FLAVONOID AND ANTHOCYANIN BIOPRODUCTION USING MICROORGANISM HOSTS

Information

  • Patent Application
  • 20220333122
  • Publication Number
    20220333122
  • Date Filed
    April 13, 2022
    2 years ago
  • Date Published
    October 20, 2022
    2 years ago
Abstract
The invention is directed to methods involved in the production of flavonoids, anthocyanins and other organic compounds. The invention provides cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.
Description
II. SEQUENCE LISTING

This application contains a sequence listing filed in electronic form as an ASCII.txt file entitled DEBU-009-02-US-Sequence-Listing.txt, created on Mar. 21, 2022, last modified Apr. 13, 2022, and having a size of 448 KB. The content of the sequence listing is incorporated herein its entirety.


III. FIELD OF THE INVENTION

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


IV. BACKGROUND OF THE INVENTION

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


V. SUMMARY OF THE INVENTION

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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


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


Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. Strategies to increase heme supply include overexpression of the genes that synthesize the precursor ALA. In an E. coli host, ALA is formed from the 5-carbon skeleton of glutamate (the C5 pathway). The three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (gltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include a mutated hemA (inserting two lysine residuals between Thr-2 and Leu-3 at N terminus of hemA gene from Salmonella typhimurium (EC:1.1.1.70). Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway (ALS is synthesized by the condensation of glycine and succinyl-CoA). Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Bradyrhizobium japonicum (EC: 2.3.1.37), ALAS of Rhodobacter capsulatus, or an ALAS having at least 50% sequence identity to a naturally occurring ALAS. Further, one or more of the downstream genes (e.g., in E. coli hemB, hemC, hemD, hemE, hemF, hemG, 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′-hydroxlase (F3′H) activity or flavonoid 3′,5′-hydroxylase (F3′5′H), an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase (DFR) activity, and/or an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase (LAR) activity.


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









TABLE 1







Tyrosine ammonia-lyase










Organism
GenBank Accession Number








Rhodotorula glutini

AGZ04575.1




Flavobacterium johnsoniae

WP_012023194.1




Herpetosiphon aurantiacus

ABX02653.1




Rhodobacter capsulatus

ADE83766.1




Saccharothrix espanaensis

AKE50820.1




Trichosporon cutaneum

AKE50834.1










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


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


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









TABLE 2







4-coumarate-CoA ligases










Organism
GenBank Accession Number








Petroselinum crispum

CAA31697.1




Camellia sinensis

ASU87409.1




Capsicum annuum

KAF3620173.1




Castanea mollissima

KAF3954751.1




Daucus carota

AIT52344.1




Gynura bicolor

BAJ17664.1




Ipomoea purpurea

AHJ60263.1




Lonicera japonica

AGE10594.1




Lycium chinense

QDL52638.1




Nelumbo nucifera

XP_010265453.1




Nyssa sinensis

KAA8540582.1




Solanum lycopersicum

NP_001333770.1




Striga asiatica

GER48539.1










The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS, SEQ 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 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97% at least 98%, or at least 99% amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida) that have the activity of a chalcone synthase.









TABLE 3







Chalcone synthases










Organism
GenBank Accession Number








Petunia hybrida

AAF60297.1




Acer palmatum

AWN08245.1




Callistephus chinensis

CAA91930.1




Camellia japonica

BAI66465.1




Capsicum annuum

XP_016566084.1




Coffea arabica

XP_027118978.1




Curcuma alismatifolia

ADP08987.1




Dendrobium catenatum

ALE71934.1




Garcinia mangostana

ACM62742.1




Iochroma calycinum

AIY22758.1




Iris germanica

BAE53636.1




Lilium speciosum

BAE79201.1




Lonicera caerulea

ALU09326.1




Lycium ruthenicum

ATB56297.1




Magnolia liliiflora

AHJ60259.1




Matthiola incana

BBM96372.1




Morus alba var. multicaulis

AHL83549.1




Nelumbo nucifera

NP_001305084.1




Nyssa sinensis

KAA8548459.1




Paeonia lactiflora

AEK70334.1




Panax notoginseng

QKV26463.1




Ranunculus asiaticus

AYV99476.1




Rosa chinensis

AEC13058.1




Theobroma cacao

XP_007032052.2










Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ 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









TABLE 8







Dihydroflavonol 4-reductases










Organism
GenBank Accession Number








Eustoma grandiflorum

BAD34461.1




Anthurium andraeanum

AAP20866.1




Camellia sinensis

AAT66505.1




Morelia rubra

KAB1203810.1




Dendrobium moniliforme

AEB96144.1




Fragaria × ananassa

AHL46451.1




Rosa chinensis

XP_024167119.1




Acer palmatum

AWN08247.1




Nyssa sinensis

KAA8531902.1




Vitis amurensis

I82380.1




Abrus precatorius

XP_027329642.1




Angelonia angustifolia

AHM27144.1




Pyrus pyrifolia

Q84KP0.1




Theobroma cacao

XP_017985307




Theobroma cacao

XP_007051597.2




Brassica oleracea var. capitata

QKO29328.1




Rubus idaeus

AXK92786.1




Citrus sinensis

AAY87035.1




Gerbera hybrida

P51105.1




Cephalotus follicularis

GAV76940.1




Ginkgo biloba

AGR34043.1




Dryopteris erythrosora

QFQ61498.1




Dryopteris erythrosora

QFQ61499.1




Cephalotus follicularis

GAV76942.1










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


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


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


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


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


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









TABLE 9







Acetyl-CoA carboxylases










Organism
GenBank Accession Number








Lipomyces starkeyi

AJT60321.1




Rhodotorula toruloides

GEM08739.1




Ustilago maydis

XP_011390921.1




Mucor circinelloides

EPB82652.1




Kalaharituber pfeilii

KAF8466702.1




Aspergillus fumigatus

KEY77072.1




Rhodotorula diobovata

TNY18634.1




Leucosporidium creatinivorum

ORY74050.1




Microbotryum intermedium

SCV70467.1




Mixia osmundae

GAA98306.1




Puccinia graminis

KAA1079218.1




Suillus occidentalis

KAG1764021.1




Gymnopilus junonius

KAF8909366.1










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


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


Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. For example, a cell engineered to produce a flavonoid or an anthocyanin as provided herein can include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from malonate. Acyl-CoA synthetase catalyzes the conversion of a carboxylic acid to its acyl-CoA thioester through an ATP-dependent two-step reaction. In the first step, the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate. In the second step, the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA product. Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced sequences. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. In Rhizobium trifolii, the 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 a 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-coumaryol-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and/or ybgC can be downregulated, disrupted, or deleted.


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


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


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


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


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


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


Host Cells

A host cell as provided herein can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and 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 (PIF) 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), dihydromyricetin (DHM), eriodictyol (EDL), and/or pentahydroxyflavone (PIF) 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:









Enzyme:
Sequence:
SEQ ID:





Tyrosine ammonia-
MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD
 1


lyase (TAL)
EEIVRMGASARTIEEYLKSDKPIYGLTQGFGPLVLFDA




Saccharothrix

DSELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNM




espanaensis

RKGYSAVSPVFWQKLADLWNKGFTPAIPRHGTVSAS



Accession:
GDLQPLAHAALAFTGVGEAWTRDADGRWSTVPAVD



ABC88669.1
ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR




SALRLVRACAVLSARLATLLGANPEHYDVGHGVARG




QVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCA




PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL




LHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGL




LVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRI




RQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEAL




ELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGI




CPPLDADRPLGAEVRAARDLLSAHADQLLVDEADGK




DFG






Phenylalanine
MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK
 2


ammonia-lyase
GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV



(PAL)
TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD




Brevibacillus

EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG




laterosporus LMG

ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE



15441
REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC



Accession:
LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ



WP_003337219.1
KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD




SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK




WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA




MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP




RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS




VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH




LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD




RALDGDIEKVVQLIRSGNLKKEIHDQNVND






Cinnamate-4-
MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLP
 3


hydroxylase (C4H)
VPIFGNWLQVGDDLNHRNLTDLAKKFGEIFLLRMGQ




Helianthusannuus

RNLVVVS SPDLAKEVLHTQGVEFGSRTRNVVFDIFTG




L.

KGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYR



Accession:
YGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQLMM



QJC72299.1
YNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQS




FEYNYGDFIPILRP




FLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV




DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAI




ETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQ




VTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHD




AKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRP




ERFFEEESKVEANGNDFRYLPFGVGRRSCPGIILALPIL




GITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKHS




TIVAKPRAL






4-coumarate-CoA
MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKV
 4


ligase (4CL)
GDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQ




Petroselinum

QGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTS




crispum

AEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIIC



Accession:
IDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPY



P14912.1
SSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYM




HSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKF




DIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLS




SVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGM




TEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIV




DPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTI




DEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVA




PAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRT




NGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSG




KILRKDLRARIASGDLPK






Chalcone synthase
MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTY
 5


(CHS)
PDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLT




Petunia x hybrida

EEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAA



Accession:
QKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKL



AAF60297.1
LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK




GARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGA




GAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHL




REVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNS




LFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNY




GNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVL




FGFGPGLTVETVVLHSVAT






Chalcone isomerase
MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERG
 6


(CHI)
LTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELL




Medicago sativa

ETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENC



Accession:
VAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASV



P28012.1
FYRQSPDGILGLSFSPDTSIPEKEAALIENKAVSSAVLE




TMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN






Flavanone 3-
MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFS
 7


hydroxylase (F3H)
NEIPIISLSGIDEVEGRRAEICNKIVEACEDWGVFQIVD




Rubus occidentalis

HGVDAKLISEMTRLARDFFALPPEEKLRFDMSGGKKG



Accession:
GFIVSSHLQGEAVQDWREIVTYFSYPVRHRDYSRWPD



ACM17897.1
KPEGWRAVTQQYSDELMGLACKLLEVLSEAMGLEKE




ALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDP




GTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVV




NLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQ




NPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSK




DLELARLKKLAKEQQPEDSEKAKLEVKQVDDIFA






Flavonoid 3′
MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIG
 8


hydroxylase (F3′H)
NLPHMGPKPHQTLAAMVTTYGPILHLRLGFADVVVA




Brassica napus

ASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDL



Accession:
VFAPYGQRWRMLRKISSVHLFSAKALEDFKHVRQEE



ABC58723.1
VGTLMRELARANTKPVNLGQLVNMCVLNALGREMI




GRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFV




PALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAM




KNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKA




LLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEE




LDSVVGRGRPINESDLSQLPYLQAVIKENFRLHPPTPLS




LPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDP




LTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAG




LSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEET




YGITLQRAVPLVVHPKLRLDMSAYGLGSA






Cytochrome P450
MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPA
 9


reductase (CPR)
VMAMLLENKELVMILTTSVAVLIGCVVVLIWRRSSGS




Catharanthus

GKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGT




roseus

AEGFAKALAEEAKARYEKAVIKVIDIDDYAADDEEYE



Accession:
EKFRKETLAFFILATYGDGEPTDNAARFYKWFVEGND



Q05001
RGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVA




EQGGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDN




LLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANG




HANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRS




CTHLDFDIAGTGLSYGTGDHVGVYCDNLSETVEEAER




LLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTA




LTRYADLLNTPKKSALLALAAYASDPNEADRLKYLAS




PAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFA




AIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGR




IHKGVCSTWMKNAIPLEESRDCSWAPIFVRQSNFKLP




ADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGT




AVFFFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSR




EGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDA




KGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQM




TGRYLRDVW






Flavonoid 3′, 5′-
MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKG
10


hydroxylase
WPVVGALPMLGNMPHAALADLSRRYGPIVYLKLGSR



(F3′5′H)
GMVVASTPDSARAFLKTQDLNFSNRPTDAGATHIAYN




Delphinium

SQDMVFADYGPRWKLLRKLSSLHMLGGKAVEDWAV




grandiflorum

VRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAMAN



Accession:
MLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFN



BAO66642
VGDFIPSIAWMDLQGIVRGMKRLHKKFDALLDKILRE




HTATRRERKEKPDLVDVLMDNRDNKSEQERLTDTNI




KALLLNLFSAGTDTSSSTIEWALTEMIKNPSIFGRAHA




EMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHPSTP




LNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNVW




ENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRIC




AGTRMGIVLVEYILGSLVHAFEWKLRDGETLNMEETF




GIALQKAVPLAAVVTPRLPPSAYVV






Dihydroflavonol 4-
MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATV
11


reductase (DFR)
RDPSNMKKVKHLLDLPGAANRLTLWKADLVDEGSFD




Anthurium

EPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNV




andraeanum

LRSCARASSTVRRVVFTSSAGTVSIHEGRRHLYDETS



Accession:
WSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK



AAP20866.1
NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPH




YSILNPVQFVHLDDLCNAHIFLFECPDAKGRYICSSHD




VTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKL




TDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYA




TEQLIATGQDNGH






Leucoanthocyanidin
MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYP
12


reductase (LAR)
TFLLVRPGPVSPSKAVIIKTFQDKGAKVIYGVINDKEC




Desmodium

MEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIK




uncinatum

RFLPSEFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEY



Accession:
GIPFTNICCNSIASWPYYDNCHPSQVPPPMDQFQIYGD



Q84V83.1
GNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRPSSN




CYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENII




PESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKF




RSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQT




GTIEEINKDIKTLVETQPNEEIKKDMKALVEAVPISAM




G






Anthocyanin
MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFD
13


dioxygenase (ANS)
EEKKDEGPQVPTIDLRDIDSDDQQVRQRCRDELKKAA




Carica papaya

VDWGVMHLVNHGIPDHLIDRVKKAGQAFFELPVEVK



Accession:
EKYANDQASGNIQGYGSKLANNASGQLEWEDYYFHL



XP_021901846.1
IFPEEKRDLAIWPNNPADYIEVTSEYARQLRRLVSKIL




GVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCP




QPELALGVEAHTDISALTFILHNMVPGLQLFYEGKWV




TAKCVPNSIVMHVGDTIEILSNGKYKSILHRGLVNKEK




VRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQ




HIQHKLFRKNQENLEAK






Anthocyanidin-3-
MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPH
14


O-glycotransferase
AVFSFFSTSESNASIFHDSMHTMQCNIKSYDVSDGVPE



(3GT)
GYVFTGRPQEGIDLFMRAAPESFRQGMVMAVAETGR




Vitis labrusca

PVSCLVADAFIWFAADMAAEMGVAWLPFWTAGPNS



Accession:
LSTHVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFR



ABR24135
DLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFE




ELDDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCL




QWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASR




VPFIWSLRDKARMHLPEGFLEKTRGHGMVVPWAPQA




EVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFF




GDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQI




LSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTL




VDLVSKPKDV






Acetyl-CoA
MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHT
15


carboxylase (ACC)
VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF




Mucor

TVMATPEDLKANAEYIRMADNFVEVPGGSNNNNYAN




circinelloides

VELIVDVAERTAVHAVWAGWGHASENPRLPEMLAKS



1006PhL
KHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTMGW



Accession:
SGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKA



EPB82652.1
AEKIGFPVMIKASEGGGGKGIRMVKDGSNFAQLFAQV




QGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFG




RDCSVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLG




KLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPT




TEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNS




ASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAG




FKPSSGIMQELNFRSSTNVWGYFSVVSAGGLHEYADS




QFGHIFAYGENRQQARKNMVIALKELSIRADFRSTVE




YIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTML




AVFCGAVTKAHMASLDCFQQYKQSLEKGQVPSKGSL




KTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQV




GIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGK




TCLLEKESDPTQLRSPSPGKLVNLLVENGDHLNAGDA




YAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDII




GILSLDDPSRVKHALPFNGTVPAFGAPHITGDKPVQRF




NATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLP




YSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP




AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFAR




YRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVIL




SLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLI




NPVSTGSALDKYFTPILKRLSEIESRATQKVTLKAREL




LILCQLPSYEERQAQMYQILKNSVTESVYGGGSEYRTP




SYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEVY




CRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANG




IDSNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLAD




LQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNI




AVRADEDMDDTAFRTKICEMITANADVFRQAHLRRL




SVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMA




YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC




RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE




IVSHEYKNSDCNHLFINFIPTFAIEADDVEHALKDFVD




RHGKRLWKLRVTGAEIRFNVQSKKPDAPIIPMRFTVD




NVSGFILKVEVYQEVKTEKSGWILKSVNKIPGAMHM




QPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQ




SVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVL




QEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDI




TFKIGSFGVAEDQVFYKASELARALGIPRIYLSANSGA




RIGLADELISQFRAAWKDASNPTAGFKYLYLTPAEYD




VLAQQGDAKSVLVEEIQDEGETRLRITDVIGHTDGLG




VENLKGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYL




VRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQL




GGTQIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVR




NAPVSMRLGADPIDRDIEYTPPKGPSDPRFFLAGKSEN




GKWLSGFFDQDSFVETLSGWARTVVVGRARLGGIPM




GVVSVETRTVENIVPADPANSDSTEQVFMEAGGVWFP




NSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRD




MYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGA




WVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKY




RKPALLATMERLDATYASLKKQLAEEGKTDEEKAAL




KVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKG




VIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATS




AAPMPREQMLDLVKQWFTNDNETVNFEDADELVSE




WFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIE




GFSQLIENLSEDARAEILRKLNSRF






Acetyl-CoA
MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTF
16


synthase (ACS)
WGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGT




Salmonella

LNLAANCLDRHLQENGDRTAIIWEGDDTSQSKHISYR




typhimurium

ELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAV



Accession:
AMLACARIGAVHSVIFGGFSPEAVAGRIIDSSSRLVITA



NP_463140.1
DEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRT




GSDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPL




FILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYH




PGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEG




VPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEG




DKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE




KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFF




GVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLF




GDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITG




RVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIP




HAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIG




PLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNL




GDTSTLADPGVVEKLLEEKQAIAMPS






Malonyl-CoA
MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAA
17


synthase (matB)
AGATAGRIGGAGRVAVWATPAMETGVAVVAALLAG




Streptomyces

VAAVPLNPKSGDKELAHILSDSAPSLVLAPPDAELPPA




coelicolor

LGALERVDVDVRARGAVPEDGADDGDPALVVYTSGT



Accession:
TGPPKGAVIPRRALATTLDALADAWQWTGEDVLVQG



WP_011028356
LPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAAREL




NDGATMLFGVPTMYHRIAETLPADPELAKALAGARL




LVSGSAALPVHDHERIAAATGRRVIERYGMTETLMNT




SVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDG




ESVGEIQVRGPNLFTEYLNRPDATAAAFTEDGFFRTG




DMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENA




LLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPP




ALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKI




MKRALNRD






Malonate
MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT
18


transporter (matC)
LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT




Streptomyces

TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV




coelicolor

SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG



Accession:
GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA



NP_626686.1
AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS




GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG




AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT




AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY




VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV




SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV




SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY




GGIVVAAVPALAWLVLVVPGFG






Malonate CoA-
MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARA
19


transferase (MdcA)
IELLETVIASGDRVCLEGNNQKQADFLSKCLSQCNPD




Acinetobacter

AVNDLHIVQSVLALPSHIDVFEKGIASKVDFSFAGPQS




calcoaceticus

LRLAQLVQQQKISIGSIHTYLELYGRYFIDLTPNICLITA



Accession:
HAADREGNLYTGPNTEDTPAIVEATAFKSGIVIAQVNE



AAB97627.1
IVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQI




TEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIEL




LLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDS




VHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQ




TAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAP




NMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVV




QTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMI




YGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTP




VGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLA




AKSVKDLVKWSGGLYSPPSRFRNW






Pantothenate kinase
MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVE
20


(CoaX)
QLTSQQALPVRACRLVSVRSEQETSQLVARLEQLFPV




Pseudomonas

SALVASSGKQLAGVRNGYLDYQRLGLDRWLALVAA




aeruginosa

HHLAKKACLVIDLGTAVTSDLVAADGVHLGGYICPG



Accession:
MTLMRSQLRTHTRRIRYDDAEARRALASLQPGQATA



Q9HWCL1
EAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGG




DAELVRDELAGARIMPDLVFVGLALACPIE






glutamyl-tRNA
MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSL
21


reductase (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




PAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVR




SNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPS




GKILRKDLRAKLAAGFPN






4-coumarate-CoA
MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSS
36


ligase (4CL)
KPCLINGSTGKVYTYSDVELTSRKVAAGFHNLGIQQR




Camellia sinensis

DTIMLLLPNCPEFVFAFLGASYLGAIITMANPFFTPAET



Accession:
IKQAKASNSKLIITQSSYTSKVLDYSSENNVKIICIDSPP



ASU87409.1
DGCLHFSELIQSNETQLPEVEIDSNEVVALPYSSGTTGL




PKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMM




CVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKL




IQRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMS




GAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLA




MCLAFAKKPFEIKSGACGTVVRNAEMKIVDPDAGFSL




PRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLH




TGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEAL




LLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVT




ENEIKKYISEQVVFYKINRVYFVDAIPKAPSGKILRK




DLRARLAAGIPS






Chaicone synthase
MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTY
37


(CHS)
PDYYFRITNSEHKTELKEKFKRMCEKSMIKTRYMHLT




Capsicum annuum

EEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEA



Accession:
AQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLA



XP_016566084.1
KLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN




NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGD




GAAAIIMGSDPIPGVERPLFQLVSAAQTLLPDSEGAID




GHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISD




WNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVL




SNYGNMSSACVLFILDEMRKASTKEGLGTSGEGLEW




GVLFGFGPGLTVETVVLHSVAI






Chalcone synthase
MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYP
38


(CHS)
DYYFRITKSEHKAELKEKFQRMCDKSMIKKRYMYLT




Rosa chinensis

EEILKENPSMCEYMAPSLDARQDMVVVEIPKLGKEAA



Accession:
TKAIKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKL



AEC13058.1
LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK




GARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGA




AAIIVGSDPLPEVEKPLFELVSAAQTILPDSDGAIDGHL




REVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWN




SLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEY




GNMSSACVLFILDEVRRKSAANGHKTTGEGLEWGVL




FGFGPGLTVETVVLHSVAA






Cha:cone synthase
MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQAD
39


(CHS)
YPDYYFRITNSDHMTDLKDKFKRMCEKSMITKRHMY




Morusalba var.

LTEEILKENPKMCEYMAPSLDARQDIVVVEVPKLGKE




multicaulis

AAAKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLT



Accession:
KLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENN



AHL83549.1
KGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDG




AAAVILGADPDTSVERPIFELVSAAQTILPDSEGAIDGH




LREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWN




SIFWIAHPGGPAILDQVEAKLGLKQEKLSATRHVLSEY




GNMSSACVLFILDEVRKKSVEEGKATTGEGLEWGVLF




GFGPGLTVETIVLHSLPAV






Chalcone synthase
MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQAD
40


(CHS)
YPDYYFRITKSEHLTELKEKFKQMCDKSMIRKRYMYL




Dendrobium

TEEILKENPNICAFMAPSLDARQDIVVTEVPKLAREAS




catenatum

ARAIKEWGQPKSRITHLIFCTTSGVDMPGADYQLTRL



Accession:
LGLRPSVNRIMLYQQGCFAGGTVLRLAKDLAENNAG



ALE71934.1
ARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAA




AIIVGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLRE




MGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSI




FWIAHPGGPAILDQVEIKLGLKEEKLASSRNVLAEYG




NMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLF




GFGPGLTVETVVLRSVPIAGAV






Chalcone isomerase
MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTI
41


(CHI)
EGNFIKFTAIGVYLEDVAVASLATKWKGKSSEELLET



Trifolium pratense
LDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVTENCVA



Accession:
HLKSVGTYGDAEVEAMEKFVEAFKPINFPPGASVFYR



PNX83855.1
QSPDGILGVSISIHFFP






Chalcone isomerase
MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGV
42


(CHI)
RGLTIEGNFIKFTGIGVYLEDQAVASLATKWKGKSSEE




Abrus precatorius

LVESLDFFRDIISGPFEKLIRGSKIRQLSGPEYSKKVME



Accession:
NCVAHMKSVGTYGDAEAAGIEEFAQAFKPVNFPPGA



XP_027366189.1
SVFYRQSPDGVLGLSFSQDATIPEEEAAVIKNKPVSAA




VLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN






Chalcone isomerase
MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGER
43


(CHI)
GLTIDGKFIKFTGIGVYLEDKAVPSLAGKWKDKSSQQ




Arachis duranensis

LLQTLHFYRDIISGPFEKLIRGSKILALSGVEYSRKVME



Accession:
NCVAHMKSVGTYGDAEAEAIQQFAEAFKNVNFKPGA



XP_015942246.1
SVFYRQSPLGHLGLSFSQDGNIPEKEAAVIENKPLSSA




VLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQH




N






Chalcone isomerase
MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGER
44


(CHI)
GLEIQGKFIKFTAIGVYLEGDAVASLAVKWKGKSKEE




Cephalotus

LTDSVEFFRDIVTGPFEKFTQVTTILPLTGQQYSEKVSE




follicularis

NCVAFWKSVGIYTDAEAKAIEKFIEVFKEETFPPGSSIL



Accession:
FTQSPNGALTIAFSKDGVIPEVGKAVIENKLLAEGLLE



GAV77263.1
SIIGKHGVSPVAKQCLATRLSELL






Flavanone 3-
MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV
45


hydroxylase (F3H)
RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD




Abrus precatorius

EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI



Accession:
MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC



XP_027329642.1
WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN




NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI




IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI




HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL




GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK






Flavanone 3-
MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLER
46


hydroxylase (F3H)
GYIVRATVRDPANLKKVKHLLDLPKADTNLTLWKAD




Camellia sinensis

LNEEGSFDEAIEGCSGVFHVATPMDFESKDPENEVIKP



Accession:
TINGVLSIIRSCTKAKTVKRLVFTSSAGTVNVQEHQQP



AAT66505.1
VFDENNWSDLHFINKKKMTGWMYFVSKTLAEKAAW




EAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRN




EGHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSS




HDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSF




SSKKLIGMGFEFKYSLEDMFRGAIDTCREKGLLPHSFA




ENPVNGNKV






Flavanone 3-
MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYI
47


hydroxylase (F3H)
VRATVRDPANMKKVKHLQELEKADKNLTLWKADLT




Nyssa sinensis

EEGSFDEAIKGCSGVFHVATPMDFESKDPENEVIKPTI



Accession:
NGVLSIVRSCVKAKTVKRLVFTSSAGTVNLQEHQQLV



KAA8531902.1
YDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAWE




ATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEA




HYSIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSH




DATIYDLAKMIREKWPEYNVPTELKGIEKDLQTVSFSS




KKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHE




TPANANANANANVKKNQNENTEI






Flavanone 3-
MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV
48


hydroxylase (F3H)
RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD




Rosa chinensis

EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI



Accession:
MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN



XP_024167119.1
WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN




NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII




KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH




EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE




TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE




VDESSVVGVKVTG






Flavonoid 3′
MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIG
49


hydroxylase (F3′H)
NLPHMGQMPHHSLAAMARTYGPLMHLRLGFVDVIV




Cephalotus

AASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDL




follicularis

VFAPYGPRWRMLRKISSVHLFSGKALDDYRHVRQEE



Accession:
VAVLIRALARAESKQAVNLGQLLNVCTANALGRVML



GAV84063.1
GRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIG




DFIPALDWLDLQGVAAKMKNLHKRFDTFLTGLLEEH




KKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDT




EIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQV




LKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHP




STPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDP




KEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAG




RRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE




DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY






Flavonoid 3′
MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPII
50


hydroxylase (F3′H)
GNLPHMGPVPHHSLAALAKVYGPLMHLRLGFVDVV




Theobroma cacao

VAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQ



Accession:
DLVFAPYGPRWRMLRKISSVHLFSGKALDDFRHVRQ



EOY22049.1
DEVGVLVRALADAKTKVNLGQLLNVCTVNALGRVM




LGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNI




GDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEH




KIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKA




LLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVRKEL




DSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLS




LPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWK




DPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRIC




AGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLN




MEEAFGLTLQRAAPLMVHPRPRLSPRAY






Flavonoid 3′
MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPW
51


hydroxylase (F3′H)
PVVGNLPHLGTIPHHSLAAMAKKYGPLMHLRLGFVD




Gerbera hybrida

VVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYN



Accession:
YQDLVFAPYGPRWRMLRKICSVHLFSTKALDDFRHV



ABA64468.1
RQEEVAILARALVGAGKSPVKLGQLLNVCTTNALAR




VMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIG




DFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHK




TGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEI




KALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARK




EMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPL




SLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW




ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRIC




VGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM




EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV






Flavonoid 3′
MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGP
52


hydroxylase (F3′H)
RGWPVLGNLPQLGPKPHHTLHALSRAHGPLFRLRLGS




Phoenix dactylifera

VDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIA



Accession:
YNYQDLVFAPYGPGWRARRKLLNVHLFSGKALEDLR



XP_008791304.2
PVREGELALLVRALRDRAGANELVDLGRAANKCATN




ALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAG




VFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDG




LIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGE




GAKLTDTDIKALLLNLFTAGTDTTSSTVEWALSELVK




HPDILRKAQLELDSVVGGDRLVSESDLPNLPFMQAIIK




ETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNV




WAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGND




FGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWD




LPEGQMPEKLDMEEAYGLTLQRATPLMVHPVPRLAP




TAYQS






Cytochrome P450
MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI
53


reductase (CPR)
IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE




Camellia sinensis

AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK



Accession:
AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG



XP_028084858
DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG




NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ




CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA




AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG




SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE




SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV




ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA




KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK




KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA




WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA




KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE




DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA




NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI




SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL




LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD




LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED




YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL




QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV




CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI




IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG




CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK




EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR




DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR




DVW






Cytochrome P450
MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI
54


reductase (CPR)
IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE




Cephalotus

AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK




follicularis

AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG



Accession:
DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG



GAV59576.1
NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ




CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA




AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG




SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE




SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV




ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA




KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK




KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA




WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA




KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE




DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA




NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI




SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL




LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD




LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED




YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL




QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV




CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI




IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG




CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK




EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR




DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR




DVW






Cytochrome P450
MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAE
55


reductase (CPR)
LSSMLIENRQFAMIISTSIAVLIGCIVMLLWRRSGGSGS




Brassica napus

SKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGT



Accession:
AEGFAKALGEEARARYEKTRFKIVDLDDYAADDDEY



XP_013706600.1
EEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEG




DDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDI




LVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPEL




DTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKN




LANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCT




HLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLL




DMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTR




YACLLSSPKKSALLALAAHASDPTEAERLKHLASPAG




KDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAV




APRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIH




KGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSD




SKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVL




FFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREGP




TKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKG




MARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGR




YLRDVW






Flavonoid 3′, 5′-
MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPG
56


hydroxylase
PKGWPFVGALPLLGTMPHVALAQMAKKYGPVMYLK



(F3′5′H)
MGTCGMVVASTPDAARAFLKTLDLNFSNRPPNAGAT




Cephalotus

HLAYNAQDMVFADYGPRWKLLRKLSNLHMLGGKAL




follicularis

EDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLTY



Accession:
AMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTS



GAV62131
AGYFNIGDFIPSIAWMDLQGIERGMKKLHKRFDALLT




KMFEEHMATAHERKGNPDLLDIVMANRDNSEGERLT




TTNIKALLLNLFSAGTDTSSSIIEWSLAEMLKNPSILKR




AHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHP




STPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP




EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGR




RICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMD




EAFGLALQKAVPLAAIVSPRLAPTAYVV






Flavonoid 3′, 5′-
MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGA
57


hydroxylase
LPFIGPMPHSGLALLARRYGPIMFLKMGIRRVVVASSS



(F3′5′H)
TAARTFLKTFDSHFSDRPSGVISKEISYNGQNMVFADY




Dendrobium

GPKWKLLRKVSSLHLLGSKAMSRWAGVRRDEALSMI




moniliforme

QFLKKHSDSEKPVLLPNLLVCAMANVIGRIAMSKRVF



Accession:
HEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGWL



AEB96145
DPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQ




GNPDLLDLVVASEVKGEDGEGLCEDNIKGFISDLFVA




GTDTSAIVIEWAMAEMLKNPSILRRAQEETDRVIGRH




RLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEP




CEVEGYHIPGETWLLVNIWAIGRDPDVWENPLVFDPE




RFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMV




MVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPK




AVPLAVMATPRLPAAAYGLL






Dihydroflavonol 4-
MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVR
58


reductase (DFR)
DPDNEKKVKHLVELPKAKTHLTLWKADLSDEGSFDE




Acer palmatum

AIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIM



Accession:
KACKKAKTVKRLVFTSSAGTVDVEEHKKPVYDENSW



AWN08247.1
SDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEENSI




DFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAII




KQGNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATI




LELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKIL




DLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHV




AAKGLIPNNSDEIHVAAAEKTTATA






Dihydroflavonol 4-
MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV
59


reductase (DFR)
RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD




Abrus precatorius

EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI



Accession:
MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC



XP_027329642.1
WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN




NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI




IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI




HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL




GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK






Dihydroflavonol 4-
MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRA
60


reductase (DFR)
TVRDPTNLKKVKPLLDLPRSNELLSIWKADLDGIEGSF




Dendrobium

DEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGI




moniliforme

LRSCKNAGSVQRVIFTSSAGTVNVEEHQAAAYDETC



Accession:
WSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFVKD



AEB96144.1
NHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHY




SILKQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDST




IYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKL




MELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVL




AAEKFEEVKEQIAVK






Dihydroflavonol 4-
MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV
61


reductase (DFR)
RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD




Rosa chinensis

EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI



Accession:
MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN



XP_024167119.1
WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN




NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII




KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH




EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE




TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE




VDESSVVGVKVTG






Leucoanthocyanidin
MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTT
62


reductase (LAR)
FLLVRSLDKGAIPSKSKTINSLHDKGAILIHGVIEDQEF




Camellia sinensis

VEGILKDHKIDIVISAVGGANILNQLTIVKAIKAVGTIK



Accession:
RFLPSEFGHDVDRANPVEPGLAMYKEKRMVRRLIEES



XP_028127206.1
GVPYTYICCNSIASWPYYDNTHPSEVIPPLDRFQIYGD




GTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHFRPSC




NFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAE




NIIPQSIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPD




ETFRTMDECFDDFVMKMDRWN






Leucoanthocyanidin
MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGK
63


reductase (LAR)
STYILSRAAARCPSKARAIKALEDQGAISIHGSVNDQE




Coffea arabica

FMEKTLKEHEIDIVISAVGGGNLLEQVILIRAMKAVGT



Accession:
IKRFLPSEFGHDVDRAEPVEPGLTMYNEKRRVRRLIEE



XP_027097479.1
SGVPYTYICCNSIASWPYYDNTHPSEVSPPLDQFQIYG




DGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHFRPS




CNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEE




NIIPQSVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYP




DEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRL




VDVLTAPKLCA






Leucoanthocyanidin
MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEA
64


reductase (LAR)
SLDSGRPTYILARSSSNSPSKASTIKFLQDRGATVIYGSI




Theobroma cacao

TDKEFMEKVLKEHKIEVVISAVGGGSILDQFNLIEAIR



Accession:
NVDTVKRFLPSEFGHDTDRADPVEPGLTMYEQKRQIR



ADD51357.1
RQIEKSGIPYTYICCNSIAAWPYHDNTHPADVLPPLDR




FKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKTVH




FQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQM




AKEMRIPQSVVAALTHDIFINGCQINFSLDKPTDVEVC




SLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAI




FVPTAKPGALPITAICT






Leucoanthocyanidin
MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLP
65


reductase (LAR)
TYVLVRPGPSRPSKSDTIKSLKDRGAIILHGVMSDKPL




Fragaria x

MEKLLKEHEIEIVISAVGGATILDQITLVEAITSVGTVK




ananassa

RFLPSEFGHDVDRADPVEPGLTMYLEKRKVRRAIEKS



Accession:
GVPYTYICCNSIASWPYYDNKHPSEVVPPLDQFQIYGD



ABH07785.2
GTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFRPSS




NLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAEN




RIPESIVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEE




SFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVE




PMAITATCA






Anthocyanin
MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRL
66


dioxygenase (ANS)
NEELTSMGNVFEEEKKEEGSQVPTIDIKDIASEDPEVR




Chenopodium

GKAIQELKRAAMEWGVMHLVNHGISDELIDRVKVAG




quinoa

QTFFELPVEEKEKYANDQASGNVQGYGSKLANSASG



Accession:
RLEWEDYYFHLSYPEDKRDLSIWPETPADYIPAVSEYS



XP_021735950.1
KELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQ




FKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPG




LQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSIL




HRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEE




PARYPPRTFAQHVQYKLFRKTQGPQTTITKN






Anthocyanin
MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDD
67


dioxygenase (ANS)
SLGDALEEIKKTEEGPQIPIVDLRGFDSGDEKERLHCM




Iris sanguinea

EEVKEAAVEWGVMHIVNHGIAPELIERVRAAGKGFFD



Accession:
LPVEAKERYANNQSEGKIQGYGSKLANNASGQLEWE



QCI56004.1
DYFFHLIFPSDKVDLSIWPKEPADYTEVMMEFAKQLR




VVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKI




NYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQV




FHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVL




HRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKR




SPAKYPPRTFAQHIQHKLFKKAQEQLAGGVHIPEAIQN






Anthocyanin
MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGD
68


dioxygenase (ANS)
VFEEEKKLEGPQIPVVDLMGLEWENEEVFKKVEEDM




Magnolia sprengeri

KKAASEWGVMHIFNHGISMELMDRVRIAGKAFFDLPI



Accession:
EEKEMYANDQASGKIAGYGSKLANNASGQLEWEDYF



AHU88620.1
FHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQLRGLV




TKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYY




PKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFD




DKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLV




NKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFT




PRTFSQHVRQKLFKKQQDALENLKSE






Anthocyanin
MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIG
69


dioxygenase (ANS)
NVFEEEKKEGPEVPTIDLTEIESEDEVVRARCHETLKK




Prosopis alba

AAQEWGVMNLVNHGIPEELLNQLRKAGETFFSLPIEE



Accession:
KEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFH



XP_028787846.1
LVFPEDKCDLSIWPRTPSDYIEVTSEYARQLRGLATKI




LGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPIC




PQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQ




WITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNK




EKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRT




FAQHIQHKLFRKCQEGLSK






Anthocyanidin-3-
MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPN
70


O-glycotransferase
THFSFLNTSQSNNSIFSSDVYNRQPNLKAHNVWDGVP



(3GT)
EGYVFVGKPQESIELFVKAAPETFRKGVEAAVAETGR




Cephalotus

KVSCLVTDAFFWFAAEIAGELGVPWVPFWTAGPCSLS




follicularis

THVYTDLIRKTIGVGGIEGREDESLEFIPGMSQVVIRDL



Accession:
QEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEEL



GAV66155.1
DLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLP




WLDQQKVASVAYISFGTVMAPSPPELVALAEALEASK




IPFIWSLGEKLKVHLPKGFLDKTRTHGIVVPWAPQSDV




LENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQ




RLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLILS




QARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALL




NLVRLSI






Anthocyanidin-3-
MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRR
71


O-glycotransferase
LAAASPNTLFSFFSTSQSNNSLFSNTITNLPRNIKVFDV



(3GT)
ADGVPDGYVFAGKPQEDIELFMKAAPHNFTTSLDTCV




Prunus cerasifera

AHTGKRLTCLITDAFLWFGAHLAHDLGVPWLPLWLS



Accession:
GLNSLSLHVHTDLLRHTIGTQSIAGRENELITKNVNIPG



AKV89253.1
MSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN




AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAA




SPPLPEAPTAADDVTGCLSWLDKQKAASSVVYVSFGS




VARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLN




ELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWN




SLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTV




EDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLL




AQQPVEPKGSSAQNFKLLLDVISGSTKV






Anthocyanidin-3-
MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFS
72


O-glycotransferase
FFNSAVSNSTLFNNGVLDSYDNIRVYHVWDGTPQGQ



(3GT)
AFTGSHFEAVGLFLKASPGNFDKVIDEAEVETGLKISC




Scutellaria

LITDAFLWFGYDLAEKRGVPWLAFWTSAQCALSAHM




baicalensis

YTHEILKAVGSNGVGETAEEELIQSLIPGLEMAHLSDL



Accession:
PPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEIDP



A0A482AQV3
IITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLD




SQTRPKSVVYISFGTVITPPENELAALSEALETCNYPFL




WSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLA




HRSVGVFVTHCGWNSILESICSGVPLICRPFFGDQKLN




SRMVEDSWKIGVRLEGGVLSKTATVEALGRVMMSEE




GEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEIINAP




QSS






Anthocyanidin-3-
MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPH
73


O-glycotransferase
AVFSFFSTSQSNASIFHDSMHTMQCNIKSYDISDGVPE



(3GT)
GYVFAGRPQEDIELFTRAAPESFRQGMVMAVAETGRP




Vitis vinifera

VSCLVADAFIWFAADMAAEMGLAWLPFWTAGPNSLS



Accession:
THVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFRDL



P51094
QEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL




DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQ




WLKERKPTSVVYISFGTVTTPPPAEVVALSEALEASRV




PFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAE




VLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFG




DQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQIL




SQEKGKKLRENLRALRETADRAVGPKGSSTENFITLV




DLVSKPKDV






Acetyl-CoA
MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHS
74


carboxylase (ACC)
VITKVLICNNGIAAVKEIRSIRKWAYETFGDERAIEFTV




Ustilago maydis

MATPEDLKVNADYIRMADQYVEVPGGSNNNNYANV



521
DLIVDVAERAGVHAVWAGWGHASENPRLPESLAASK



Accession:
HKIIFIGPPGSAMRSLGDKISSTIVAQHADVPCMPWSG



XP_011390921.1
TGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAE




KIGYPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLG




EVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGR




DCSVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAK




LVGYVSAGTVEWLYSPESGEFAFLELNPRLQVEHPTT




EMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRG




NEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDT




GFKPGMGALTELNFRSSTSTWGYFSVGTSGALHEYAD




SQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVE




YLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADL




AVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIK




TVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRTLV




QLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSK




TCLIEQENDPTQIRSPSPGKLVRFLVDSGDHVKANQAI




AEIEVMKMYLPLVAAEDGVVSFVKTAGVALSPGDIIG




ILSLDDPSRVQHAKPFAGQLPDFGMPVIVGNKPHQRY




TALVEVLNDILDGYDQSFRMQAVIKELIETLRNPELPY




GQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEFPA




ARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYA




GGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELR




LQADGDLDKVVALQTSRNGINRKNALLLTLLDKHIKG




TSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVS




LDADMPSLADRSAQMQAILRGSVTSSKYGGDDEYHA




PSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALC




TYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQL




PRSASSLKERERQVSISDLSMMDNNRRARPIRELRTGA




MTSCADVADIPELLPKVLKFFKSSAGASGAPINVLNV




AVVDQTDFVDAEVRSQLALYTNACSKEFSAARVRRV




TYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALA




YQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADT




RFFVRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEV




ALGQPEYRTADASHIFMSFIYQLDVSLVDVQKAIAGFL




ERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNET




GLVVRYETYEETVADDGSVILRGIEPQGKDATLNAQS




AHFPYTTKVALQSRRSRAHALQTTFVYDFIDVLGQAV




RASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRA




PGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQ




AGSFGPVEDRFFAAASKLARELGVPRLYISANSGARIG




LATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQA




KAPNSVMTKPVQAADGSVHNIITDIIGKPQGGLGVEC




LSGSGLIAGETSRAKDQIFTATIITGRSVGIGAYLARLG




ERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQ




IMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGP




LPIMPTTDSWDRAVTYQPPRGPYDPRWLINGTKAEDG




TKLTGLFDEGSFVETLGGWATSVVTGRARLGGIPVGV




IAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNS




AYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMYD




EILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVV




VDSAINDNGMIEMSADVNSARGGVLEASGLVEIKYRA




DKQRATMERLDSVYAKLSKEAAEATDFTAQTTARKA




LAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRS




ALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLK




HVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLA




ASKQLKAQYILAQISTLDPELRAQLAASLK






Acetyl-CoA
MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHT
75


carboxylase (ACC)
VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF




Hesseltinella

TVMATPEDLKANAEYIRMADNYIEVPGGTNNNNYAN




vesiculosa

VELIVDVAERTGVHAVWAGWGHASENPRLPEMLAKS



Accession:
KNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTMGW



ORX57605.1
SGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLA




SAEKIGFPVMIKASEGGGGKGIRKVEDPSTFKQAFAQ




VQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLF




GRDCSVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLG




KLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPT




TEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN




SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPD




AGFKPSGGVMQELNFRSSTNVWGYFSVVSSGAMHEY




ADSQFGHIFAYGENRQQARKNMVIALKELSIRGDFRT




TVEYIIRLLETPDFTDNTINTGWLDMLISKKLTAERPDT




MLAVFCGAVTKAHLASVECWQQYKNSLERGQIPSKE




SLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKT




QVGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMID




GKTCLLEKESDPTQLRSPSPGKLVSLLLENGDHIRTGQ




AYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGD




IIGILSLDDPSRVKHALPFTGQVPKYGLPHLTGDKPHQ




RFTHLKQTLEYVLQGYDNQGLIQTIVKELSEVLNNPEL




PYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKG




FPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIA




ASYSNGLKQHEHSVYVDLMEQYYNVEVLFNSNQSRD




EEVILALRDQHKDDLEKVINIILSHAKVNIKNNLILMLL




DIIYPATSSEALDRCFLPILKHLSEIDSRGTQKVTLKAR




EYLILCQLPSLEERQSQMYNILKSSVTESVYGGGTEYR




TPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALE




VYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSA




GGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTL




EELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMEN




ILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALR




RITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALA




YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC




RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE




IVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDR




HGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNV




SGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQP




LSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAI




HNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA




IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDI




TFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSG




ARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEY




DALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGL




GVENLRGSGLIAGATSRAYDDIFTITLVTCRSVGIGAY




LVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNL




QLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPE




CRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQ




ENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGI




PMGVVAVETRTVENIVPADPANADSQEQVVMEAGGV




WFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGG




QRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGEL




RGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGI




VEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKE




QADELIAQVEAREQALLPVYQQVAIQFADLHDRSGR




MEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRK




MDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVA




WFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAV




VEGFSKLMESLSVDQRKEVLHKLATRF






Acetyl-CoA
MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVF
76


carboxylase (ACC)
MHGLGSSTSFWEAPFSRSNLSSRFRLIRYDFDGHGLSP




Rhodotorula

VSLLDAADDGAMIPLVDLVEDLAAVMEWTGVDKVA




toruloides

GIVGHSMSGLVASTFAAKYPQKVEKLVLLGAMRSLN



NBRC10032
PTVQTNMLKRADTVLESGLSAIVAQVVSAALSDKSKQ



Accession:
DSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDY



GEM08739.1
STIKAKTLVVSGESDYLSNKETTEALVNDIPGAKEVQ




MDGVGHWHAVEDPAGLAKILDGFFLQGKFSGEAKA




VNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPPS




NVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKW




AYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVE




VPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGH




ASENPRLPESLAASKHKIVFIGPPGSAMRSLGDKISSTI




VAQHAEVPCMDWSGQGVDQVTQSLEGYVTVADDVY




QQACVHDADEGLARASRIGYPVMIKASEGGGGKGIR




KVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLE




VQVLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAK




PDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDK




FAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGV




PLHRIRDIRTLYGKAPNGSSEIDFEFENPESAKTQRKPS




PKGHVVAVRITAENPDAGFKPSMGTLQELNFRSSTNV




WGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKN




MVVALKELSIRGDFRTTVEYLIKLLETDAFEQNTITTA




WLDSLISARLTAERPDTTLAIICGAVTKAHLASEANIA




EYKRILEKGQSPPKELLATVVPLEFVLEDVKYRATASR




SSPSSWSIYVNGSNVSVGIRPLADGGLLILLDGRSYTC




YAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSPGKL




VRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQ




FIKQPGATLEAGDILGILSLDDPSRVHHAKPFDGQLPA




LGLPSIIGTKPHQRFAYLKDVLSNILMGYDNQAIMQSS




IKELISVLRNPELPYGEANAVLSTLSGRIPAKLEQTLRQ




YIDSAHESGAEFPSAKCRKAIDTTLEQLRPAEAQTVRN




FLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKP




FSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKN




SLVLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKA




TTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPT




VYGEPGHDRTPRGEVLKDVIDSRFTVFDVLPSFFQHQ




DQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEP




ATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLT




FLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFP




DSNKGKLQQQPAASSSQEQWNVINVALTVPASAQVD




EDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQ




YPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSN




FHLEPCPVENRQVHIYYATAKGNSSDCRFFVRALVRP




GRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRR




AADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRF




WRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYE




AYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEW




LQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGK




TAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVG




WIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFY




AVTQLARQLGLPRVYLSANSGARLGIAEELVDLFSVA




WADSSRPEKGFKYLYLTAEKLGELKNKGEKSVITKRI




EDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAY




DDIFTITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTG




AGALNKVLGREVYSSNLQLGGTQIMYKNGVSHLTAA




NDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSI




DYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSF




QETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIP




ADPANPLSNEQKIMEAGQVWYPNSSFKTGQAIFDFNR




EGLPLIIFANWRGFSGGQQDMFDEVLKRGSLIVDGLS




AYKQPVFVYIVPNGELRGGAWVVLDPSINAEGMMEM




YVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTY




HALRVKSTDASLSPTDAAQAKTELAAREKQLMPIYQQ




VALQFADSHDKAGRILSKGCAREALEWSNARRYFYA




RLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQ




GVDLNNDLAAAAAFEQGAAAITERVKLARATTVAST




LAQLAQDDKEAFAASLQQVLGDKLTAADLARILA






Malonyl-CoA
MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVAR
77


synthase (matB)
SGRMANVLVARGVKPGDRVAAQAEKSVAALVLYLA




Rhodopseudomonas

TVRAGAVYLPLNTAYTLHELDYFIGDAEPKLVVCDPA




palustris

KREGIAALAQKVGAGVETLDAKGQGSLSEAAAQASV



Accession:
DFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNLAS



WP_011661926.1
NSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLF




ARASMIFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQ




SDGLTKEAARHMRLFISGSAPLLADTHREWASRTGHA




VLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSL




RVVDPETGAELSPGEIGMIEVKGPNVFQGYWRMPEKT




KAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGG




FNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAV




VVRDKGASVDEAAVLGALQGQLAKFKMPKRVLFVD




DLPRNTMGKVQKNVLREAYAKLYAK






Malonyl-CoA
MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCS
78


synthase (matB)
ARITHVLVKLGVSPGDRVAVQVEKSAQALMLYLGCL




Rhizobium

RAGAVYLPLNTAYTPAELEYFLGDATPKLVVVSPCAA



sp. BUS003
EQLEPLARRVGTRLLTLGVNGDGSLMDMASLEPVEF



Accession:
ADIERKADDLAAILYTSGTTGRSKGAMLTHDNLLSNA



NKF42351.1
QTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAG




GAIYLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQD




ERLNKANTRHMRLFISGSAPLLAETHRLFEEYTGHAIL




ERYGMTETNMITSNPCDGARVPGTVGYALPGVSVRIT




DPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKE




EFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNI




YPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVG




QPKAHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNT




MGKVQKNVLRDRYRDLYLK






Malonyl-CoA
MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMT
79


synthase (matB)
GTARYANVLVGLGVKPGDRVAVQVEKSAGAIFLYLA




Ochrobactrum sp.

CVRAGAVFLPLNTAYTLTEIEYFLGDAEPALVVCDPA



3-3
RRDGITEVAKKTGVPAVETLGKGQDGSLFDKAAAAP



Accession:
ETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDNLA



WP_114216069.1
SNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTI




LVAGASMLFLPKFDADKVFELMPRATTMMGVPTFYV




RLVQDARLTREATKHMRLFISGSAPLLAETHKLFREK




TGVSILERYGMTETNMNTSNPYDGDRVAGTVGFPLPG




VALRVADPETGAAIPQGEIGVIEVKGPNVFSGYWRMP




EKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLV




ISGGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGE




GVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVI




VVDDLPRNTMGKVQKNLLRDAYKGLYGG






Malonate
MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT
80


transporter (matC)
LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT




Rhizobiales

TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV




bacterium

SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG



Accession:
GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA



MBN8942514.1
AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS




GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG




AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT




AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY




VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV




SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV




SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY




GGIVVAAVPALAWLVLVVPGFG






Malonate
MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMI
81


transporter (matC)
IGMKTNEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL




Rhizobium

VECAVRLVRGRIGLIPWVMFLVAAIITGFGALGPAAV




leguminosarum

AILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSPI



Accession:
SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVFF



AAC83457.1
VFGGARVMKHDPASLGPLPELHPEGVSASIRGHGGTP




AKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIG




ALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSW




STVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLV




ALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAI




GVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVL




RQLLIYSALIAIIGPIVAWLVFVVPGLV






Malonate
MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLII
82


transporter (matC)
GMKPADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWL




Agrobacterium vitis

VERSVRMVRGRVGWIPWVMFLVAAIITGFGALGPAA



Accession:
VAILAPVALSFAVQYRIHPVLMGLMVIHGAQAGGFSPI



WP_180575084.1
SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLIFFI




FGGLSILKQRSSVKGPLPELHPEGISASIKGHGGTPAKP




FREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL




VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTV




LLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALL




LCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGV




VAAIAISTTIVDTSPFSTNGALVVANAPDDQRDKVMR




QMLIYSALIALIGPVIAWLVFVVPGII






Malonate
MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLI
83


transporter (matC)
IGMKTSDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL




Neorhizobium sp.

VECAVRMVRGHVAWIPWVMFVVAAITGFGALGPAA



Accession:
VAILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSP



WP_105370917.1
ISVYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVF




FVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGT




PAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGI




GALVFKFNVGLVAITVAVALALISPKTQKAAVDKVS




WSTVLLIAGIITYVGVLEKAGTVNYVANGISSLGMPLL




VALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHIS




AVGVVAAIAISTTIVDTSPFSTNGALVVANAPDETREQ




VLRQLLIYSALIAIIGPVVAWLVFVVPGLV






Malonate CoA-
MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALL
84


transferase (MdcA)
ETVIDRGDRVCLEGNNQKQADFLSKSLSSCNPDIVNG




Moraxella

LHIVQSVLALPSHIDVFERGIASKVDFSFAGPQSLRLAQ




catarrhalis

LVQAQKITIGAIHTYLELYGRYFIDLTPNVALITAHAA



Accession:
DKRGNLYTGANTEDTPAIVEATTFKSGIVIAQVNEIVD



WPO64617969.1
ELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITEIQ




ILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTY




AESLGLKGEICTHWALNPHPTLIPAIESGFIHSVHSFGS




EVGMENYVKARSDVFFTGADGSMRSNRAFSQTAGLY




ACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSD




PHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVE




TYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDD




VTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLG




RDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKS




VKDLVRWSDRLYEPPSRFRNW






Malonate CoA-
MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEI
85


transferase (MdcA)
PVDRIIDLLEAVIQPGDRVCLEGNNQKQADFLSESLAD




Dechloromonas

CDPARINHLSMVQSVLALPSHVDLFERGLATRLDFSFS




aromatica

GPQGARLAKLVQEQRIEIGAIHTYLELFGRYFMDLTPN



Accession:
VALIAAQAADAEGNLYLGPNTEDTPAIVEATAFKGGI



WP_011289741.1
VIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPL




FTRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIG




FDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPA




IEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMR




SNRAFSQTAGLYACDMFIGSTLQMDLAGNSSTATLGR




ITGFGGAPNMGSDPHGRRHASPAWLKAGREAYGPQA




IRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASM




GSDLPPIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAI




RGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDP




RQASRDLLAARSIKDLVRCSGGLYAPPSRFRNW






Malonate CoA-
MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVV
86


transferase (MdcA)
ALLEAVIEPGDRVCLEGNNQKQADFLARCLTEVDPAR




Pseudomonas

VHDLHMVQSVLSLAAHLDVFERGIAKRLDFSFSGPQA




cissicola

ARLAGLVSEGRIEIGAIHTYLELFGRYFIDLTPRIALVT



Accession:
AQAADRHGNLYTGPNTEDTPVIVEATAFKGGIVIAQV



WP_078590875.1
NEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPA




QISEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIE




LLLPTYAQSLGLKGKICRHWALNPHPALIPAIESGFVQ




SVHSFGSELGMENYIAARPDIFFTGADGSMRSNRALS




QTAGLYACDMFIGSTLQIDLQGNSSTATRDRIAGFGG




APNMGSDARGRRHASAAWLKAGREAATPGEMPRGR




KLVVQMVETFREHMAPAFVDRLDAWELAERANMPL




PPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGV




SGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRD




ATRDLLAARTVKDLVRWSGGLYDPPKRFRNW






Malonate CoA-
MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTE
87


transferase (MdcA)
KIVEALTALIFPGDRVVIEGNNQKQASFLSKALSQVNP




Geobacillus

EKVNGLHIIMSSVSRPEHLDLFEKGIARKIDFSYAGPQS




subterraneus

LRMSQMLEDGKLVIGEIHTYLELYGRLFIDLTPSVALV



Accession:
AADKADASGNLYTGPNTEETPTLVEATAFRDGIVIAQ



WP_184319829.1
VNELADELPRVDIPGSWIDFVVAADHPYELEPLFTRDP




RLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIE




LLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVE




SIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQ




VAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGA




PNMGHDPRGRRHSSPAWLDMITSDHPAAKGRKLVVQ




MVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGE




DVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLE




RDPRKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSI




EELVEWSEGLYEPPARFRSW






Pantothenate kinase
MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADE
88


(CoaX)
LAVLLQGLMGTHPLLGMELGEGIDGIAICSTVPAVLH




Streptomyces sp.

ELREVSRRYYGDVPAILVEPGVKTGVPILMDNPKEVG



CLI2509
TDRIINAVAAQHLYGGPAIVVDFGTATTFDAVSARGE



Accession:
YTGGVIAPGIEISVEALGLRGAQLRKIELARPRSVIGKS



WP_095682415.1
TVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVT




VIATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERN




AGRR






Pantothenate kinase
MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQA
89


(CoaX)
PAELLKGLTFRLERLNPDFIGVSSVLGQAVNNHVAESL




Streptomyces

ERLQKPFEFAQVHAKHALMSSDYNPAQLGVDRWLQ




cinereus

MLGIIEPSKKQCVIGCGTAVTIDLVDQGHHLGGYIFPSI



Accession:
YLQRESLFSGTRQISIIDGTFDSIDSGTNTQDAVHHGIM



WP_188874884.1
LSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI




RQDLVLAGLQRFFAAKNNTKNQN






Pantothenate kinase
MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTAD
90


(CoaX)
ELAVLMQGLMGRQPGGAGRERVDGLAICSSVPAVLH




Kitasatospora

ELREVTRRYYGDLPAVLVAPGVKTGVHVLMDNPKEV




kifunensis

GADRIVNALAANHLYGGPCIVVDFGTATTFDAINERG



Accession:
DYVGGAIAPGIEISVEALGVRGAQLRKIELAKPRNVIG



WP_184936930.1
KNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPE




DVQVIATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYE




RNTAS






glutamyl-tRNA
MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQA
91


reductase (hemA)
LPMVQGGVVLSTCNRTEIYLSVEEQDNLREALIRWLC




Citrobacter

EYHNLNEEDLRNSLYWHQDNDAVSHLMRVASGLDS




freundii

LVLGEPQILGQVKKAFADSQKGHQNASALERMFQKS



Accession:
FSVAKRVRTETDIGSSAVSVAFAACTLARQIFESLSTV



NTY05430.1
TVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQ




VLADEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKG




MVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAY




LYSVDDLQSIISHNLAQRKAAAVEAETIVEQEASEFMA




WLRAQGASDTIREYRSQSEQIRDELTAKALAALQQGG




DAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDSER




LNILRDSLGLE






glutamyl-tRNA
MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQP
92


reductase (hemA)
LVQGGVVLSTCNRTELYLSVEQQENLQEQLVKWLCD




Pseudomonas

YHHLSADEVRKSLYWHQDNAAVSHLMRVASGLDSL




reactans

VVGEPQILGQVKKAFAESQHGQAVSGELERLFQKSFS



Accession:
VAKRVRTETDIGASAVSVAFAACTLARQIFESLSDVSV



NWA43040.1
LLVGAGETIELVARHLREHKVRHMMIANRTRERAQV




LASEVGAEVITLQDIDARLADADIIISSTASPLPIIGKGM




VERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYL




YSVDDLHSIIQNNMAQRKAAAVQAESIVEQESSNFMA




WLRSQGAVEIIRDYRSRADLVRAEAEAKALAAIAQGA




DVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDVERL




QILRDSLGLDQQ






glutamyl-tRNA
MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQ
93


reductase (hemA)
QPAVRGGVVLSTCNRTELYLSMEDKENSHEQLIRWLC




Gamma-

QYHQIEPNELQSSIYWHQDNQAVSHLMRVASGLDSL




proteobacteria

VLGEPQILGQVKKAFADSQNYDSLSSELERLFQKSFSV



Accession:
AKRVRTETQIGANAVSVAFAACTLARQIFESLSSLTILL



WP_193016510.1
VGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE




VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVER




ALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLYSV




DDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWL




RAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADA




EKVIQQLSHQLMNRLIHTPTKSLQQAASDGDIERLNLL




RESLGITHN






5-aminolevulinic
MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITI
94


acid synthase
PPNATVEMCPHAKAARDAARIAEDLAAAAASKQQPA



(ALAS)
KKAGGCPFHAAQAQAQAKPAAAPKETVATADKKGK




Schizophyllum

SPRAAGGFDYEKFYEEELDKKHQDKSYRYFNNINRLA




commune H4-8

ARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLET



Accession:
MHRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLH



XP_003036856.1
RKEGALVFTSCYVANDATLSTLGSKMPGCVIFSDRMN




HASMIQGIRHSGTKKVIFEHNDLADLEKKLAEYPKETP




KIIAFESVYSMCGSIGPIKEICDLAEKYGAITFLDEVHA




VGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMD




RVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIF




TTSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREV




KRRFAELDIPVVPGPSHIVPVLVGDAALAKQASDKLL




AEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDH




LIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQD




FVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAVV




AARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQ




SSGLKLPVEEMTMGQTIAVAA






5-aminolevulinic
MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTAL
95


acid synthase
TERATKCPVMGPALNVRSKEIVAGYASVAANSDVALI



(ALAS)
HKEKGVFPPPGATVEMCPHASAARAAARMADDLAA




Crassisporium

AAEKKKGHFTSAAPRDEAAQAAAAGCPFHVKAAAD




junariophilum

AAAARKAAAAPAPVKAKEDGGFNYESFYVNELDKK



Accession:
HQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCA



KAF8165006.1
NDYLGMGNNPVVLETMHRTLDKYGHGAGGTRNIAG




NGAMHLSLEQELATLHRKPAALVFSSCYVANDATLST




LGAKLPGCIFFSDTMNHASMIQGMRHSGAKRVLFKH




NDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKE




ICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDY




DAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGG




YIAGSDDFVDMIRSYAPGFIFTTSLPPATVAGARASVV




YQKHYVGDRQLKQVNVREVKRRFAELDVPVVPGPSH




IVPVLVGDAALAKAASDKLLAEHNIYVQSINYPTVAR




GEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA




DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLD




GTSPRTLRNGEAAVVDAMAVGQARAVFDNLLGPISG




KLQSERSVLASSTPAAANPARPAARKVVKMKTGGVP




MSEDIPLPPPDVSASA






5-aminolevulinic
MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSIS
96


acid synthase
ILTERATKCPVMGPALNVRSKEITAGYASVAGSSEVD



(ALAS)
QIHKQQGVTVPVNATVEMCPHASAARAAARMADDL




Dendrothele

AAAAAQKKVGSGASSAKAAAAGCPFHKSVAAGASA




bispora CBS

STASKPSAPIHKASVPGGFDYDNFYNNELEKKHKDKS



962.96
YRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLG



Accession:
MGNNPVVLETMHRTLDKYGHGAGGTRNIAGNGALH



THV05492.1
LGLEQELAALHRKEAALVFSSCYVANDATLSTLGSKL




PGCILFSDKMNHASMIQGMRHSGAKKVIFNHNDLEDL




ENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEK




YGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAA




GKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSM




DFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYL




GDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGD




AALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRIT




VTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAG




GRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQR




FLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD




DELKVPLPTLVAAAA






5-aminolevulinic
MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHAL
97


acid synthase
WHSPKGKRDVVIWCSNDYLGMGQHPKVVGAMVETA



(ALAS)
TRVGTGAGGTRNIAGTHHPLVQLEAELADLHGKEASL




Bradyrhizobium

LFTSGYVSNQTGIATIAKLIPNCLILSDELNHNSMIEGIR




japonicum

QSGCERVVFRHNDLADLEEKLKAAGPNRPKLIACESL



Accession:
YSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMY



A0A0A3YXD2
GPRGGGIAERDGVMHRIDILEGTLAKAFGCLGGYIAA




NGQIIDAVRSYAPGFIFTTALPPAICSAATAAIRHLKTS




NWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFI




GDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRI




TPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKSLAAE






Cytochrome b5
MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLE
98



Petunia x hybrida.

EHPGGEEVLIESAGKDATKEFQDIGHSKAAKNLLFKY



Accession:
QIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAY



AAD10774.1
VIKEDPKPKYLTFVEYLLPFLAAAFYLYYRYLTGALQ




F
















TABLE 12







Glossary of abbreviations










Abbreviation
Full Name







3GT
anthocyanidin-3-O-glycotransferase



4CL
4-coumarate-CoA ligase



ACC
acetyl-CoA carboxylase



ACOT
acyl-CoA thioesterase



acpP
acyl carrier protein



ACS
acetyl-CoA synthase



adhE
aldehyde-alcohol dehydrogenase



ADP
adenosine diphosphate



ALA
5-aminolevulinic acid



ALAS
ALA synthase



ANS
anthocyanin dioxygenase



aroG
DAHP synthase



aroK
shikimate kinase



aroL
shikimate kinase



ATP
adenosine triphosphate



C3G
cyanidin-3-O-glycoside



C4H
cinnimate-4-hydroxylase



CHI
chalcone isomerase



CHS
chalcone synthase



CoA
coenzyme A



CPR
cytochrome P450 Reductase



DAD
diode array detector



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



DctPQM
a malonate transporter



DFR
dihydroflavonol 4-reductase



DHL
dihydrokaempferol



DHM
dihydromyricein



DHQ
dihydroquercetin



DMSO
dimethyl sulfoxide



E4P
erythrose-4-phosphate



F3′H
flavonoid 3′ hydroxylase



F3H
flavanone 3-hydroxylase



fabB
beta-ketoacyl-ACP synthase I



fabD
malonyl-coA-ACP transacylase



fabF
beta-ketoacyl-ACP synthase II



FadA
3-ketoacyl-CoA thiolase



FadB
fatty acid oxidation complex subunit alpha



FadE
acyl-CoA dehydrogenase



GltX
glutamyl-tRNA synthetase



hemA
glutamyl-tRNA reductase



hemL
glutamate-1-semialdehyde aminotransferase



HPLC
high performance liquid chromatography



ldhA
lactate dehydrogenase



LAR
leucoanthocyanidin reductase



matB
malonyl-CoA synthase



matC
malonate transporter



mdcA
malonate coA-transferase



mdcC
acyl-carrier protein, subunit of mdc



mdcD
malonyl-CoA decarboxylase, subunit of mdc



mdcE
co-decarboxylase, subunit of mdc



pABA
para-aminobenzoic acid



PAL
phenylalanine ammonia-lyase



PanK
pantothenase kinase



Pdh
pyruvate dehydrogenase



PEP
phosphoenolpyruvate



pHBA
para-hydroxybenzoic acid



PHE
phenylalanine



pheA
chorismate mutase/prephenate dehydrogenase



poxB
pyruvate dehydrogenase



ppsA
phosphoenolpyruvate synthase



TAL
tyrosine ammonia-lyase



TCA
tricarboxylic acid cycle



tesA
thioesterase I



tesB
thioesterase II



tktA
transketolase



TRP
tryptophan



TYR
tyrosine



TyrA
chorismate mutase



tyrR
transcriptional regulator



ybgC
a thioesterase



yciA
a thioesterase



ydiB
QUIN/shikamate dehydrogenase



ackA-pta
Acetate kinase-phosphate acetyltransferase









Claims
  • 1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production and/or availability of malonyl-CoA.
  • 2. The engineered host cell of claim 1, wherein the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.
  • 3. The engineered host cell of claim 1, wherein the engineered host cell comprises one or more genetic modifications selected from a group consisting of: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase.
  • 4. The engineered host cell of claim 1, wherein the engineered host cell is E. coli.
  • 5. The engineered host cell of claim 4, wherein the E. coli further comprises genes from fungi.
  • 6. The engineered host cell of claim 3, wherein the acetyl-CoA carboxylase is from a species selected from the group consisting of 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.
  • 7. The engineered host cell of claim 1, wherein the one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • 8. The engineered host cell of claim 1, wherein the one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 9. The engineered host cell of claim 8, wherein the acetyl-CoA synthase is selected from the group consisting of, 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.
  • 10. The engineered host cell of claim 1, wherein the 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.
  • 11. The engineered host cell of claim 10, wherein the malonyl-CoA synthetase is selected from the group consisting 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.
  • 12. The engineered host cell of claim 7, wherein the wherein one or more genetic modifications to decrease fatty acid biosynthesis is selected from the group consisting of: (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.
  • 13. The engineered host cell of claim 1, wherein the engineered host cell comprises peptides selected from a group consisting of: (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.
  • 14. 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.
  • 15. The method of claim 14, wherein the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.
  • 16. The method of claim 14, wherein the engineered host cell comprises one or more genetic modifications selected from a group consisting of: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase.
  • 17. The method of claim 14, wherein the engineered host cell is E. coli.
  • 18. The method of claim 17, wherein the E. coli further comprises genes from fungi.
  • 19. The method of claim 14, wherein the acetyl-CoA carboxylase is from a species selected from the group consisting of 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.
  • 20. The method of claim 14, wherein the one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis.
  • 21. The method of claim 14, wherein the one or more genetic modification is overexpression of acetyl-CoA synthase (ACS).
  • 22. The method of claim 21, wherein the acetyl-CoA synthase (ACS) is selected from the group consisting of, 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.
  • 23. The method of claim 14, wherein the 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.
  • 24. The method of claim 23, wherein the malonyl-CoA synthetase is selected from the group consisting 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.
  • 25. The method of claim 20, wherein the wherein one or more genetic modifications to decrease fatty acid biosynthesis is selected from the group consisting of: (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.
  • 26. The method of claim 14, wherein the engineered host cell comprises peptides selected from a group consisting of: (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.
I. RELATED APPLICATIONS

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

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