US20220333118A1 - Flavonoid and anthocyanin bioproduction using microorganism hosts - Google Patents

Flavonoid and anthocyanin bioproduction using microorganism hosts Download PDF

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US20220333118A1
US20220333118A1 US17/720,031 US202217720031A US2022333118A1 US 20220333118 A1 US20220333118 A1 US 20220333118A1 US 202217720031 A US202217720031 A US 202217720031A US 2022333118 A1 US2022333118 A1 US 2022333118A1
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coa
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flavonoid
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Jingyi Li
Nicholas Brideau
Joshua Britton
Erik Holtzapple
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Debut Biotechnology Inc
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Debut Biotechnology Inc
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Definitions

  • the invention related to materials (including engineered cells and cell lines) and methods involved in the production of flavonoids, anthocyanins and other organic compounds.
  • 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.
  • malonyl-Cofactor-A malonyl-Cofactor-A
  • a range of flavonoids and anthocyanins including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, and cyaninidin-3-glucoside are biomanufactured using a modified microbial host.
  • 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.
  • 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.
  • 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.
  • 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).
  • feed molecules include, but are not limited to, acetate, malonate, tyrosine, phenylalanine, pantothenate, coumarate, etc.
  • the feed molecules may be of reduced or low purity.
  • glycerol as a feed molecule may be crude glycerol, including a biomass comprising glycerol, for example, glycerol obtained as a byproduct of biodiesel processing.
  • 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.
  • 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.
  • 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.
  • 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:
  • 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.
  • ACC acetyl-CoA carboxylase
  • 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.
  • acetyl-CoA carboxylase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin
  • 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).
  • acetyl-CoA synthase ACS
  • 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.
  • an engineered host cell that overexpresses a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA.
  • PDH pyruvate dehydrogenase
  • a variant of the Lpd subunit of PDH can be expressed that includes a mutation (E354K) that reduces inhibition of PDH by NADH.
  • 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.
  • malonyl-CoA synthetases examples 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.
  • 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.
  • 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.
  • 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.
  • CoA coenzyme A
  • Strategies for increasing CoA supply include upregulating endogenous pantothenate kinase (PanK) (EC:2.7.1.33) that produces CoA from pantothenate.
  • 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.
  • 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.
  • lactate dehydrogenase lactate dehydrogenase
  • pyruvate oxidase acetyl phosphate transferase and acetate kinase.
  • 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).
  • adhE aldehyde-alcohol dehydrogenase
  • ldhA lactate dehydrogenase
  • poxB pyruvate oxidase
  • ackA-pta enzyme acetate kinase phosphate acetyltransferase
  • 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-coumaroyl-CoA.
  • thioesterase genes tesA, tesB, yciA, and ybgC can be downregulated, disrupted, or deleted.
  • genes encoding enzymes of the tricarboxylic acid cycle 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.
  • TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
  • 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.
  • 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.
  • the Phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.
  • an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme.
  • Cytochrome P450 CYPs
  • CYPs 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.
  • 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).
  • gltX glutamyl-tRNA synthetase
  • hemA glutamyl-tRNA reductase
  • hemL glutamate-1-semialdehyde aminotransferase
  • 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).
  • 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).
  • ALS is synthesized by the condensation of glycine and succinyl-CoA.
  • 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.
  • one or more of the downstream genes e.g., in E.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • the production of flavonoid or anthocyanin from glycerol occurs through enzymatic transformation.
  • the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
  • 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.
  • 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.
  • one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors.
  • one or more genetic modifications cause reduction of formation of byproducts.
  • 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.
  • 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.
  • 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.
  • 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
  • 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.
  • the engineered cell is E. coli .
  • one or more genetic modifications decreases fatty acid biosynthesis.
  • 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.
  • 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-Co
  • 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.
  • the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
  • 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.
  • 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.
  • one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors.
  • 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.
  • 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.
  • 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.
  • 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.
  • the engineered cell is E. Coli .
  • one or more genetic modifications decreases fatty acid biosynthesis.
  • 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.
  • 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
  • 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.
  • the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
  • 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.
  • 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.
  • one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors.
  • one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors.
  • one or more genetic modifications cause reduction of formation of byproducts.
  • 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.
  • 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.
  • 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.
  • 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.
  • at least one the engineered host cell is E. coli .
  • one or more genetic modifications decreases fatty acid biosynthesis.
  • 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 naring
  • 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
  • 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.
  • the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
  • 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.
  • 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.
  • one or more genetic modifications lead to increase metabolic flux to flavonoid precursors or cofactors.
  • 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.
  • 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.
  • 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.
  • 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.
  • at least one the engineered host cell is E. coli .
  • one or more genetic modifications decreases fatty acid biosynthesis.
  • 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 naring
  • 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
  • the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA.
  • the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.
  • 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.
  • the engineered host cell is an E. coli .
  • the E. coli cell further comprises genes from fungi.
  • 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.
  • one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 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 .
  • 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.
  • PDH pyruvate dehydrogenase
  • PanK pantothenate kinase
  • 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.
  • 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.
  • 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: 80,
  • 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.
  • the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.
  • 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.
  • the engineered host cell is an E. coli . In certain embodiments, the E.
  • coli cell further comprises genes from fungi.
  • 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.
  • one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 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 .
  • 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.
  • PDH pyruvate dehydrogenase
  • PanK pantothenate kinase
  • 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.
  • 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.
  • 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: 80,
  • the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.
  • one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
  • 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.
  • one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
  • 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.
  • 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.
  • 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.
  • one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
  • 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.
  • 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.
  • 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.
  • 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).
  • one or more genetic modifications comprises overexpression of anthocyanin synthase.
  • 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.
  • one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT).
  • 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.
  • 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.
  • 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).
  • one or more genetic modifications comprises overexpression of anthocyanin synthase.
  • 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.
  • one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT).
  • 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.
  • 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.
  • 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.
  • one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT).
  • 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.
  • 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.
  • 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).
  • DHQ dihydroquercetin
  • DLM dihydromyricein
  • EDL eriodictoyl
  • PPF pentahydroxyflayaone
  • 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′-hydroxy
  • the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK).
  • 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.
  • the engineered host cell produces eriodictyol or taxifolin.
  • the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H).
  • the engineered host cell produces pentahydroxyflavone or dihydromyricetin.
  • flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7.
  • flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • flavonoid 3′,5′-hydroxylase 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.
  • the engineered host cell further comprises cytochrome b5.
  • cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
  • the flavanone-3-hydroxylase 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.
  • 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).
  • DHQ dihydroquercetin
  • DLM dihydromyricein
  • EDL eriodictoyl
  • PPF pentahydroxyflayaone
  • the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and/or pentahydroxyflayanone (PHF) is naringenin and/or dihydrokaempferol (DHK).
  • 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.
  • the engineered host cell produces eriodictyol or taxifolin.
  • the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H).
  • the engineered host cell produces pentahydroxyflavone or dihydromyricetin.
  • flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7.
  • flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • flavonoid 3′,5′-hydroxylase 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.
  • the engineered host cell further comprises cytochrome b5.
  • cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
  • the flavanone-3-hydroxylase 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.
  • 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.
  • 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.
  • flavonoids flavonoids
  • flavonoids product or flavonoids compound
  • flavonoids compound a member of a diverse group of phytonutrients found in almost all fruits and vegetables.
  • flavonoid flavonoid product
  • 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.
  • 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,
  • 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.
  • flavonoid precursor 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • TAL exogenous nucleic acid sequence encoding tyrosine ammonia lyase
  • PAL phenylalanine ammonia-lyase
  • C4H cinnamate-4-hydroxylase
  • 4CL 4-coumarate-Co
  • 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′-hydroxylase (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.
  • F3H flavanone-3-hydroxylase
  • F3′H flavonoid 3′-hydroxylase
  • F3′5′H flavonoid 3′,5′-hydroxylase
  • CPR cytochrome P450 reductase
  • DFR
  • Tyrosine ammonia-lyase 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.
  • 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.
  • phenylalanine ammonia-lyase 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).
  • 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 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 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.
  • SEQ ID NO: 3 C4H, Helianthus annuus L.
  • 4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester.
  • 4CLs of Petroselinum crispum SEQ ID NO: 4
  • 4CLs with SEQ ID NOS: 33-36 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.
  • the chalcone synthase can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA to produce the naringenin precursor naringenin chalcone or naringenin.
  • An exemplary chalcone synthase is the chalcone synthase of Petuniax hybrida (CHS, SEQ TD NO: 5).
  • CHSs with SEQ ID: 37-40 are also considered for use in the engineered cells provided herein.
  • CHS homologs and variants having at least 5000, at least 5500, at least 600%, at least 650%, at least 700%, at least 7500 at least 800%, at least 850%, at least 900%, at least 9500 at least 960%, at least 9700 at least 980%, or at least 9900 amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida ) that have the activity of a chalcone synthase.
  • Chalcone isomerase (CHI, also referred to as chalcone flavanone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones.
  • CHI of Medicago sativa SEQ TD NO: 6
  • CHI of Table 4 CHIs with SEQ TD NOS: 41-44
  • 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.
  • 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 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).
  • F3H of Rubus occidentalis SEQ ID NO: 7
  • F3Hs with SEQ ID NOS: 45-48 F3Hs listed in Table 5
  • 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.
  • Flavonoid 3′-hydroxylases 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.
  • 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.
  • CPR cytochrome P450 reductase
  • 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.
  • 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.
  • 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.
  • the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin).
  • 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.
  • 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 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).
  • 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.
  • Leucoanthocyanidin reductase catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin.
  • LAR Leucoanthocyanidin reductase
  • SEQ ID NO: 12 Desmodium uncinatum
  • 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.
  • 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.
  • ANS anthocyanin synthase
  • 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.
  • 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- ⁇ -D-glucose to anthocyanidins to form glycosylated anthocyanins.
  • 3GT flavonoid-3-glucosyl transferase
  • 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.
  • 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.
  • 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-coumaroyl-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 thi
  • Malonyl-CoA is the direct precursor for chalcone synthase to perform sequential condensations with p-coumaroyl-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 CO 2 donor. In the second reaction, the carboxyl-group is transferred from biotin to acetyl-CoA to form malonyl-CoA.
  • ACC acetyl-CoA carboxylase
  • 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.
  • Mucor circinelloides SEQ ID NO: 15
  • acetyl-CoA carboxylase can be introduced into the host cells.
  • 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.
  • 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.
  • 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 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.
  • acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin
  • 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.
  • 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) 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.
  • a variant of the Lpd subunit of PDH can be expressed that includes one or more mutations that reduces inhibition of PDH by NADH.
  • a LPD variant in E. coli that contains E354K mutation, and the mutated enzyme was less sensitive to NADH inhibition than the native LPD.
  • 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.
  • the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate.
  • the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA product.
  • 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.
  • Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase.
  • the matB gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter.
  • An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, 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:18 Streptomyces coelicolor
  • SEQ ID NO:80 Rhizobiales bacterium
  • SEQ ID NO:81 Rhizobium leguminosarum
  • Agrobacterium vitis SEQ ID NO: 82
  • 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.
  • 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.
  • malonate CoA-transferase EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase
  • the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity.
  • the mdcA gene product, the ⁇ subunit is malonate CoA-transferase
  • mdcD gene product, the ⁇ subunit is a malonyl-CoA decarboxylase.
  • the mdcE gene product, the ⁇ subunit may play a role in subunit interaction to form a stable complex or as a codecarboxylase.
  • the mdcC gene product, the ⁇ subunit was an acyl-carrier protein, which has a unique CoA-like prosthetic group.
  • the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply.
  • 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.
  • 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.
  • CoA coenzyme A
  • 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.
  • 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.
  • PanK-I PanK-II
  • PanK-III also known as CoaX (found in bacteria).
  • 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.
  • 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.
  • 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.
  • 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.
  • the gene beta-ketoacyl-ACP synthase II E. coli fabF
  • 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.
  • genes that are downregulated, disrupted, or deleted can include adhE, ldhA, poxB, and ackA-pta.
  • 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-coumaroyl-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.
  • ACOTs Acyl-CoA thioesterase enzymes
  • acyl-CoAs short-, medium-, long- and very long-chain
  • bile acid-CoAs bile acid-CoAs
  • methyl branched-CoAs methyl branched-CoAs
  • 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.
  • 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.
  • genes encoding enzymes of the tricarboxylic acid cycle can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes.
  • TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
  • 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.
  • TRP tryptophan
  • TRR tyrosine
  • PHE phenylalanine
  • 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.
  • 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.
  • the subsequent five steps are catalyzed by single enzymes.
  • 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.
  • an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme.
  • Cytochrome P450 CYPs
  • CYPs Cytochrome P450
  • 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.
  • C4 pathway Shemin pathway
  • 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.
  • E E.
  • the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL).
  • GltX glutamyl-tRNA synthetase
  • hemA glutamyl-tRNA reductase
  • hemL glutamate-1-semialdehyde aminotransferase
  • 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.
  • heterologous ALAS gene can be introduced to produce ALA via the C4 pathway.
  • 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.
  • one or more of the downstream genes E.
  • 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.
  • 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 cyanobacterial 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.
  • 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.
  • 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.
  • 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.
  • 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.
  • tyrosine and coumaric acid are provided in the culture medium.
  • 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.
  • 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.
  • 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.
  • 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.
  • glycerol is used as a carbon feedstock.
  • the glycerol is crude glycerol.
  • the method comprises isolating naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside.
  • 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%.
  • 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%).
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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%.
  • 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.
  • 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 ⁇ M 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.
  • 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 ⁇ M 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.
  • 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.
  • 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 ⁇ M 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.
  • 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.
  • the HPLC method was as follows: An Agilent 1200 HPLC was fitted with an Ascentis C18 Column 150 mm ⁇ 4.6 mm, 3 ⁇ m, equipped with a R-18 (3 ⁇ m) guard column. The column was heated to 30° C. with the sample block being maintained at 25° C. For each sample, 5 ⁇ L 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:
  • 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.
  • 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 ⁇ m 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 ⁇ m, equipped with a LiChrospher 100 RP-8 (5 ⁇ m) LiChroCART 4-4 guard column. The column was heated to 25° C. with the sample block being maintained at 25° C.
  • 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 .
  • 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.
  • the production of flavonoid or anthocyanin from a carbon source that can also be an energy source occurs through enzymatic transformation.
  • 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.
  • 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.
  • one or more genetic modifications lead to increase in metabolic flux to flavonoid precursors or cofactors. As shown in FIG.
  • one or more of the genetic modifications cause reduction of formation of byproducts.
  • 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.
  • 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.
  • 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.
  • a 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.
  • 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.
  • 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, wherein flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase, wherein flavanone-3-hydroxylase, where
  • 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.
  • 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
  • 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-
  • 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 ).
  • 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.
  • 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 ).
  • 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.
  • 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.
  • 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.
  • the engineered host cell is an E. coli .
  • 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.
  • one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 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 .
  • 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.
  • PDH pyruvate dehydrogenase
  • PanK pantothenate kinase
  • 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.
  • 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.
  • 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: 80,
  • 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.
  • 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.
  • the engineered host cell is an E. coli .
  • 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.
  • one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 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 .
  • 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.
  • PDH pyruvate dehydrogenase
  • PanK pantothenate kinase
  • 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.
  • 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.
  • 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: 80,
  • 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.
  • 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).
  • DHQ dihydroquercetin
  • DLM dihydromyricein
  • EDL eriodictyol
  • PPF pentahydroxyflayaone
  • the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK).
  • 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.
  • the engineered host cell produces eriodictyol or taxifolin.
  • the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H).
  • the engineered host cell produces pentahydroxyflavone or dihydromyricetin.
  • flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7.
  • flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • flavonoid 3′,5′-hydroxylase 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.
  • the engineered host cell further comprises cytochrome b5.
  • cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 98.
  • 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).
  • DHQ dihydroquercetin
  • DLM dihydromyricein
  • EDL eriodictyol
  • PPF pentahydroxyflayaone
  • the precursor for increase in production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PHF) is naringenin and/or dihydrokaempferol (DHK).
  • 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.
  • the engineered host cell produces eriodictyol or taxifolin.
  • the engineered host cell further comprises flavonoid 3′,5′-hydroxylase (F3′5′H).
  • the engineered host cell produces pentahydroxyflavone or dihydromyricetin.
  • flavonoid 3′-hydroxylase (F3′H) is truncated to remove the N-terminal leader sequence.
  • cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.
  • flavonoid 3′-hydroxylase (F3′H) is fused with cytochrome P450 reductase (CPR).
  • flavonoid 3′,5′-hydroxylase (F3′5′H) is fused with cytochrome P450 reductase (CPR).
  • flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 7.
  • flavanone-3′-hydroxylase (F3′H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 8.
  • cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO. 9.
  • flavonoid 3′,5′-hydroxylase 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.
  • the engineered host cell further comprises cytochrome b5.
  • 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.
  • 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).
  • one or more genetic modifications comprises overexpression of anthocyanin synthase.
  • 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.
  • one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT).
  • 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.
  • 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).
  • 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.
  • 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).
  • one or more genetic modifications comprises overexpression of anthocyanin synthase.
  • 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.
  • one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT).
  • 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.
  • 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).
  • 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.
  • 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.
  • the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has
  • 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).
  • F6P fructose-6-phosphate
  • E4P erythrose-4-phosphate
  • tktA is overexpressed to upregulate tyrosine.
  • Step 20 conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP).
  • PEP phosphoenolpyruvate
  • DAHP deoxy-d-arabino-heptulosonate-7-phosphate
  • Step 21 conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) to dehydroquinate (DHQ).
  • DAHP deoxy-d-arabino-heptulosonate-7-phosphate
  • DHQ dehydroquinate
  • E4P erythrose-4-phosphate
  • 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.
  • DHS 3-dehydroshikimate
  • SHK shikimic acid
  • 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).
  • ESP 5-enolpyruvylshikimate-3-phosphate
  • CHA chorismic acid
  • 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).
  • HPP 4-hydroxy-phenylpyruvate
  • POPP phenylpyruvate
  • Phe phenylalanine
  • 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.
  • one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
  • 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.
  • one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
  • one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).
  • ppsA exogenous phosphoenolpyruvate synthase
  • tktA exogenous transketolase
  • the one or more genetic modifications comprises disruption of tyrR gene.
  • 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.
  • one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
  • 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.
  • 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 coumaric 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 6 amino levulinic acid (ALA). hemL is overexpressed to upregulate ALA.
  • GSA glutamate semialdehyde
  • ALA 6 amino levulinic acid
  • Step 34 conversion of 6 amino levulinic acid (ALA) to porphobilinogen (PBG).
  • ALA 6 amino levulinic acid
  • PBG porphobilinogen
  • 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.
  • PPPIX protoporphyrinogen IX
  • 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.
  • SEQ Enzyme Sequence: ID: Tyrosine ammonia- MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD 1 lyase (TAL) EEIVRMGASARTIEEYLKSDKPIYGLTQGFGPLVLFDA Saccharothrix DSELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNM espanaensis RKGYSAVSPVFWQKLADLWNKGFTPAIPRHGTVSAS Accession: GDLQPLAHAALAFTGVGEAWTRDADGRWSTVPAVD ABC88669.1 ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR SALRLVRACAVLSARLATLLGANPEHYDVGHGVARG QVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCA PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL LHGGNFHAMPVGFASDQ

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Abstract

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

Description

    I. RELATED APPLICATIONS
  • This application claims priority to U.S. Provisional Application No. 63/174,403, filed on Apr. 13, 2021. The content of U.S. Provisional Application No. 63/174,403 is hereby incorporated by reference in its entirety.
    • II. SEQUENCE LISTING
  • This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled DEBU-009-03-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-flavanone 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, acetyl-CoA, and/or p-coumaroyl-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, hemL, 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′-hydroxylase (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-coumaroyl-CoA to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petuniax hybrida (CHS, SEQ TD 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 5000, at least 5500, at least 600%, at least 650%, at least 700%, at least 7500 at least 800%, at least 850%, at least 900%, at least 9500 at least 960%, at least 9700 at least 980%, or at least 9900 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 flavanone 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 TD NO: 6), CHI of Table 4, CHIs with SEQ TD 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-coumaroyl-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-coumaroyl-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 matB gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, for example of Streptomyces coelicolor (SEQ ID NO:18), of Rhizobiales bacterium (SEQ ID NO:80), of Rhizobium leguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ ID NO: 82), of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a naturally-occurring malonate transporter. Cell cultures of a host cell engineered to express a malonyl-CoA synthetase and a malonate transporter can include a culture medium that includes malonate.
  • In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase (EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase) that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity. The mdcA gene product, the α subunit, is malonate CoA-transferase, and mdcD gene product, the β subunit, is a malonyl-CoA decarboxylase. The mdcE gene product, the γ subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the δ subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. When the α subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an α subunit to exchange the acetyl group for a malonyl group. As the thioester transfer should be thermodynamically favorable, the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply. Examples of mdcAs that can be expressed in an engineered cell as provided herein include, without limitation, mdcA of Acinetobacter calcoaceticus (SEQ ID NO: 19), mdcAs of Table 10, mdcAs with SEQ ID NOS: 84-87, or a transferase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally occurring malonate CoA-transferases.
  • TABLE 10
    Malonate CoA-transferases (malonate decarboxylase subunit alpha)
    Organism GenBank Accession Number
    Acinetobacter calcoaceticus AAB97627.1
    Geobacillus sp. QNU36929.1
    Acinetobacter johnsonii WP_087014029.1
    Acinetobacter marinus WP_092618543.1
    Acinetobacter rudis WP_016655668.1
    Psychrobacter sp. G WP_020444454.1
    Moraxella catarrhalis WP_064617969.1
    Zoogloea sp. MBL0283742.1
    Dechloromonas sp. KAB2923906.1
    Stenotrophomonas rhizophila WP_123729366.1
    Xanthomonas cucurbitae WP_159407614.1
  • In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA supply include expressing or overexpressing at least one enzyme of a CoA biosynthesis pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4′-phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-limiting step in the biosynthesis of CoA. Three distinct types of PanK have been identified—PanK-I (found in bacteria), PanK-II (mainly found in eukaryotes, but also in the Staphylococci) and PanK-III, also known as CoaX (found in bacteria). In E. coli, pantothenate kinase is competitively inhibited by CoA itself, as well as by some CoA esters. The type III enzymes CoaX are not subject to feedback inhibition by CoA. In some embodiments, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, without limitation, CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33, SEQ ID NO: 20), CoaX of Streptomyces sp. CLI2509 (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-coumaroyl-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and/or ybgC can be downregulated, disrupted, or deleted.
  • In further embodiments, a cell engineered for the production of flavonoids or anthocyanins can have one or more of fatty acid degradation genes downregulated, disrupted, or deleted to improve precursor supply to the flavonoid pathway. In E. coli, for example, the acyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and/or gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted, or deleted.
  • Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
  • Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from feedstock such as glucose or glycerol is necessary to make biological production economically viable. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The shikimate pathway is the central metabolic route leading to formation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), this pathway exclusively exists in plants and microorganisms. It starts with the condensation of intermediates of glycolysis and pentosephosphate-pathway, phosphoenolpyruvate (PEP), and erythrose-4-phosphate (E4P), respectively, which enter the pathway through a series of condensation and redox reactions via 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the central branch point metabolite chorismate is obtained via shikimate-3-phosphate under ATP hydrolysis and introduction of a second PEP. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. In E. coli three DAHP synthase isozymes exist (aroF, aroG, aroH), which are each feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), in contrast the two DAHP synthases of plants are not subject to feedback-inhibition. In plants and bacteria, the subsequent five steps are catalyzed by single enzymes. From the central intermediate chorismate the pathway branches off to anthranilate and prephenate leading to aromatic amino acid, para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA) synthesis, the latter being a precursor for folate metabolism. Strategies to increase L-tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the ppsA, aroG, and/or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.
  • Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There exist two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA synthase (ALAS). The C4 pathway is restricted to mammals, fungi and purple nonsulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions, including assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chains, and incorporation of reduced iron into the molecule, are required to convert ALA to heme. In an E. coli host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemA with SEQ ID NOS: 91-93. Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway. Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Rhodobacter capsulatus (SEQ ID:22), ALAS with SEQ ID NOS: 94-97, or an ALAS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally-occurring ALAS. Further, one or more of the downstream genes (E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemL, 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 cyanobacterial 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, 3 μm, equipped with a R-18 (3 μm) guard column. The column was heated to 30° C. with the sample block being maintained at 25° C. For each sample, 5 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 coumaric 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 6 amino levulinic acid (ALA). hemL is overexpressed to upregulate ALA.
  • Step 34: conversion of 6 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:
    SEQ
    Enzyme: Sequence: ID:
    Tyrosine ammonia- MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD 1
    lyase (TAL) EEIVRMGASARTIEEYLKSDKPIYGLTQGFGPLVLFDA
    Saccharothrix DSELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNM
    espanaensis RKGYSAVSPVFWQKLADLWNKGFTPAIPRHGTVSAS
    Accession: GDLQPLAHAALAFTGVGEAWTRDADGRWSTVPAVD
    ABC88669.1 ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR
    SALRLVRACAVLSARLATLLGANPEHYDVGHGVARG
    QVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCA
    PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL
    LHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGL
    LVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRI
    RQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEAL
    ELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGI
    CPPLDADRPLGAEVRAARDLLSAHADQLLVDEADGK
    DFG
    Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 2
    ammonia-lyase GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV
    (PAL) TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD
    Brevibacillus EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
    laterosporus LMG ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE
    15441 REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC
    Accession: LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ
    WP_003337219.1 KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD
    SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK
    WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA
    MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
    RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS
    VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH
    LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD
    RALDGDIEKVVQLIRSGNLKKEIHDQNVND
    Cinnamate-4- MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLP 3
    hydroxylase (C4H) VPIFGNWLQVGDDLNHRNLTDLAKKFGEIFLLRMGQ
    Helianthus annuus RNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFTG
    L. KGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYR
    Accession: YGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQLMM
    QJC72299.1 YNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQS
    FEYNYGDFIPILRP
    FLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV
    DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAI
    ETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQ
    VTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHD
    AKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRP
    ERFFEEESKVEANGNDFRYLPFGVGRRSCPGIELALPIL
    GITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKHS
    TIVAKPRAL
    4-coumarate-CoA MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKV 4
    ligase (4CL) GDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQ
    Petroselinum QGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTS
    crispum AEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIIC
    Accession: IDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPY
    P14912.1 SSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYM
    HSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKF
    DIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLS
    SVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGM
    TEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIV
    DPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTI
    DEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVA
    PAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRT
    NGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSG
    KILRKDLRARIASGDLPK
    Chalcone synthase MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTY 5
    (CHS) PDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLT
    Petunia x hybrida EEBLKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAA
    Accession: QKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKL
    AAF60297.1 LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
    GARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGA
    GAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHL
    REVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNS
    LFWIAHPGGPADLDQVEIKLGLKPEKLKATRNVLSNY
    GNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVL
    FGFGPGLTVETVVLHSVAT
    Chalcone isomerase MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERG 6
    (CHI) LTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELL
    Medicago sativa ETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENC
    Accession: VAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASV
    P28012.1 FYRQSPDGILGLSFSPDTSIPEKEAALIENKAVSSAVLE
    TMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN
    Flavanone 3- MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFS 7
    hydroxylase (F3H) NEIPIISLSGIDEVEGRRAEICNKIVEACEDWGVFQIVD
    Rubus occidentalis HGVDAKLISEMTRLARDFFALPPEEKLRFDMSGGKKG
    Accession: GFIVSSHLQGEAVQDWREIVTYFSYPVRHRDYSRWPD
    ACM17897.1 KPEGWRAVTQQYSDELMGLACKLLEVLSEAMGLEKE
    ALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDP
    GTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVV
    NLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQ
    NPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSK
    DLELARLKKLAKEQQPEDSEKAKLEVKQVDDIFA
    Flavonoid 3′ MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIG 8
    hydroxylase (F3′H) NLPHMGPKPHQTLAAMVTTYGPILHLRLGFADVVVA
    Brassica napus ASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDL
    Accession: VFAPYGQRWRMLRKISSVHLFSAKALEDFKHVRQEE
    ABC58723.1 VGTLMRELARANTKPVNLGQLVNMCVLNALGREMI
    GRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFV
    PALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAM
    KNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKA
    LLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEE
    LDSVVGRGRPINESDLSQLPYLQAVIKENFRLHPPTPLS
    LPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDP
    LTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAG
    LSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEET
    YGITLQRAVPLVVHPKLRLDMSAYGLGSA
    Cytochrome P450 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPA 9
    reductase (CPR) VMAMLLENKELVMILTTSVAVLIGCVWLIWRRSSGS
    Catharanthus GKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGT
    roseus AEGFAKALAEEAKARYEKAVIKVIDIDDYAADDEEYE
    Accession: EKFRKETLAFFILATYGDGEPTDNAARFYKWFVEGND
    Q05001 RGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVA
    EQGGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDN
    LLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANG
    HANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRS
    CTHLDFDIAGTGLSYGTGDHVGVYCDNLSETVEEAER
    LLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTA
    LTRYADLLNTPKKSALLALAAYASDPNEADRLKYLAS
    PAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFA
    AIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGR
    IHKGVCSTWMKNAIPLEESRDCSWAPIFVRQSNFKLP
    ADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGT
    AVFFFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSR
    EGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDA
    KGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQM
    TGRYLRDVW
    Flavonoid 3′, 5′- MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKG 10
    hydroxylase WPVVGALPMLGNMPHAALADLSRRYGPIVYLKLGSR
    (F3′5′H) GMVVASTPDSARAFLKTQDLNFSNRPTDAGATHIAYN
    Delphinium SQDMVFADYGPRWKLLRKLSSLHMLGGKAVEDWAV
    grandiflorum VRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAMAN
    Accession: MLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFN
    BA066642 VGDFIPSIAWMDLQGIVRGMKRLHKKFDALLDKILRE
    HTATRRERKEKPDLVDVLMDNRDNKSEQERLTDTNI
    KALLLNLFSAGTDTSSSTIEWALTEMIKNPSIFGRAHA
    EMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHPSTP
    LNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNVW
    ENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRIC
    AGTRMGIVLVEYILGSLVHAFEWKLRDGETLNMEETF
    GIALQKAVPLAAVVTPRLPPSAYVV
    Dihydroflavonol 4- MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATV 11
    reductase (DFR) RDPSNMKKVKHLLDLPGAANRLTLWKADLVDEGSFD
    Anthurium EPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNV
    andraeanum LRSCARASSTVRRVVFTSSAGTVSIHEGRRHLYDETS
    Accession: WSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK
    AAP20866.1 NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPH
    YSILNPVQFVHLDDLCNAHIFLFECPDAKGRYICSSHD
    VTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKL
    TDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYA
    TEQLIATGQDNGH
    Leucoanthocyanidin MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYP 12
    reductase (LAR) TFLLVRPGPVSPSKAVIIKTFQDKGAKVIYGVINDKEC
    Desmodium MEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIK
    uncinatum RFLPSEFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEY
    Accession: GIPFTNICCNSIASWPYYDNCHPSQVPPPMDQFQIYGD
    Q84V83.1 GNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRPSSN
    CYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENII
    PESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKF
    RSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQT
    GTIEEINKDIKTLVETQPNEEIKKDMKALVEAVPISAM
    G
    Anthocyanin MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFD 13
    dioxygenase (ANS) EEKKDEGPQVPTIDLRDIDSDDQQVRQRCRDELKKAA
    Carica papaya VDWGVMHLVNHGIPDHLIDRVKKAGQAFFELPVEVK
    Accession: EKYANDQASGNIQGYGSKLANNASGQLEWEDYYFHL
    XP_021901846.1 IFPEEKRDLAIWPNNPADYIEVTSEYARQLRRLVSKIL
    GVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCP
    QPELALGVEAHTDISALTFILHNMVPGLQLFYEGKWV
    TAKCVPNSIVMHVGDTIEILSNGKYKSILHRGLVNKEK
    VRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQ
    HIQHKLFRKNQENLEAK
    Anthocyanidin-3 - MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPH 14
    O-glycotransferase AVFSFFSTSESNASIFHDSMHTMQCNIKSYDVSDGVPE
    (3 GT) GYVFTGRPQEGIDLFMRAAPESFRQGMVMAVAETGR
    Vitis labrusca PVSCLVADAFIWFAADMAAEMGVAWLPFWTAGPNS
    Accession: LSTHVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFR
    ABR24135 DLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFE
    ELDDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCL
    QWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASR
    VPFIWSLRDKARMHLPEGFLEKTRGHGMVVPWAPQA
    EVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFF
    GDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQI
    LSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTL
    VDLVSKPKDV
    Acetyl-CoA MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHT 15
    carboxylase (ACC) VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF
    Mucor TVMATPEDLKANAEYIRMADNFVEVPGGSNNNNYAN
    circinelloides VELIVDVAERTAVHAVWAGWGHASENPRLPEMLAKS
    1006PhL KHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTMGW
    Accession: SGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKA
    EPB82652.1 AEKIGFPVMIKASEGGGGKGIRMVKDGSNFAQLFAQV
    QGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFG
    RDCSVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLG
    KLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPT
    TEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNS
    ASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAG
    FKPSSGIMQELNFRSSTNVWGYFSVVSAGGLHEYADS
    QFGHIFAYGENRQQARKNMVIALKELSIRADFRSTVE
    YIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTML
    AVFCGAVTKAHMASLDCFQQYKQSLEKGQVPSKGSL
    KTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQV
    GIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGK
    TCLLEKESDPTQLRSPSPGKLVNLLVENGDHLNAGDA
    YAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDII
    GILSLDDPSRVKHALPFNGTVPAFGAPHITGDKPVQRF
    NATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLP
    YSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP
    AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFAR
    YRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVIL
    SLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLI
    NPVSTGSALDKYFTPILKRLSEIESRATQKVTLKAREL
    LILCQLPSYEERQAQMYQILKNSVTESVYGGGSEYRTP
    SYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEVY
    CRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANG
    IDSNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLAD
    LQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNI
    AVRADEDMDDTAFRTKICEMITANADVFRQAHLRRL
    SVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMA
    YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
    RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE
    IVSHEYKNSDCNHLFINFIPTFAIEADDVEHALKDFVD
    RHGKRLWKLRVTGAEIRFNVQSKKPDAPIIPMRFTVD
    NVSGFILKVEVYQEVKTEKSGWILKSVNKIPGAMHM
    QPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQ
    SVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVL
    QEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDI
    TFKIGSFGVAEDQVFYKASELARALGIPRIYLSANSGA
    RIGLADELISQFRAAWKDASNPTAGFKYLYLTPAEYD
    VLAQQGDAKSVLVEEIQDEGETRLRITDVIGHTDGLG
    VENLKGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYL
    VRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQL
    GGTQIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVR
    NAPVSMRLGADPIDRDIEYTPPKGPSDPRFFLAGKSEN
    GKWLSGFFDQDSFVETLSGWARTVVVGRARLGGIPM
    GVVSVETRTVENIVPADPANSDSTEQVFMEAGGVWFP
    NSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRD
    MYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGA
    WVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKY
    RKPALLATMERLDATYASLKKQLAEEGKTDEEKAAL
    KVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKG
    VIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATS
    AAPMPREQMLDLVKQWFTNDNETVNFEDADELVSE
    WFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIE
    GFSQLIENLSEDARAEILRKLNSRF
    Acetyl-CoA MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTF 16
    synthase (ACS) WGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGT
    Salmonella LNLAANCLDRHLQENGDRTAIIWEGDDTSQSKHISYR
    typhimurium ELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAV
    Accession: AMLACARIGAVHSVIFGGFSPEAVAGRIIDSSSRLVITA
    NP_463140.1 DEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRT
    GSDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPL
    FILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYH
    PGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEG
    VPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEG
    DKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE
    KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFF
    GVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLF
    GDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITG
    RVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIP
    HAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIG
    PLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNL
    GDTSTLADPGVVEKLLEEKQAIAMPS
    Malonyl-CoA MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAA 17
    synthase (matB) AGATAGRIGGAGRVAVWATPAMETGVAVVAALLAG
    Streptomyces VAAVPLNPKSGDKELAHILSDSAPSLVLAPPDAELPPA
    coelicolor LGALERVDVDVRARGAVPEDGADDGDPALVVYTSGT
    Accession: TGPPKGAVIPRRALATTLDALADAWQWTGEDVLVQG
    WP_011028356 LPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAAREL
    NDGATMLFGVPTMYHRIAETLPADPELAKALAGARL
    LVSGSAALPVHDHERIAAATGRRVIERYGMTETLMNT
    SVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDG
    ESVGEIQVRGPNLFTEYLNRPDATAAAFTEDGFFRTG
    DMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENA
    LLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPP
    ALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKI
    MKRALNRD
    Malonate MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT 18
    transporter (matC) LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT
    Streptomyces TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV
    coelicolor SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG
    Accession: GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA
    NP_626686.1 AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS
    GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG
    AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
    AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY
    VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
    SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV
    SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY
    GGIVVAAVPALAWLVLVVPGFG
    Malonate CoA- MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARA 19
    transferase (MdcA) IELLETVIASGDRVCLEGNNQKQADFLSKCLSQCNPD
    Acinetobacter AVNDLHIVQSVLALPSHIDVFEKGIASKVDFSFAGPQS
    calcoaceticus LRLAQLVQQQKISIGSIHTYLELYGRYFIDLTPNICLITA
    Accession: HAADREGNLYTGPNTEDTPAIVEATAFKSGIVIAQVNE
    AAB97627.1 IVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQI
    TEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIEL
    LLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDS
    VHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQ
    TAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAP
    NMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVV
    QTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMI
    YGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTP
    VGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLA
    AKSVKDLVKWSGGLYSPPSRFRNW
    Pantothenate kinase MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVE 20
    (CoaX) QLTSQQALPVRACRLVSVRSEQETSQLVARLEQLFPV
    Pseudomonas SALVASSGKQLAGVRNGYLDYQRLGLDRWLALVAA
    aeruginosa HHLAKKACLVIDLGTAVTSDLVAADGVHLGGYICPG
    Accession: MTLMRSQLRTHTRRIRYDDAEARRALASLQPGQATA
    Q9HWCL1 EAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGG
    DAELVRDELAGARIMPDLVFVGLALACPIE
    glutamyl-tRNA MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSL 21
    reductase (hemAm) LAQPMVQGGVVLSTCNRTELYLSVEEQDNLQEALIR
    Salmonella WLCDYHNLNEDDLRNSLYWHQDNDAVSHLMRVASG
    typhimurium LDSLVLGEPQILGQVKKAFADSQKGHLNASALRRMF
    Accession: QKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL
    AAA88610.1 STVTVLLVGAGETIELVARHLREHKVQKMIIANRTRE
    RAQALADEVGAEVISLSDIDARLQDADIIISSTASPLPII
    GKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLA
    NAYLYSVDDLQSIISHNLAQRQAAAVEAETIVEQEASE
    FMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQ
    QGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDGD
    DERLNILRDSLGLE
    5-aminolevulinic MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQ 22
    acid synthase WNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHE
    (ALAS) ALEAVGAGSGGTRNISGTTAYHRRLEAEIADLHGKEA
    Rhodobacter ALVFSSAYIANDATLSTLRLLFPGLIIYSDSLNHASMIE
    capsulatus GIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFE
    Accession: SVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMY
    CAA37857 GPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIA
    ASAKMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLK
    TAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHI
    VPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRG
    TERLRFTPSPVHDLKQIDGLVHAMDLLWARCA
    Tyrosine ammonia- MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTP 23
    lyase (TAL) ALRERCARAHARLEHAIAEQRHIYGITTGFGPLANRLI
    Rhodobacter GADQGAELQQNLIYHLATGVGPKLSWAEARALMLAR
    capsulatus SB 1003 LNSILQGASGASPETIDRIVAVLNAGFAPEVPAQGTVG
    Accession: ASGDLTPLAHMVLALQGRGRMIDPSGRVQEAGAVM
    ADE84832.1 DRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEA
    ARAIDAALRHSAVLMEVLSGHAEAWHPAFAELRPHP
    GQLRATERLAQALDGAGRVCRTLTAARRLTAADLRP
    EDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCE
    LNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDAL
    NAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQA
    GLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNG
    ANQDVVSMGTIAARRARAQLLPLSQIQAILALALAQA
    MDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRP
    LAGHIEAVAQGLRHPSAAADPPA
    Tyrosine ammonia- MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQ 24
    lyase (TAL) ATKTVVIDGHNMKVGDVVAVARHGAKVELAASVAG
    Trichosporon PVQASVDFKESKKHTSIYGVTTGFGGSADTRTSDTEA
    cutaneum LQISLLEHQLCGYLPTDPTYEGMLLAAMPIPIVRGAM
    Accession: AVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVPLR
    XP_018276715 GTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLT
    APEAIAKYKLKTVRLASKEGLGLVNGTAVSAAAGAL
    ALYDAECLAMMSQTNTALTVEALDGHVGSFAPFIQEI
    RPHVGQIEAAKNIRHMLSNSKLAVHEEPELLADQDAG
    ILRQDRYALRTSAQWIGPQLEMLGLARQQIETELNSTT
    DNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQN
    LGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCK
    GLDIHCGAYCAELGFLANPMSNHVQSTEMHNQSVNS
    MAFASARKTMEANEVLSLLLGSQMYCATQALDLRV
    MEVKFKMAIVKLLNDTLTKHFSTFLTPEQLAKLNTTA
    AITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDALM
    VNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADG
    RNDLEPAEYLGQTRAVYEAIRSDLGVKVRRGDVAEG
    KSGKSIGSNVARIVEAMRDGRLMGAVSKMFF
    Tyrosine ammonia- MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNF 25
    lyase (TAL) LKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLI
    Flavobacterium RSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSV
    johnsoniae INLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLI
    Accession: GEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALING
    WP_012023194 TSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAY
    DDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKR
    EDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETI
    NNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDY
    ISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFV
    NLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSI
    PNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIV
    QAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYP
    FVQKVKDHLINN
    Tyrosine ammonia- MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILA 26
    lyase (TAL) QIEASCAYINQAVKEHQPVYGVTTGFGGMANVIISPEE
    Herpetosiphon AAELQNNAIWYHKTGAGKLLPFTDVRAAMLLRANSH
    aurantiacus DSM MRGASGIRLEIIQRMVTFLNANVTPHVREFGSIGASGD
    785 LVPLISITGALLGTDQAFMVDFNGETLDCISALERLGL
    Accession: PRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLA
    ABX04526.1 LALEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWA
    ADHMLELLQGSQLSRNELDGSHDYRDGDLIQDRYSL
    RCLPQFLGPIIDGMAFISHHLRVEINSANDNPLIDTASA
    ASYHGGNFLGQYIGVGMDQLRYYMGLMAKHLDVQI
    ALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTANS
    IMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLA
    RQTIQTLQQYIAITLMFGVQAVDLRTHKLAGHYNAAE
    LLSPLTAKIYHAVRSIVKHPPSPERPYIWNDDEQVLEA
    HISALAHDIANDGSLVSAVEQTLSGLRSIILFR
    Phenylalanine MHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAME 27
    ammonia-lyase CSHLEEIKRMVDTYQNATQVMIEGATLTVPQVAAIAR
    (PAL) RPEVHVVLDAANARSRVDESSNWVLDRIMGGGDIYG
    Physcomitrella VTTGFGATSHRRTQQGVELQRELIRFLNAGVLSKGNS
    patens LPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKLL
    Accession: NAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKA
    XP_001758374.1 VTEDGREVSALEALRIAGVEKPFELAPKEGLALVNGT
    AVGSALASTVCYDANIMVLLAEVLSALFCEVMQGKP
    EFADPLTHKLKHHPGQMEAAAVMEWVLDGSSFMKA
    AAKFNETDPLRKPKQDRYALRTSPQWLGPQVEVIRNA
    THAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSM
    DNMRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSG
    GPNPSLDYGMKGAEIAMASYLSEINYLANPVTTHVQS
    AEQHNQDVNSLGLVSARKTEEAMEILKLMSATFLVG
    LCQAIDLRHVEETMQSAVKQVVTQVAKKTLFMGSDG
    SLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLMEK
    LRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSR
    VVPAVREAYDTKGLSLVPNRIQDCRTYPLYKLVRGDL
    KTQLLSGQRTVSPGQEIEKVFNAISAGQLVAPLLECVQ
    GWTGTPGPFSARASC
    Phenylalanine MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNEL 28
    ammonia-lyase LEFLQKGRDQLENKLKENVAIYGINTGFGGNGDLIIPF
    (PAL) DKLDYHQSNLLDFLTCGTGDFFNDQYVRGIQFIIIIALS
    Dictyostelium RGWSGVRPMVIQTLAKHLNKGIIPQVPMHGSVGASG
    discoideum AX4 DLVPLSYIANVLCGKGMVKYNEKLMNASDALKITSIE
    Accession: PLVLKSKEGLALVNGTRVMSSVSCISINKFETIFKAAIG
    XP_644510.1 SIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNK
    YFNTSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRC
    APQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVL
    SGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMH
    SEFSKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQE
    AAPACIHSLTTEQFNQDIVSLGLHSANGAASMLIKLCD
    IVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIKIIPI
    LENDRRTDIDVREITNAILQDKLDFFNLNL
    Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 29
    ammonia-lyase GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV
    (PAL) TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD
    Brevibacillus EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG
    laterosporus LMG ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE
    15441 REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC
    Accession: LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ
    WP_003337219.1 KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD
    SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK
    WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA
    MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP
    RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS
    VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH
    LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD
    RALDGDIEKVVQLIRSGNLKKEIHDQNVND
    Cinnamate-4- MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGP 30
    hydroxylase (C4H) IPVPVFGNWLQVGDDLNHRNLTEMAKKFGEVFMLR
    Rubus sp. SSL-2007 MGQRNLVWSSPDLAKEVLHTQGVEFGSRTRNVVFDI
    Accession: FTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQ
    ABX74781.1 QYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQ
    LMMYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSR
    LAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLF
    KDYFVDERKKLSSTQATTNEGLKCAIDHILDAQQKGE
    INEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQ
    KKLRDELDTVLGRGVQITEPEIQKLPYLQAVVKETLR
    LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
    ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
    GVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQTQL
    DTTEKGGQFSLHILKHSPIVMKPRT
    Cinnamate-4- MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPV 31
    hydroxylase (C4H) PVFGNWLQVGDDLNHRNLTDMAKKFGDVFMLRMG
    Fragaria vesca QRNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFT
    Accession: GKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQY
    XP_004294725.1 RHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQLM
    MYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLA
    QSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKD
    YFVDERKKLASTQVTTNEGLKCAIDHILDAQQKGEIN
    EDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQK
    KLRDELDTVLGHGVQVTEPELHKLPYLQAVVKETLR
    LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL
    ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF
    GVGRRSCPGIILALPILGVTLGRLVQNFEMLPPPGQTQ
    LDTTEKGGQFSLHILKHSTIVMKPRA
    Cinnamate-4- MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVP 32
    hydroxylase (C4H) VFGNWLQVGDDLNHRNLTEYAKKFGDVFLLRMGQR
    Solanum tuberosum NLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGK
    Accession: GQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRG
    ABC69046.1 GWESEAASVVEDVKKNPESATNGIVLRKRLQLMMYN
    NMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFE
    YNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVD
    ERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNV
    LYIVENFNVAAIETTLWSIEWGIAELVNHPHIQKKLRD
    EIDTVLGPGMQVTEPDMPKLPYLQAVIKETLRLRMAI
    PLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNP
    AHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGR
    RSCPGIILALPILGITLGRLVQNFEMLPPPGQSKLDTSE
    KGGQFSLHILKHSTIVMKPRSF
    4-coumarate-CoA MGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKV 33
    ligase (4CL) SDRACLINGATGETFSYAQVELISRRVASGLNKLGIHQ
    Daucus carota GDTMMILLPNTPEYFFAFLGASYRGAVSTMANPFFTS
    Accession: PEVIKQLKASQAKLIITQACYVEKVKEYAAENNITVVC
    AIT52344.1 IDEAPRDCLHFTTLMEADEAEMPEVAIDSDDVVALPY
    SSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIHS
    EDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIV
    PFLELIQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVR
    TVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEA
    GPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPE
    THASLPRNQSGEICIRGDQIMKGYLNDPESTKTTIDEE
    GWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAPAEI
    EALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGS
    TTTEEEIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKIL
    RKELRARIASGDLPK
    4-coumarate-CoA MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGS 34
    ligase (4CL) RPCLINGSTGEILTYDQVELASRRVGSGLHRLGIRQGD
    Striga asiatica TIMLLLPNSPEFVLAFLGASHIGAVSTMANPFFTPAEV
    Accession: VKQAAASRAKLIVTQACHVDKVRDYAAEHGVKVVC
    GER48539.1 VDGAPPEECLPFSEVASGDEAELPAVKISPDDVVALPY
    SSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHS
    DDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIV
    PFLELIQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRT
    VMSGAAPLGRELEDAVRLKFPNAKLGQGYGMTEAGP
    VLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDTETG
    ASLGRNQPGEICIRGDQIMKGYLNDPESTERTIDKEGW
    LHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEA
    LLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTI
    TEDEIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKD
    LRARLAAGVPN
    4-coumarate-CoA MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISE 35
    ligase (4CL) FSSRPCLINGANNQIYTYADVELNSRKVAAGLHKQFGI
    Capsicum annuum QQKDTIMILLPNSPEFVFAFLGASYLGAISTMANPLFTP
    Accession: AEVVKQVKASNAEIIVTQACHVNKVKDYALENDVKI
    KAF3620179.1 VCIDSAPEGCVHFSELIQADEHDIPEVQIKPDDVVALP
    YSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYI
    HSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKF
    DIVPFLELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLS
    SVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMT
    EAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVD
    PDTGNSLHRNQSGEICIRGDQIMKGYLNDPEATAGTID
    KEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQVA
    PAELEALLLNHPNISDAAWPMKDEQAGEVPVAFVVR
    SNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPS
    GKDLRKDLRAKLAAGFPN
    4-coumarate-CoA MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSS 36
    ligase (4CL) KPCLINGSTGKVYTYSDVELTSRKVAAGFHNLGIQQR
    Camellia DTIMLLLPNCPEFVFAFLGASYLGAIITMANPFFTPAET
    sinensis IKQAKASNSKLIITQSSYTSKVLDYSSENNVKIICIDSPP
    Accession: DGCLHFSELIQSNETQLPEVEIDSNEVVALPYSSGTTGL
    ASU87409.1 PKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMM
    CVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKL
    IQRYKVTIGPFVPPIVLAIAKSEWDDYDLSTIRTMMS
    GAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLA
    MCLAFAKKPFEEKSGACGTVVRNAEMKIVDPDAGFSL
    PRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLH
    TGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEAL
    LLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVT
    ENEIKKYISEQVVFYKKINRVYFVDAIPKAPSGKILRK
    DLRARLAAGIPS
    Chalcone synthase MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTY 37
    (CHS) PDYYFRITNSEHKTELKEKFKRMCEKSMIKTRYMHLT
    Capsicum annuum EEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEA
    Accession: AQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLA
    XP_016566084.1 KLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN
    NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGD
    GAAAIIMGSDPIPGVERPLFQLVSAAQTLLPDSEGAID
    GHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISD
    WNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVL
    SNYGNMSSACVLFILDEMRKASTKEGLGTSGEGLEW
    GVLFGFGPGLTVETVVLHSVAI
    Chalcone synthase MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYP 38
    (CHS) DYYFRITKSEHKAELKEKFQRMCDKSMIKKRYMYLT
    Rosa chinensis EEILKENPSMCEYMAPSLDARQDMVVVEIPKLGKEAA
    Accession: TKAIKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKL
    AEC13058.1 LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK
    GARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGA
    AAllVGSDPLPEVEKPLFELVSAAQTILPDSDGADDGHL
    REVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWN
    SLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEY
    GNMSSACVLFILDEVRRKSAANGHKTTGEGLEWGVL
    FGFGPGLTVETVVLHSVAA
    Chalcone synthase MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQAD 39
    (CHS) YPDYYFRITNSDHMTDLKDKFKRMCEKSMITKRHMY
    Morus alba var. LTEEILKENPKMCEYMAPSLDARQDIVVVEVPKLGKE
    multicaulis AAAKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLT
    Accession: KLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENN
    AHL83549.1 KGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDG
    AAAVILGADPDTSVERPIFELVSAAQTILPDSEGAIDGH
    LREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWN
    SIFWIAHPGGPAILDQVEAKLGLKQEKLSATRHVLSEY
    GNMSSACVLFILDEVRKKSVEEGKATTGEGLEWGVLF
    GFGPGLTVETIVLHSLPAV
    Chalcone synthase MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQAD 40
    (CHS) YPDYYFRITKSEHLTELKEKFKQMCDKSMIRKRYMYL
    Dendrobium TEEILKENPNICAFMAPSLDARQDIVVTEVPKLAREAS
    catenatum ARAIKEWGQPKSRITHLIFCTTSGVDMPGADYQLTRL
    Accession: LGLRPSVNRIMLYQQGCFAGGTVLRLAKDLAENNAG
    ALE71934.1 ARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAA
    AIIVGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLRE
    MGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSI
    FWIAHPGGPAILDQVEIKLGLKEEKLASSRNVLAEYG
    NMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLF
    GFGPGLTVETVVLRSVPIAGAV
    Chalcone isomerase MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTI 41
    (CHI) EGNFIKFTAIGVYLEDVAVASLATKWKGKSSEELLET
    Trifolium pratense LDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVTENCVA
    Accession: HLKSVGTYGDAEVEAMEKFVEAFKPINFPPGASVFYR
    PNX83855.1 QSPDGILGVSISIHFFP
    Chalcone isomerase MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGV 42
    (CHI) RGLTIEGNFIKFTGIGVYLEDQAVASLATKWKGKSSEE
    Abrus precatorius LVESLDFFRDIISGPFEKLIRGSKIRQLSGPEYSKKVME
    Accession: NCVAHMKSVGTYGDAEAAGIEEFAQAFKPVNFPPGA
    XP_027366189.1 SVFYRQSPDGVLGLSFSQDATIPEEEAAVIKNKPVSAA
    VLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN
    Chalcone isomerase MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGER 43
    (CHI) GLTIDGKFIKFTGIGVYLEDKAVPSLAGKWKDKSSQQ
    Arachis duranensis LLQTLHFYRDIISGPFEKLIRGSKILALSGVEYSRKVME
    Accession: NCVAHMKSVGTYGDAEAEAIQQFAEAFKNVNFKPGA
    XP_015942246.1 SVFYRQSPLGHLGLSFSQDGNIPEKEAAVIENKPLSSA
    VLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQH
    N
    Chalcone isomerase MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGER 44
    (CHI) GLEIQGKFIKFTAIGVYLEGDAVASLAVKWKGKSKEE
    Cephalotus LTDSVEFFRDIVTGPFEKFTQVTTILPLTGQQYSEKVSE
    follicularis NCVAFWKSVGIYTDAEAKAIEKFIEVFKEETFPPGSSIL
    Accession: FTQSPNGALTIAFSKDGVIPEVGKAVIENKLLAEGLLE
    GAV77263.1 SIIGKHGVSPVAKQCLATRLSELL
    Flavanone 3- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 45
    hydroxylase (F3H) RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD
    Abrus precatorius EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
    Accession: MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
    XP_027329642.1 WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN
    NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI
    IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI
    HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL
    GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK
    Flavanone 3- MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLER 46
    hydroxylase (F3H) GYIVRATVRDPANLKKVKHLLDLPKADTNLTLWKAD
    Camellia sinensis LNEEGSFDEAIEGCSGVFHVATPMDFESKDPENEVIKP
    Accession: TINGVLSIIRSCTKAKTVKRLVFTSSAGTVNVQEHQQP
    AAT66505.1 VFDENNWSDLHFINKKKMTGWMYFVSKTLAEKAAW
    EAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRN
    EGHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSS
    HDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSF
    SSKKLIGMGFEFKYSLEDMFRGAIDTCREKGLLPHSFA
    ENPVNGNKV
    Flavanone 3- MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYI 47
    hydroxylase (F3H) VRATVRDPANMKKVKHLQELEKADKNLTLWKADLT
    Nyssa sinensis EEGSFDEAIKGCSGVFHVATPMDFESKDPENEVIKPTI
    Accession: NGVLSIVRSCVKAKTVKRLVFTSSAGTVNLQEHQQLV
    KAA8531902.1 YDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAWE
    ATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEA
    HYSIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSH
    DATIYDLAKMIREKWPEYNVPTELKGIEKDLQTVSFSS
    KKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHE
    TPANANANANANVKKNQNENTEI
    Flavanone 3- MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV 48
    hydroxylase (F3H) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
    Rosa chinensis EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
    Accession: MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN
    XP_024167119.1 WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN
    NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII
    KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH
    EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE
    TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
    VDESSVVGVKVTG
    Flavonoid 3′ MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIG 49
    hydroxylase (F3′H) NLPHMGQMPHHSLAAMARTYGPLMHLRLGFVDVIV
    Cephalotus AASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDL
    follicularis VFAPYGPRWRMLRKISSVHLFSGKALDDYRHVRQEE
    Accession: VAVLIRALARAESKQAVNLGQLLNVCTANALGRVML
    GAV84063.1 GRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIG
    DFIPALDWLDLQGVAAKMKNLHKRFDTFLTGLLEEH
    KKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDT
    EIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQV
    LKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHP
    STPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDP
    KEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAG
    RRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE
    DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY
    Flavonoid 3′ MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPII 50
    hydroxylase (F3′H) GNLPHMGPVPHHSLAALAKVYGPLMHLRLGFVDVV
    Theobroma cacao VAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQ
    Accession: DLVFAPYGPRWRMLRKISSVHLFSGKALDDFRHVRQ
    EOY22049.1 DEVGVLVRALADAKTKVNLGQLLNVCTVNALGRVM
    LGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNI
    GDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEH
    KIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKA
    LLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVRKEL
    DSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLS
    LPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWK
    DPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRIC
    AGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLN
    MEEAFGLTLQRAAPLMVHPRPRLSPRAY
    Flavonoid 3′ MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPW 51
    hydroxylase (F3′H) PVVGNLPHLGTIPHHSLAAMAKKYGPLMHLRLGFVD
    Gerbera hybrida VVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYN
    Accession: YQDLVFAPYGPRWRMLRKICSVHLFSTKALDDFRHV
    ABA64468.1 RQEEVAILARALVGAGKSPVKLGQLLNVCTTNALAR
    VMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIG
    DFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHK
    TGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEI
    KALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARK
    EMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPL
    SLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW
    ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRIC
    VGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM
    EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV
    Flavonoid 3′ MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGP 52
    hydroxylase (F3′H) RGWPVLGNLPQLGPKPHHTLHALSRAHGPLFRLRLGS
    Phoenix dactylifera VDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIA
    Accession: YNYQDLVFAPYGPGWRARRKLLNVHLFSGKALEDLR
    XP_008791304.2 PVREGELALLVRALRDRAGANELVDLGRAANKCATN
    ALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAG
    VFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDG
    LIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGE
    GAKLTDTDIKALLLNLFTAGTDTTSSTVEWALSELVK
    HPDILRKAQLELDSVVGGDRLVSESDLPNLPFMQAIIK
    ETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNV
    WAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGND
    FGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWD
    LPEGQMPEKLDMEEAYGLTLQRATPLMVHPVPRLAP
    TAYQS
    Cytochrome P450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI 53
    reductase (CPR) IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE
    Camellia sinensis AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK
    Accession: AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
    XP_028084858 DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG
    NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ
    CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
    AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG
    SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE
    SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV
    ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA
    KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
    KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
    WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA
    KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
    DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA
    NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI
    SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL
    LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD
    LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED
    YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL
    QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV
    CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI
    IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
    CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK
    EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR
    DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR
    DVW
    Cytochrome P450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI 54
    reductase (CPR) IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE
    Cephalotus AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK
    follicularis AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG
    Accession: DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG
    GAV59576.1 NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ
    CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA
    AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG
    SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE
    SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV
    ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA
    KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK
    KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA
    WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA
    KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE
    DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA
    NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI
    SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL
    LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD
    LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED
    YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL
    QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV
    CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI
    IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG
    CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK
    EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR
    DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR
    DVW
    Cytochrome P450 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAE 55
    reductase (CPR) LSSMLIENRQFAMIISTSIAVLIGCIVMLLWRRSGGSGS
    Brassica napus SKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGT
    Accession: AEGFAKALGEEARARYEKTRFKIVDLDDYAADDDEY
    XP_013706600.1 EEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEG
    DDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDI
    LVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPEL
    DTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKN
    LANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCT
    HLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLL
    DMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTR
    YACLLSSPKKSALLALAAHASDPTEAERLKHLASPAG
    KDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAV
    APRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIH
    KGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSD
    SKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVL
    FFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREGP
    TKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKG
    MARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGR
    YLRDVW
    Flavonoid 3′, 5′- MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPG 56
    hydroxylase PKGWPFVGALPLLGTMPHVALAQMAKKYGPVMYLK
    (F3′5′H) MGTCGMVVASTPDAARAFLKTLDLNFSNRPPNAGAT
    Cephalotus HLAYNAQDMVFADYGPRWKLLRKLSNLHMLGGKAL
    follicularis EDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLTY
    Accession: AMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTS
    GAV62131 AGYFNIGDFIPSIAWMDLQGIERGMKKLHKRFDALLT
    KMFEEHMATAHERKGNPDLLDIVMANRDNSEGERLT
    TTNIKALLLNLFSAGTDTSSSIIEWSLAEMLKNPSILKR
    AHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHP
    STPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP
    EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGR
    RICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMD
    EAFGLALQKAVPLAAIVSPRLAPTAYVV
    Flavonoid 3′, 5′- MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGA 57
    hydroxylase LPFIGPMPHSGLALLARRYGPIMFLKMGIRRVVVASSS
    (F3′5′H) TAARTFLKTFDSHFSDRPSGVISKEISYNGQNMVFADY
    Dendrobium GPKWKLLRKVSSLHLLGSKAMSRWAGVRRDEALSMI
    moniliforme QFLKKHSDSEKPVLLPNLLVCAMANVIGRIAMSKRVF
    Accession: HEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGWL
    AEB96145 DPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQ
    GNPDLLDLVVASEVKGEDGEGLCEDNIKGFISDLFVA
    GTDTSAIVIEWAMAEMLKNPSILRRAQEETDRVIGRH
    RLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEP
    CEVEGYHIPGETWLLVNIWAIGRDPDVWENPLVFDPE
    RFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMV
    MVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPK
    AVPLAVMATPRLPAAAYGLL
    Dihydroflavonol 4- MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVR 58
    reductase (DFR) DPDNEKKVKHLVELPKAKTHLTLWKADLSDEGSFDE
    Acer palmatum AIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIM
    Accession: KACKKAKTVKRLVFTSSAGTVDVEEHKKPVYDENSW
    AWN08247.1 SDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEENSID
    FISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAI
    IKQGNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATI
    LELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKIL
    DLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHV
    AAKGLIPNNSDEIHVAAAEKTTATA
    Dihydroflavonol 4- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 59
    reductase (DFR) RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD
    Abrus precatorius EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI
    Accession: MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC
    XP_027329642.1 WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN
    NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI
    IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI
    HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL
    GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK
    Dihydroflavonol 4- MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRA 60
    reductase (DFR) TVRDPTNLKKVKPLLDLPRSNELLSIWKADLDGIEGSF
    Dendrobium DEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGI
    moniliforme LRSCKNAGSVQRVIFTSSAGTVNVEEHQAAAYDETC
    Accession: WSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFVKD
    AEB96144.1 NHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHY
    SILKQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDST
    IYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKL
    MELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVL
    AAEKFEEVKEQIAVK
    Dihydroflavonol 4- MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV 61
    reductase (DFR) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD
    Rosa chinensis EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI
    Accession: MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN
    XP_024167119.1 WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN
    NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII
    KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH
    EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE
    TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE
    VDESSVVGVKVTG
    Leucoanthocyanidin MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTT 62
    reductase (LAR) FLLVRSLDKGAIPSKSKTINSLHDKGAILIHGVIEDQEF
    Camellia sinensis VEGILKDHKIDIVISAVGGANILNQLTIVKAIKAVGTIK
    Accession: RFLPSEFGHDVDRANPVEPGLAMYKEKRMVRRLIEES
    XP_028127206.1 GVPYTYICCNSIASWPYYDNTHPSEVIPPLDRFQIYGD
    GTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHFRPSC
    NFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAE
    NIIPQSIVASFTHDIFEKGCQVNFSIDGPNEVEVSNLYPD
    ETFRTMDECFDDFVMKMDRWN
    Leucoanthocyanidin MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGK 63
    reductase (LAR) STYILSRAAARCPSKARAIKALEDQGAISIHGSVNDQE
    Coffea arabica FMEKTLKEHEIDIVISAVGGGNLLEQVILIRAMKAVGT
    Accession: IKRFLPSEFGHDVDRAEPVEPGLTMYNEKRRVRRLIEE
    XP_027097479.1 SGVPYTYICCNSIASWPYYDNTHPSEVSPPLDQFQIYG
    DGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHFRPS
    CNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEE
    NIIPQSVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYP
    DEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRL
    VDVLTAPKLCA
    Leucoanthocyanidin MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEA 64
    reductase (LAR) SLDSGRPTYILARSSSNSPSKASTIKFLQDRGATVIYGSI
    Theobroma cacao TDKEFMEKVLKEHKIEVVISAVGGGSILDQFNLIEAIR
    Accession: NVDTVKRFLPSEFGHDTDRADPVEPGLTMYEQKRQIR
    ADD51357.1 RQIEKSGIPYTYICCNSIAAWPYHDNTHPADVLPPLDR
    FKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKTVH
    FQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQM
    AKEMRIPQSVVAALTHDIFINGCQINFSLDKPTDVEVC
    SLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAI
    FVPTAKPGALPITAICT
    Leucoanthocyanidin MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLP 65
    reductase (LAR) TYVLVRPGPSRPSKSDTIKSLKDRGAIILHGVMSDKPL
    Fragaria x MEKLLKEHEIEIVISAVGGATILDQITLVEAITSVGTVK
    ananassa RFLPSEFGHDVDRADPVEPGLTMYLEKRKVRRAIEKS
    Accession: GVPYTYICCNSIASWPYYDNKHPSEVVPPLDQFQIYGD
    ABH07785.2 GTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFRPSS
    NLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAEN
    RIPESIVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEE
    SFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVE
    PMAITATCA
    Anthocyanin MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRL 66
    dioxygenase (ANS) NEELTSMGNVFEEEKKEEGSQVPTIDIKDIASEDPEVR
    Chenopodium GKAIQELKRAAMEWGVMHLVNHGISDELIDRVKVAG
    quinoa QTFFELPVEEKEKYANDQASGNVQGYGSKLANSASG
    Accession: RLEWEDYYFHLSYPEDKRDLSIWPETPADYIPAVSEYS
    XP_021735950.1 KELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQ
    FKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPG
    LQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSIL
    HRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEE
    PARYPPRTFAQHVQYKLFRKTQGPQTTITKN
    Anthocyanin MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDD 67
    dioxygenase (ANS) SLGDALEEIKKTEEGPQIPIVDLRGFDSGDEKERLHCM
    Iris sanguinea EEVKEAAVEWGVMHIVNHGIAPELIERVRAAGKGFFD
    Accession: LPVEAKERYANNQSEGKIQGYGSKLANNASGQLEWE
    QCI56004.1 DYFFHLIFPSDKVDLSIWPKEPADYTEVMMEFAKQLR
    VVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKI
    NYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQV
    FHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVL
    HRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKR
    SPAKYPPRTFAQHIQHKLFKKAQEQLAGGVHIPEAIQN
    Anthocyanin MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGD 68
    dioxygenase (ANS) VFEEEKKLEGPQIPVVDLMGLEWENEEVFKKVEEDM
    Magnolia sprengeri KKAASEWGVMHIFNHGISMELMDRVRIAGKAFFDLPI
    Accession: EEKEMYANDQASGKIAGYGSKLANNASGQLEWEDYF
    AHU88620.1 FHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQLRGLV
    TKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYY
    PKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFD
    DKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLV
    NKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFT
    PRTFSQHVRQKLFKKQQDALENLKSE
    Anthocyanin MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIG 69
    dioxygenase (ANS) NVFEEEKKEGPEVPTIDLTEIESEDEVVRARCHETLKK
    Prosopis alba AAQEWGVMNLVNHGIPEELLNQLRKAGETFFSLPIEE
    Accession: KEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFH
    XP_028787846.1 LVFPEDKCDLSIWPRTPSDYIEVTSEYARQLRGLATKI
    LGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPIC
    PQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQ
    WITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNK
    EKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRT
    FAQHIQHKLFRKCQEGLSK
    Anthocyanidin-3- MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPN 70
    O-glycotransferase THFSFLNTSQSNNSIFSSDVYNRQPNLKAHNVWDGVP
    (3 GT) EGYVFVGKPQESIELFVKAAPETFRKGVEAAVAETGR
    Cephalotus KVSCLVTDAFFWFAAEIAGELGVPWVPFWTAGPCSLS
    follicularis THVYTDLIRKTIGVGGIEGREDESLEFIPGMSQVVIRDL
    Accession: QEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEEL
    GAV66155.1 DLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLP
    WLDQQKVASVAYISFGTVMAPSPPELVALAEALEASK
    IPFIWSLGEKLKVHLPKGFLDKTRTHGIVVPWAPQSDV
    LENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQ
    RLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLILS
    QARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALL
    NLVRLSI
    Anthocyanidin-3- MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRR 71
    O-glycotransferase LAAASPNTLFSFFSTSQSNNSLFSNTITNLPRNIKVFDV
    (3 GT) ADGVPDGYVFAGKPQEDIELFMKAAPHNFTTSLDTCV
    Prunus cerasifera AHTGKRLTCLITDAFLWFGAHLAHDLGVPWLPLWLS
    Accession: GLNSLSLHVHTDLLRHTIGTQSIAGRENELITKNVNIPG
    AKV89253.1 MSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN
    AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAA
    SPPLPEAPTAADDVTGCLSWLDKQKAASSVVYVSFGS
    VARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLN
    ELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWN
    SLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTV
    EDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLL
    AQQPVEPKGSSAQNFKLLLDVISGSTKV
    Anthocyanidin-3- MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFS 72
    O-glycotransferase FFNSAVSNSTLFNNGVLDSYDNIRVYHVWDGTPQGQ
    (3 GT) AFTGSHFEAVGLFLKASPGNFDKVIDEAEVETGLKISC
    Scutellaria LITDAFLWFGYDLAEKRGVPWLAFWTSAQCALSAHM
    baicalensis YTHEILKAVGSNGVGETAEEELIQSLIPGLEMAHLSDL
    Accession: PPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEIDP
    A0A482AQV3 IITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLD
    SQTRPKSVVYISFGTVITPPENELAALSEALETCNYPFL
    WSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLA
    HRSVGVFVTHCGWNSILESICSGVPLICRPFFGDQKLN
    SRMVEDSWKIGVRLEGGVLSKTATVEALGRVMMSEE
    GEIIRENVNEMNEKAKIAVEPKGSSFKNFNKLLEIINAP
    QSS
    Anthocyanidin-3- MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPH 73
    O-glycotransferase AVFSFFSTSQSNASIFHDSMHTMQCNIKSYDISDGVPE
    (3 GT) GYVFAGRPQEDIELFTRAAPESFRQGMVMAVAETGRP
    Vitis vinifera VSCLVADAFIWFAADMAAEMGLAWLPFWTAGPNSLS
    Accession: THVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFRDL
    P51094 QEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL
    DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQ
    WLKERKPTSVVYISFGTVTTPPPAEVVALSEALEASRV
    PFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAE
    VLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFG
    DQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQIL
    SQEKGKKLRENLRALRETADRAVGPKGSSTENFITLV
    DLVSKPKDV
    Acetyl-CoA MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHS 74
    carboxylase (ACC) VITKVLICNNGIAAVKEIRSIRKWAYETFGDERAIEFTV
    Ustilago maydis MATPEDLKVNADYIRMADQYVEVPGGSNNNNYANV
    521 DLIVDVAERAGVHAVWAGWGHASENPRLPESLAASK
    Accession: HKIIFIGPPGSAMRSLGDKISSTIVAQHADVPCMPWSG
    XP_011390921.1 TGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAE
    KIGYPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLG
    EVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGR
    DCSVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAK
    LVGYVSAGTVEWLYSPESGEFAFLELNPRLQVEHPTT
    EMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRG
    NEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDT
    GFKPGMGALTELNFRSSTSTWGYFSVGTSGALHEYAD
    SQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVE
    YLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADL
    AVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIK
    TVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRTLV
    QLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSK
    TCLIEQENDPTQIRSPSPGKLVRFLVDSGDHVKANQAI
    AEIEVMKMYLPLVAAEDGVVSFVKTAGVALSPGDIIG
    ILSLDDPSRVQHAKPFAGQLPDFGMPVIVGNKPHQRY
    TALVEVLNDILDGYDQSFRMQAVIKELIETLRNPELPY
    GQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEFPA
    ARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYA
    GGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELR
    LQADGDLDKVVALQTSRNGINRKNALLLTLLDKHIKG
    TSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVS
    LDADMPSLADRSAQMQAILRGSVTSSKYGGDDEYHA
    PSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALC
    TYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQL
    PRSASSLKERERQVSISDLSMMDNNRRARPIRELRTGA
    MTSCADVADIPELLPKVLKFFKSSAGASGAPINVLNV
    AVVDQTDFVDAEVRSQLALYTNACSKEFSAARVRRV
    TYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALA
    YQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADT
    RFFVRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEV
    ALGQPEYRTADASHIFMSFIYQLDVSLVDVQKAIAGFL
    ERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNET
    GLVVRYETYEETVADDGSVILRGIEPQGKDATLNAQS
    AHFPYTTKVALQSRRSRAHALQTTFVYDFIDVLGQAV
    RASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRA
    PGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQ
    AGSFGPVEDRFFAAASKLARELGVPRLYISANSGARIG
    LATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQA
    KAPNSVMTKPVQAADGSVHNIITDIIGKPQGGLGVEC
    LSGSGLIAGETSRAKDQIFTATIITGRSVGIGAYLARLG
    ERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQ
    IMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGP
    LPIMPTTDSWDRAVTYQPPRGPYDPRWLINGTKAEDG
    TKLTGLFDEGSFVETLGGWATSVVTGRARLGGIPVGV
    IAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNS
    AYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMYD
    EILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVV
    VDSAINDNGMIEMSADVNSARGGVLEASGLVEIKYRA
    DKQRATMERLDSVYAKLSKEAAEATDFTAQTTARKA
    LAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRS
    ALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLK
    HVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLA
    ASKQLKAQYILAQISTLDPELRAQLAASLK
    Acetyl-CoA MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHT 75
    carboxylase (ACC) VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF
    Hesseltinella TVMATPEDLKANAEYIRMADNYIEVPGGTNNNNYAN
    vesiculosa VELIVDVAERTGVHAVWAGWGHASENPRLPEMLAKS
    Accession: KNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTMGW
    ORX57605.1 SGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLA
    SAEKIGFPVMIKASEGGGGKGIRKVEDPSTFKQAFAQ
    VQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLF
    GRDCSVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLG
    KLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPT
    TEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN
    SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPD
    AGFKPSGGVMQELNFRSSTNVWGYFSVVSSGAMHEY
    ADSQFGHIFAYGENRQQARKNMVIALKELSIRGDFRT
    TVEYIIRLLETPDFTDNTINTGWLDMLISKKLTAERPDT
    MLAVFCGAVTKAHLASVECWQQYKNSLERGQIPSKE
    SLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKT
    QVGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMID
    GKTCLLEKESDPTQLRSPSPGKLVSLLLENGDHIRTGQ
    AYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGD
    IIGILSLDDPSRVKHALPFTGQVPKYGLPHLTGDKPHQ
    RFTHLKQTLEYVLQGYDNQGLIQTIVKELSEVLNNPEL
    PYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKG
    FPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIA
    ASYSNGLKQHEHSVYVDLMEQYYNVEVLFNSNQSRD
    EEVILALRDQHKDDLEKVINIILSHAKVNIKNNLILMLL
    DIIYPATSSEALDRCFLPILKHLSEIDSRGTQKVTLKAR
    EYLILCQLPSLEERQSQMYNILKSSVTESVYGGGTEYR
    TPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALE
    VYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSA
    GGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTL
    EELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMEN
    ILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALR
    RITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALA
    YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC
    RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE
    IVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDR
    HGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNV
    SGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQP
    LSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAI
    HNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA
    IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDI
    TFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSG
    ARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEY
    DALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGL
    GVENLRGSGLIAGATSRAYDDIFTITLVTCRSVGIGAY
    LVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNL
    QLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPE
    CRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQ
    ENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGI
    PMGVVAVETRTVENIVPADPANADSQEQVVMEAGGV
    WFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGG
    QRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGEL
    RGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGI
    VEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKE
    QADELIAQVEAREQALLPVYQQVAIQFADLHDRSGR
    MEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRK
    MDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVA
    WFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAV
    VEGFSKLMESLSVDQRKEVLHKLATRF
    Acetyl-CoA MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVF 76
    carboxylase (ACC) MHGLGSSTSFWEAPFSRSNLSSRFRLIRYDFDGHGLSP
    Rhodotorula VSLLDAADDGAMIPLVDLVEDLAAVMEWTGVDKVA
    toruloides GIVGHSMSGLVASTFAAKYPQKVEKLVLLGAMRSLN
    NBRC10032 PTVQTNMLKRADTVLESGLSAIVAQVVSAALSDKSKQ
    Accession: DSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDY
    GEM08739.1 STIKAKTLVVSGESDYLSNKETTEALVNDIPGAKEVQ
    MDGVGHWHAVEDPAGLAKILDGFFLQGKFSGEAKA
    VNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPPS
    NVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKW
    AYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVE
    VPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGH
    ASENPRLPESLAASKHKIVFIGPPGSAMRSLGDKISSTI
    VAQHAEVPCMDWSGQGVDQVTQSLEGYVTVADDVY
    QQACVHDADEGLARASRIGYPVMIKASEGGGGKGIR
    KVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLE
    VQVLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAK
    PDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDK
    FAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGV
    PLHRIRDIRTLYGKAPNGSSEIDFEFENPESAKTQRKPS
    PKGHVVAVRITAENPDAGFKPSMGTLQELNFRSSTNV
    WGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKN
    MVVALKELSIRGDFRTTVEYLIKLLETDAFEQNTITTA
    WLDSLISARLTAERPDTTLAIICGAVTKAHLASEANIA
    EYKRILEKGQSPPKELLATVVPLEFVLEDVKYRATASR
    SSPSSWSIYVNGSNVSVGIRPLADGGLLILLDGRSYTC
    YAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSPGKL
    VRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQ
    FIKQPGATLEAGDILGILSLDDPSRVHHAKPFDGQLPA
    LGLPSIIGTKPHQRFAYLKDVLSNILMGYDNQAIMQSS
    IKELISVLRNPELPYGEANAVLSTLSGRIPAKLEQTLRQ
    YIDSAHESGAEFPSAKCRKAIDTTLEQLRPAEAQTVRN
    FLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKP
    FSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKN
    SLVLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKA
    TTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPT
    VYGEPGHDRTPRGEVLKDVIDSRFTVFDVLPSFFQHQ
    DQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEP
    ATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLT
    FLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFP
    DSNKGKLQQQPAASSSQEQWNVINVALTVPASAQVD
    EDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQ
    YPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSN
    FHLEPCPVENRQVHIYYATAKGNSSDCRFFVRALVRP
    GRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRR
    AADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRF
    WRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYE
    AYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEW
    LQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGK
    TAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVG
    WIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFY
    AVTQLARQLGLPRVYLSANSGARLGIAEELVDLFSVA
    WADSSRPEKGFKYLYLTAEKLGELKNKGEKSVITKRI
    EDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAY
    DDIFTITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTG
    AGALNKVLGREVYSSNLQLGGTQIMYKNGVSHLTAA
    NDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSI
    DYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSF
    QETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIP
    ADPANPLSNEQKIMEAGQVWYPNSSFKTGQAIFDFNR
    EGLPLIIFANWRGFSGGQQDMFDEVLKRGSLIVDGLS
    AYKQPVFVYIVPNGELRGGAWVVLDPSINAEGMMEM
    YVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTY
    HALRVKSTDASLSPTDAAQAKTELAAREKQLMPIYQQ
    VALQFADSHDKAGRILSKGCAREALEWSNARRYFYA
    RLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQ
    GVDLNNDLAAAAAFEQGAAAITERVKLARATTVAST
    LAQLAQDDKEAFAASLQQVLGDKLTAADLARILA
    Malonyl-CoA MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVAR 77
    synthase (matB) SGRMANVLVARGVKPGDRVAAQAEKSVAALVLYLA
    Rhodopseudomonas TVRAGAVYLPLNTAYTLHELDYFIGDAEPKLVVCDPA
    palustris KREGIAALAQKVGAGVETLDAKGQGSLSEAAAQASV
    Accession: DFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNLAS
    WP_011661926.1 NSLTLVEFWRFTPDDVLMALPIYFITHGLFVASNVTLF
    ARASMIFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQ
    SDGLTKEAARHMRLFISGSAPLLADTHREWASRTGHA
    VLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSL
    RVVDPETGAELSPGEIGMIEVKGPNVFQGYWRMPEKT
    KAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGG
    FNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAV
    VVRDKGASVDEAAVLGALQGQLAKFKMPKRVLFVD
    DLPRNTMGKVQKNVLREAYAKLYAK
    Malonyl-CoA MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCS 78
    synthase (matB) ARITHVLVKLGVSPGDRVAVQVEKSAQALMLYLGCL
    Rhizobium RAGAVYLPLNTAYTPAELEYFLGDATPKLVVVSPCAA
    sp. BUS003 EQLEPLARRVGTRLLTLGVNGDGSLMDMASLEPVEF
    Accession: ADIERKADDLAAILYTSGTTGRSKGAMLTHDNLLSNA
    NKF42351.1 QTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAG
    GAIYLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQD
    ERLNKANTRHMRLFISGSAPLLAETHRLFEEYTGHAIL
    ERYGMTETNMITSNPCDGARVPGTVGYALPGVSVRIT
    DPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKE
    EFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNI
    YPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVG
    QPKAHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNT
    MGKVQKNVLRDRYRDLYLK
    Malonyl-CoA MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMT 79
    synthase (matB) GTARYANVLVGLGVKPGDRVAVQVEKSAGAIFLYLA
    Ochrobactrum sp. CVRAGAVFLPLNTAYTLTEIEYFLGDAEPALVVCDPA
    3-3 RRDGITEVAKKTGVPAVETLGKGQDGSLFDKAAAAP
    Accession: ETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDNLA
    WP_114216069.1 SNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTI
    LVAGASMLFLPKFDADKVFELMPRATTMMGVPTFYV
    RLVQDARLTREATKHMRLFISGSAPLLAETHKLFREK
    TGVSILERYGMTETNMNTSNPYDGDRVAGTVGFPLPG
    VALRVADPETGAAIPQGEIGVIEVKGPNVFSGYWRMP
    EKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLV
    ISGGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGE
    GVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVI
    VVDDLPRNTMGKVQKNLLRDAYKGLYGG
    Malonate MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT 80
    transporter (matC) LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT
    Rhizobiales TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV
    bacterium SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG
    Accession: GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLL
    MBN8942514.1 IAAVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS
    GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG
    AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT
    AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY
    VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV
    SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV
    SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY
    GGIVVAAVPALAWLVLVVPGFG
    Malonate MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMI 81
    transporter (matC) IGMKTNEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL
    Rhizobium VECAVRLVRGRIGLIPWVMFLVAAIITGFGALGPAAV
    leguminosarum AILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSPI
    Accession: SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVFF
    AAC83457.1 VFGGARVMKHDPASLGPLPELHPEGVSASIRGHGGTP
    AKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIG
    ALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSW
    STVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLV
    ALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAI
    GVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVL
    RQLLIYSALIAIIGPIVAWLVFVVPGLV
    Malonate MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLI 82
    transporter (matC) IGMKPADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWL
    Agrobacterium vitis VERSVRMVRGRVGWIPWVMFLVAAIITGFGALGPAA
    Accession: VAILAPVALSFAVQYRIHPVLMGLMVIHGAQAGGFSPI
    WP_180575084.1 SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLIFF
    IFGGLSILKQRSSVKGPLPELHPEGISASIKGHGGTPAKP
    FREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL
    VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTV
    LLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALL
    LCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGV
    VAAIAISTTIVDTSPFSTNGALVVANAPDDQRDKVMR
    QMLIYSALIALIGPVIAWLVFVVPGII
    Malonate MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLI 83
    transporter (matC) IGMKTSDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL
    Neorhizobium sp. VECAVRMVRGHVAWIPWVMFVVAAITGFGALGPAA
    Accession: VAILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSP
    WP_105370917.1 ISVYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVF
    FVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGT
    PAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGI
    GALVFKFNVGLVAITVAVALALISPKTQKAAVDKVS
    WSTVLLIAGIITYVGVLEKAGTVNYVANGISSLGMPLL
    VALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHIS
    AVGVVAAIAISTTIVDTSPFSTNGALVVANAPDETREQ
    VLRQLLIYSALIAIIGPVVAWLVFVVPGLV
    Malonate CoA- MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALL 84
    transferase (MdcA) ETVIDRGDRVCLEGNNQKQADFLSKSLSSCNPDIVNG
    Moraxella LHIVQSVLALPSHIDVFERGIASKVDFSFAGPQSLRLAQ
    catarrhalis LVQAQKITIGAIHTYLELYGRYFIDLTPNVALITAHAA
    Accession: DKRGNLYTGANTEDTPAIVEATTFKSGIVIAQVNEIVD
    WPO64617969.1 ELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITEIQ
    ILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTY
    AESLGLKGEICTHWALNPHPTLIPAIESGFIHSVHSFGS
    EVGMENYVKARSDVFFTGADGSMRSNRAFSQTAGLY
    ACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSD
    PHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVE
    TYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDD
    VTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLG
    RDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKS
    VKDLVRWSDRLYEPPSRFRNW
    Malonate CoA- MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEI 85
    transferase (MdcA) PVDRIIDLLEAVIQPGDRVCLEGNNQKQADFLSESLAD
    Dechloromonas CDPARINHLSMVQSVLALPSHVDLFERGLATRLDFSFS
    aromatica GPQGARLAKLVQEQRIEIGAIHTYLELFGRYFMDLTPN
    Accession: VALIAAQAADAEGNLYLGPNTEDTPAIVEATAFKGGI
    WP_011289741.1 VIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPL
    FTRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIG
    FDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPA
    IEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMR
    SNRAFSQTAGLYACDMFIGSTLQMDLAGNSSTATLGR
    ITGFGGAPNMGSDPHGRRHASPAWLKAGREAYGPQA
    IRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASM
    GSDLPPIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAI
    RGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDP
    RQASRDLLAARSIKDLVRCSGGLYAPPSRFRNW
    Malonate CoA- MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVV 86
    transferase (MdcA) ALLEAVIEPGDRVCLEGNNQKQADFLARCLTEVDPAR
    Pseudomonas VHDLHMVQSVLSLAAHLDVFERGIAKRLDFSFSGPQA
    cissicola ARLAGLVSEGRIEIGAIHTYLELFGRYFIDLTPRIALVT
    Accession: AQAADRHGNLYTGPNTEDTPVIVEATAFKGGIVIAQV
    WP_078590875.1 NEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPA
    QISEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIE
    LLLPTYAQSLGLKGKICRHWALNPHPALIPAIESGFVQ
    SVHSFGSELGMENYIAARPDIFFTGADGSMRSNRALS
    QTAGLYACDMFIGSTLQIDLQGNSSTATRDRIAGFGG
    APNMGSDARGRRHASAAWLKAGREAATPGEMPRGR
    KLVVQMVETFREHMAPAFVDRLDAWELAERANMPL
    PPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGV
    SGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRD
    ATRDLLAARTVKDLVRWSGGLYDPPKRFRNW
    Malonate CoA- MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTE 87
    transferase (MdcA) KIVEALTALIFPGDRVVIEGNNQKQASFLSKALSQVNP
    Geobacillus EKVNGLHIIMSSVSRPEHLDLFEKGIARKIDFSYAGPQS
    subterraneus LRMSQMLEDGKLVIGEIHTYLELYGRLFIDLTPSVALV
    Accession: AADKADASGNLYTGPNTEETPTLVEATAFRDGIVIAQ
    WP_184319829.1 VNELADELPRVDIPGSWIDFVVAADHPYELEPLFTRDP
    RLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIE
    LLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVE
    SIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQ
    VAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGA
    PNMGHDPRGRRHSSPAWLDMITSDHPAAKGRKLVVQ
    MVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGE
    DVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLE
    RDPRKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSI
    EELVEWSEGLYEPPARFRSW
    Pantothenate kinase MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADE 88
    (CoaX) LAVLLQGLMGTHPLLGMELGEGIDGIAICSTVPAVLH
    Streptomyces sp. ELREVSRRYYGDVPAILVEPGVKTGVPILMDNPKEVG
    CLI2509 TDRIINAVAAQHLYGGPAIVVDFGTATTFDAVSARGE
    Accession: YTGGVIAPGIEISVEALGLRGAQLRKIELARPRSVIGK
    WP_095682415.1 STVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVT
    VIATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERN
    AGRR
    Pantothenate kinase MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQA 89
    (CoaX) PAELLKGLTFRLERLNPDFIGVSSVLGQAVNNHVAESL
    Streptomyces ERLQKPFEFAQVHAKHALMSSDYNPAQLGVDRWLQ
    cinereus MLGIIEPSKKQCVIGCGTAVTIDLVDQGHHLGGYIFPSI
    Accession: YLQRESLFSGTRQISIIDGTFDSIDSGTNTQDAVHHGIM
    WP_188874884.1 LSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI
    RQDLVLAGLQRFFAAKNNTKNQN
    Pantothenate kinase MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTAD 90
    (CoaX) ELAVLMQGLMGRQPGGAGRERVDGLAICSSVPAVLH
    Kitasatospora ELREVTRRYYGDLPAVLVAPGVKTGVHVLMDNPKEV
    kifunensis GADRIVNALAANHLYGGPCIVVDFGTATTFDAINERG
    Accession: DYVGGAIAPGIEISVEALGVRGAQLRKIELAKPRNVIG
    WP_184936930.1 KNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPE
    DVQVIATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYE
    RNTAS
    glutamyl-tRNA MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQA 91
    reductase (hemA) LPMVQGGVVLSTCNRTEIYLSVEEQDNLREALIRWLC
    Citrobacter EYHNLNEEDLRNSLYWHQDNDAVSHLMRVASGLDS
    freundii LVLGEPQILGQVKKAFADSQKGHQNASALERMFQKS
    Accession: FSVAKRVRTETDIGSSAVSVAFAACTLARQIFESLSTV
    NTY05430.1 TVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQ
    VLADEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKG
    MVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAY
    LYSVDDLQSIISHNLAQRKAAAVEAETIVEQEASEFMA
    WLRAQGASDTIREYRSQSEQIRDELTAKALAALQQGG
    DAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDSER
    LNILRDSLGLE
    glutamyl-tRNA MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQP 92
    reductase (hemA) LVQGGVVLSTCNRTELYLSVEQQENLQEQLVKWLCD
    Pseudomonas YHHLSADEVRKSLYWHQDNAAVSHLMRVASGLDSL
    reactans VVGEPQILGQVKKAFAESQHGQAVSGELERLFQKSFS
    Accession: VAKRVRTETDIGASAVSVAFAACTLARQIFESLSDVSV
    NWA43040.1 LLVGAGETIELVARHLREHKVRHMMIANRTRERAQV
    LASEVGAEVITLQDIDARLADADIIISSTASPLPIIGKGM
    VERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYL
    YSVDDLHSIIQNNMAQRKAAAVQAESIVEQESSNFMA
    WLRSQGAVEIIRDYRSRADLVRAEAEAKALAAIAQGA
    DVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDVERL
    QILRDSLGLDQQ
    glutamyl-tRNA MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQ 93
    reductase (hemA) QPAVRGGVVLSTCNRTELYLSMEDKENSHEQLIRWLC
    Gammaproteo- QYHQIEPNELQSSIYWHQDNQAVSHLMRVASGLDSL
    bacteria VLGEPQBLGQVKKAFADSQNYDSLSSELERLFQKSFSV
    Accession: AKRVRTETQIGANAVSVAFAACTLARQIFESLSSLTILL
    WP_193016510.1 VGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE
    VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVER
    ALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLYSV
    DDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWL
    RAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADA
    EKVIQQLSHQLMNRLIHTPTKSLQQAASDGDIERLNLL
    RESLGITHN
    5-aminolevulinic MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITI 94
    acid synthase PPNATVEMCPHAKAARDAARIAEDLAAAAASKQQPA
    (ALAS) KKAGGCPFHAAQAQAQAKPAAAPKETVATADKKGK
    Schizophyllum SPRAAGGFDYEKFYEEELDKKHQDKSYRYFNNINRLA
    commune H4-8 ARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLET
    Accession: MHRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLH
    XP_003036856.1 RKEGALVFTSCYVANDATLSTLGSKMPGCVIFSDRMN
    HASMIQGIRHSGTKKVIFEHNDLADLEKKLAEYPKETP
    KIIAFESVYSMCGSIGPIKEICDLAEKYGAITFLDEVHA
    VGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMD
    RVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIF
    TTSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREV
    KRRFAELDIPVVPGPSHIVPVLVGDAALAKQASDKLL
    AEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDH
    LIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQD
    FVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAVV
    AARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQ
    SSGLKLPVEEMTMGQTIAVAA
    5-aminolevulinic MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTAL 95
    acid synthase TERATKCPVMGPALNVRSKEIVAGYASVAANSDVALI
    (ALAS) HKEKGVFPPPGATVEMCPHASAARAAARMADDLAA
    Crassisporium AAEKKKGHFTSAAPRDEAAQAAAAGCPFHVKAAAD
    junariophilum AAAARKAAAAPAPVKAKEDGGFNYESFYVNELDKK
    Accession: HQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCA
    KAF8165006.1 NDYLGMGNNPVVLETMHRTLDKYGHGAGGTRNIAG
    NGAMHLSLEQELATLHRKPAALVFSSCYVANDATLST
    LGAKLPGCIFFSDTMNHASMIQGMRHSGAKRVLFKH
    NDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKE
    ICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDY
    DAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGG
    YIAGSDDFVDMIRSYAPGFIFTTSLPPATVAGARASVV
    YQKHYVGDRQLKQVNVREVKRRFAELDVPVVPGPSH
    IVPVLVGDAALAKAASDKLLAEHNIYVQSINYPTVAR
    GEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA
    DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLD
    GTSPRTLRNGEAAVVDAMAVGQARAVFDNLLGPISG
    KLQSERSVLASSTPAAANPARPAARKVVKMKTGGVP
    MSEDIPLPPPDVSASA
    5-aminolevulinic MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSI 96
    acid synthase SILTERATKCPVMGPALNVRSKEITAGYASVAGSSEVD
    (ALAS) QIHKQQGVTVPVNATVEMCPHASAARAAARMADDL
    Dendrothele AAAAAQKKVGSGASSAKAAAAGCPFHKSVAAGASA
    bispora CBS STASKPSAPIHKASVPGGFDYDNFYNNELEKKHKDKS
    962.96 YRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLG
    Accession: MGNNPVVLETMHRTLDKYGHGAGGTRNIAGNGALH
    THV05492.1 LGLEQELAALHRKEAALVFSSCYVANDATLSTLGSKL
    PGCILFSDKMNHASMIQGMRHSGAKKVIFNHNDLEDL
    ENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEK
    YGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAA
    GKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSM
    DFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYL
    GDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGD
    AALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRIT
    VTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAG
    GRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQR
    FLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD
    DELKVPLPTLVAAAA
    5-aminolevulinic MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHAL 97
    acid synthase WHSPKGKRDVVIWCSNDYLGMGQHPKVVGAMVETA
    (ALAS) TRVGTGAGGTRNIAGTHHPLVQLEAELADLHGKEASL
    Bradyrhizobium LFTSGYVSNQTGIATIAKLIPNCLILSDELNHNSMIEGIR
    japonicum QSGCERVVFRHNDLADLEEKLKAAGPNRPKLIACESL
    Accession: YSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMY
    A0A0A3YXD2 GPRGGGIAERDGVMHRIDILEGTLAKAFGCLGGYIAA
    NGQIIDAVRSYAPGFIFTTALPPAICSAATAAIRHLKTS
    NWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFI
    GDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRI
    TPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKSLAAE
    Cytochrome b5 MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLE 98
    Petunia x hybrida, EHPGGEEVLIESAGKDATKEFQDIGHSKAAKNLLFKY
    Accession: QIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAY
    AAD10774.1 VIKEDPKPKYLTFVEYLLPFLAAAFYLYYRYLTGALQ
    F
  • TABLE 12
    Glossary of abbreviations
    Abbreviation Full Name
    3GT anthocyanidin-3-O-glycotransferase
    4CL 4-coumarate-CoA ligase
    ACC acetyl-CoA carboxylase
    ACOT acyl-CoA thioesterase
    acpP acyl carrier protein
    ACS acetyl-CoA synthase
    adhE aldehyde-alcohol dehydrogenase
    ADP adenosine diphosphate
    ALA 5-aminolevulinic acid
    ALAS ALA synthase
    ANS anthocyanin dioxygenase
    aroG DAHP synthase
    aroK shikimate kinase
    aroL shikimate kinase
    ATP adenosine triphosphate
    C3G cyanidin-3-O-glycoside
    C4H cinnimate-4-hydroxylase
    CHI chalcone isomerase
    CHS chalcone synthase
    CoA coenzyme A
    CPR cytochrome P450 Reductase
    DAD diode array detector
    DAHP deoxy-d-arabino-heptulosonate-7-phosphate
    DctPQM a malonate transporter
    DFR dihydroflavonol 4-reductase
    DHL dihydrokaempferol
    DHM dihydromyricein
    DHQ dihydroquercetin
    DMSO dimethyl sulfoxide
    E4P erythrose-4-phosphate
    F3′H flavonoid 3′ hydroxylase
    F3H flavanone 3-hydroxylase
    fabB beta-ketoacyl-ACP synthase I
    fabD malonyl-coA-ACP transacylase
    fabF beta-ketoacyl-ACP synthase II
    FadA 3-ketoacyl-CoA thiolase
    FadB fatty acid oxidation complex subunit alpha
    FadE acyl-CoA dehydrogenase
    GltX glutamyl-tRNA synthetase
    hemA glutamyl-tRNA reductase
    hemL glutamate-1-semialdehyde aminotransferase
    HPLC high performance liquid chromatography
    ldhA lactate dehydrogenase
    LAR leucoanthocyanidin reductase
    matB malonyl-CoA synthase
    matC malonate transporter
    mdcA malonate coA-transferase
    mdcC acyl-carrier protein, subunit of mdc
    mdcD malonyl-CoA decarboxylase, subunit of mdc
    mdcE co-decarboxylase, subunit of mdc
    pABA para-aminobenzoic acid
    PAL phenylalanine ammonia-lyase
    PanK pantothenase kinase
    Pdh pyruvate dehydrogenase
    PEP phosphoenolpyruvate
    pHBA para-hydroxybenzoic acid
    PHE phenylalanine
    pheA chorismate mutase/prephenate dehydrogenase
    poxB pyruvate dehydrogenase
    ppsA phosphoenolpyruvate synthase
    TAL tyrosine ammonia-lyase
    TCA tricarboxylic acid cycle
    tesA thioesterase I
    tesB thioesterase II
    tktA transketolase
    TRP tryptophan
    TYR tyrosine
    TyrA chorismate mutase
    tyrR transcriptional regulator
    ybgC a thioesterase
    yciA a thioesterase
    ydiB QUIN/shikamate dehydrogenase
    ackA-pta Acetate kinase-phosphate acetyltransferase

Claims (16)

1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.
2. The engineered host cell of claim 1, wherein the one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
3. The engineered host cell of claim 1, wherein the one or more genetic modifications are selected from a group consisting of: (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.
4. The engineered host cell of claim 1, wherein the one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
5. The engineered host cell of claim 1, wherein the one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).
6. The engineered host cell of claim 1, wherein the one or more genetic modifications comprises expression of exogenous transketolase (tktA).
7. The engineered host cell of claim 1, wherein the one or more genetic modifications comprises disruption of tyrR gene.
8. The engineered host of claim 1, wherein the 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.
9. A method of increasing endogenous biosynthesis of tyrosine comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase endogenous biosynthesis of tyrosine.
10. The method of claim 9, wherein the one or more genetic modifications comprises upregulation of 3-deoxy-D-arabino-heptulosonate synthase.
11. The method of claim 9, wherein the one or more genetic modifications are selected from a group consisting of: (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.
12. The method of claim 9, wherein the one or more genetic modifications comprises downregulation of L-phenylalanine biosynthetic pathway.
13. The method of claim 9, wherein the one or more genetic modifications comprises expression of exogenous phosphoenolpyruvate synthase (ppsA).
14. The method of claim 9, wherein the one or more genetic modifications comprises expression of exogenous transketolase (tktA).
15. The method of claim 9, wherein the one or more genetic modifications comprises disruption of tyrR gene.
16. The method of claim 9, wherein the 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.
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