolpyruvate synthase



TAL
tyrosine ammonia-lyase



TCA
tricarboxylic acid cycle



tesA
thioesterase I



tesB
thioesterase II



tktA
transketolase



TRP
tryptophan



TYR
tyrosine



TyrA
chorismate mutase



tyrR
transcriptional regulator



ybgC
a thioesterase



yciA
a thioesterase



ydiB
QUIN/shikamate dehydrogenase



ackA-pta
Acetate kinase-phosphate acetyltransferase








Claims
  • 1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF), wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H).
  • 2. The cell of claim 1, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK).
  • 3. The engineered host cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • 4. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is truncated to remove the N-terminal leader sequence.
  • 5. The engineered host cell of claim 1, wherein the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • 6. The engineered host cell of claim 1, wherein the flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • 7. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • 8. The engineered host cell of claim 1, wherein the flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • 9. The engineered host cell of claim 1, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • 10. The engineered host cell of claim 1, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57.
  • 11. The engineered host cell of claim 1, wherein the engineered host cell further comprises cytochrome b5.
  • 12. The engineered host cell of claim 11, wherein the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
  • 13. The engineered host cell of claim 1, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.
  • 14. A method of increasing the production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayaone (PHF) comprising an engineered host cell, wherein the engineered host cell comprises cytochrome P450 reductase (CPR) and at least one of flavanone-3-hydroxylase (F3H), flavanone-3′-hydroxylase (F3′H), or flavonoid 3′,5′-hydroxylase (F3′5′H).
  • 15. The method of claim 14, wherein the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) are naringenin and/or dihydrokaempferol (DHK).
  • 16. The method of claim 14, wherein the flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • 17. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is truncated to remove the N-terminal leader sequence.
  • 18. The method of claim 14, wherein the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • 19. The method of claim 14, wherein the flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • 20. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • 21. The method of claim 14, wherein the flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • 22. The method of claim 14, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • 23. The method of claim 14, wherein the flavonoid 3′,5′-hydroxylase (F3′5′H) has an amino acid sequence at least 80% identical to the polypeptides selected from the group consisting of: (i) SEQ ID NO. 10, (ii) SEQ ID NO. 56, and (iii) SEQ ID NO. 57.
  • 24. The method of claim 14, wherein the engineered host cell further comprises cytochrome b5.
  • 25. The method of claim 24, wherein the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
  • 26. The method of claim 14, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptides selected from a group consisting of: (i) SEQ ID NO. 7, (ii) SEQ ID NO. 45, (iii) SEQ ID NO. 46, (iv) SEQ ID NO. 47, and (v) SEQ ID NO. 48.
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.

Provisional Applications (1)
Number Date Country
63174403 Apr 2021 US