Bioproduction of flavonoids and anthocyanins using microbial hosts

Abstract

The present invention is directed to methods for the production of flavonoids, anthocyanins and other organic compounds. The present invention provides engineered cells for the production of flavonoids, anthocyanins and other organic compounds, comprising one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and / or reducing carbon loss resulting from the production of by-products.

Description

[Technical field]

[0001] I. Related Applications This application claims priority to U.S. Provisional Patent Application No. 63 / 174,403, filed April 13, 2021. The contents of U.S. Provisional Patent Application No. 63 / 174,403 are hereby incorporated by reference in their entirety.

[0002] II. SEQUENCE LISTING This application contains a Sequence Listing, which is filed in electronic format as an ASCII.txt file entitled DEBU-009-01-WO-Sequence-Listing.txt, created on March 21, 2022, and last modified on April 12, 2022, and having a size of 448KB. The contents of the Sequence Listing are incorporated herein in their entirety.

[0003] III. FIELD OF THEINVENTION The present invention was directed to materials (including engineered cells and cell lines) and methods involved in the production of flavonoids, anthocyanins and other organic compounds. [Background technology]

[0004] IV. Background of the Invention Flavonoids and anthocyanins are natural products produced in plants that find various roles such as antioxidants, ultraviolet (UV) defense mechanisms, and color. Over the past few years, the health benefits of flavonoids and anthocyanins have been widely documented. These compounds have the ability to scavenge radicals and can act as enzyme inhibitors and anti-inflammatory agents. Given these recognized health and color benefits, much research has been put into understanding how these compounds are made in nature. Flavonoids and anthocyanins are synthesized from a phenylpropanoid starter unit and a malonyl-cofactor A (malonyl-CoA) extender unit, which then undergoes modification to create many polyphenolic compounds, such as taxifolin, naringenin, and (+)-catechin. However, in most cases, these compounds are extracted or chemically manufactured. Summary of the Invention [Means for solving the problem]

[0005] V. Summary of the Invention To move away from agricultural and chemically derived products, we have created engineered cells for the bioproduction of flavonoids and anthocyanins. This approach provides a suitable route for the rapid, safe, economical and sustainable production of a wide variety of important flavonoids.

[0006] Herein, various flavonoids and anthocyanins, including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, and cyaninidin-3-glucoside, are biomanufactured using engineered microbial hosts. Herein, the engineered cells contain one or more genetic modifications that increase the bioproduction of flavonoids and anthocyanins by increasing metabolic flux to flavonoid precursors and / or reducing carbon losses due to the production of by-products.

[0007] Provided herein is an engineered cell for the production of flavonoids, anthocyanins and other organic compounds, comprising one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and / or reducing carbon loss due to the production of by-products.As a non-limiting example, genetic modification can be a modification that allows the engineered host cell to overexpress or underexpress one or more endogenous genes, or can be a modification that allows the engineered host cell to express one or more non-native genes.The engineered cell as provided herein can comprise multiple genetic modifications.

[0008] Also provided is a cell culture for producing one or more flavonoids or anthocyanins. The cell culture comprises the engineered cells disclosed herein in a culture medium that includes a carbon source, such as glycerol, sugar, or organic acid, which can also be an energy source. 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 flavonoid precursors (e.g., tyrosine, p-coumaroyl-CoA, or malonyl-CoA). Examples of feed molecules include, but are not limited to, acetate, malonate, tyrosine, phenylalanine, pantothenate, coumarate, and the like. In some embodiments, the feed molecule can be of reduced or low purity. For example, the glycerol as a feed molecule can be crude glycerol, including biomass that contains glycerol, such as glycerol obtained as a by-product of biodiesel processing. Alternatively, or in addition, the culture medium may contain supplemental compounds that may be cofactors or precursors of cofactors used by enzymes functioning in the flavonoid pathway, such as, for example, bicarbonate, biotin, thiamine, pantothenate, alpha-ketoglutarate, ascorbate, or 5-aminolevulinic acid.

[0009] Further provided is a method for producing flavonoids and anthocyanins, comprising culturing the engineered cells for the production of flavonoids or anthocyanins as provided herein under conditions in which the cells produce flavonoids or anthocyanins.In some examples, the method comprises culturing the engineered cells in a culture medium that comprises 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 method may further comprise recovering at least one of flavonoids from the culture medium, whole culture, or cells.

[0010] In a first aspect, provided herein is a cell engineered to produce one or more flavonoids or anthocyanins, the cell comprising a nucleic acid sequence encoding any of 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, in addition to one or more genetic modifications to improve production of the flavonoid or anthocyanin. As provided herein, cells engineered to produce one or more of the flavonoids can be engineered to contain an exogenous nucleic acid sequence encoding a tyrosine ammonia lyase activity capable of forming 4-coumaric acid using tyrosine as a substrate (e.g., tyrosine ammonia lyase TAL, EC: 4.3.1.25), or alternatively or additionally, an exogenous nucleic acid sequence encoding a phenylalanine ammonia lyase activity capable of converting phenylalanine to trans-cinnamic acid, and an exogenous nucleic acid sequence encoding a cinnamic acid-4-hydroxylase activity capable of forming 4-coumaric acid from trans-cinnamic acid, from coumaric acid. an exogenous nucleic acid sequence encoding a CoA ligase activity that forms p-coumaroyl-CoA (e.g., 4-coumarate-CoA ligase, 4CL, EC: 6.2.1.12), an exogenous nucleic acid sequence encoding a 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 a chalcone isomerase activity that forms naringenin from naringenin chalcone by its cyclase activity (e.g., chalcone-flavonone isomerase, CHI, EC: 5.5.1.6), an exogenous nucleic acid sequence encoding a flavanone-3-hydroxylase activity for forming dihydrokaempferol from naringenin or taxifolin from eriodictyol (e.g., naringenin 3-dioxygenase, F3H, EC:1.14.11.9), coupled with an exogenous nucleic acid sequence encoding a cytochrome P450 reductase activity for forming taxifolin or dihydromyricetin from dihydrokaempferol or for forming eriodictyol or pentahydroxyflavone from naringenin (e.g., flavonoid 3'-monooxygenase, F3'H, EC: cytochrome P450 / NADPH--P450 reductase, EC:1.14.14.1; F3'5'H, EC:1.14.14.81), 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 leucoanthocyanidin reductase activity that forms catechin from leucocyanidin (e.g., leucoanthocyanidin reductase, LAR, EC:1.17.1.3). Optionally, cells engineered to produce anthocyanins can include an exogenous nucleic acid sequence encoding an anthocyanin synthase activity (e.g., anthocyanin synthase, ANS, EC:1.14.20) that forms cyanidin from catechin or leucocyanidin, delphinidin from leucodelphinidin, or pelargonidin from leucopelargonidin.4) and an exogenous nucleic acid sequence encoding a 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 production of flavonoid or anthocyanin products, for example, by increasing metabolic flux into the flavonoid or anthocyanin pathway or by decreasing by-product formation.

[0011] The engineered cells for producing flavonoids are 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 is a large single-chain polypeptide in most eukaryotes, including fungi, and a multi-subunit enzyme in plants and bacteria, such as E. coli, is overexpressed in the host strain. Examples of acetyl-CoA carboxylases that can be expressed in the engineered host cells for producing flavonoids or anthocyanins include, but are not limited to, the ACC gene of Mucor circinelloides, the ACC gene of Rhodotorula toruloides, the ACC gene of Lipomyces starkeyi, the ACC gene of Ustilago maydis, and the orthologues of these ACCs in other species that have at least 50% amino acid identity to these ACCs.

[0012] Further 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), which converts acetate and CoA to acetyl-CoA, is overexpressed in the host cell. The culture of engineered host cells containing an overexpressed nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in engineered host cells to produce flavonoids or anthocyanins include, but are not limited to, the ACS gene of E. coli, the ACS of Salmonella typhimurium, and orthologs of these ACS in other species that have at least 50% amino acid identity to these ACS.

[0013] In further embodiments, engineered host cells that overexpress the gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA, are also contemplated. Additionally, a variant of the Lpd subunit of PDH can be expressed in E. coli that contains a mutation (E354K) that reduces inhibition of PDH by NADH.

[0014] Alternatively, or in addition to the strategies for increasing ACC activity and for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms independent of the activity of ACC can be used.In some embodiments, the cells engineered to produce flavonoids or anthocyanins are further engineered to include an exogenous nucleic acid sequence encoding malonyl-CoA synthetase that produces malonyl-CoA from malonate to increase the cellular supply of malonyl-CoA.Examples of malonyl-CoA synthetases include Streptomyces coelicolor malonyl-CoA synthetase, Rhodopseudomonas palustris malonyl-CoA synthetase, or malonyl-CoA synthetase with at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases.Malonate can be added to the culture medium of the culture containing the cells engineered to express malonyl-CoA synthetase, if necessary. An engineered cell that contains an exogenous gene encoding a malonyl-CoA synthetase can also contain an exogenous nucleic acid sequence encoding a malonate transporter, such as the malonate transporter encoded by the matC gene of Streptomyces coelicolor or the malonate transporter encoded by DctPQM of Sinorhizobium medicae.

[0015] In a further embodiment, the engineered cell for producing flavonoids or anthocyanins is further engineered to contain an exogenous nucleic acid sequence encoding malonyl-CoA-transferase, which generates malonyl-CoA by direct transfer of CoA from acetyl-CoA. Examples of malonyl-CoA-transferases that can be expressed in engineered cells as provided herein include, but are not limited to, the alpha subunit of malonyl-CoA-decarboxylase from Acinetobacter calcoaceticus (mdcA), the alpha subunit of malonyl-CoA-decarboxylase from Geobacillus sp (mdcA), or transferases that have at least 50% identity to any of these or other naturally occurring malonyl-CoA-transferases.

[0016] In some embodiments, the engineered cells for producing flavonoids or anthocyanins are 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), which produces CoA from pantothenate. Alternatively, or in addition, the host cell can be engineered to contain a nucleic acid sequence encoding a type III pantothenate kinase (EC:2.7.1.33) that is not feedback inhibited by coenzyme A, such as the CoaX gene of pseudomonas aeruginosa. The culture of engineered cells for producing flavonoids or anthocyanins can, in some embodiments, contain a medium that contains pantothenate, a precursor of CoA biosynthesis, and can also optionally contain cysteine, which is used in CoA biosynthesis.

[0017] Further strategies for increasing malonyl-CoA flux into the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Without limiting embodiments to any particular mechanism, the malonyl-CoA supply available for flavonoid biosynthesis can be increased by limiting fatty acid biosynthesis. In some embodiments, 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 can be mutated or downregulated is the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of engineered host cells that can be downregulated include beta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and acyl carrier protein (E. coli acpP).

[0018] Additional genetic modifications that may be present in engineered host cells 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 E. coli host cells, genes that are downregulated, disrupted or deleted can include aldehyde alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), pyruvate oxidase (poxB), and the enzyme acetate kinase phosphate acetyltransferase (ackA-pta).

[0019] Additionally, engineered cells for the production of flavonoids or anthocyanins may have one or more genes encoding thioesterases that are downregulated, disrupted or deleted to prevent hydrolysis of the precursors malonyl-CoA, acetyl-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.

[0020] Alternatively or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase the supply of alpha-ketoglutarate, which functions as a cofactor for one or more of the flavonoid and anthocyanin pathway enzymes.Other TCA enzymes that can be downregulated include citrate synthase, which converts acetyl-CoA to citrate.

[0021] In further embodiments, engineered host cells for the production of flavonoids or anthocyanins are also contemplated for upregulating the endogenous biosynthesis of the amino acid tyrosine.Tyrosine is one of the precursors of 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 shikimic acid pathway.The first step of the shikimic acid pathway is catalyzed by DAHP synthase isozyme and is regulated by feedback inhibition.Strategies for increasing tyrosine production can include, but are not limited to, transcriptional deregulation, removal of 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 DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and / or the rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN) / shikimate dehydrogenase (ydiB) can be overexpressed. Additionally, phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.

[0022] In further embodiments, engineered host cells for the production of flavonoids or anthocyanins are also contemplated, which are further engineered to upregulate the endogenous biosynthesis of the cofactor heme. One of the exogenous genes in the engineered cells provided herein, cytochrome P450 (CYP), contains heme as a cofactor. Improving heme supply can be an effective strategy for increasing flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. Strategies for increasing heme supply include overexpression of genes that synthesize the precursor ALA. In E. coli hosts, ALA is formed from the five-carbon skeleton of glutamic acid (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 E. coli hosts, engineered cells provided herein can be further engineered to express or overexpress hemA or its variants and / or heML to increase production of the heme precursor ALA. Non-limiting examples of hemA genes that can be overexpressed include mutant hemA, which inserts two lysine residues between Thr-2 and Leu-3 at the N-terminus of the 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, in which ALS is synthesized by condensation of glycine with succinyl-CoA. Non-limiting examples of heterologous ALAS that can be expressed in E. coli include ALAS from Bradyrhizobium japonicum (EC:2.3.1.37), ALAS from Rhodobacter In addition, one or more of the downstream genes that catalyze the synthesis of heme from ALA (e.g., hemB, hemC, hemD, hemE, hemF, hemG, hemil, or hemH in E. coli) can be overexpressed to direct flux from ALA towards heme production.Cultures of engineered cells for the production of flavonoids or anthocyanins may, in some embodiments, contain media that includes succinate and / or glycine, which are precursors for heme biosynthesis via the C4 pathway.

[0023] In another aspect, the present invention provides a cell culture comprising the engineered cell as provided herein in a culture medium, the culture medium comprising a carbon source that is also the energy source of the cell, the carbon source can be, for example, as a non-limiting example, glycerol, sugar or organic acid.The culture medium can further comprise a feed molecule that is used to produce flavonoid or anthocyanin.The 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 comprising a carbon source and at least one supplement that is an enzyme cofactor or a precursor of an enzyme cofactor.

[0024] In yet another aspect, a method for producing flavonoids and anthocyanins, comprising incubating a culture of engineered host cells as provided herein to produce flavonoids or anthocyanins. The method may further comprise recovering at least one of the flavonoids from the cells, culture medium, or whole culture.

[0025] In yet another aspect, the present invention provides engineered host cells comprising one or more genetic modifications, which result in the engineered host cells producing flavonoids or anthocyanins via multiple chemical intermediates from a carbon source that can also be an energy source. In certain embodiments, the production of flavonoids or anthocyanins from glycerol occurs by enzymatic conversion. In certain embodiments, the production of flavonoids or anthocyanins from a carbon source that can also be an energy source occurs by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass containing glycerol, and (vi) any combination thereof. In certain embodiments, the engineered host cells are cultured in a medium comprising molecules selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass containing glycerol, and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors. In certain embodiments, the one or more genetic modifications cause the formation of reduced by-products. In certain embodiments, the one or more genetic modifications are selected from (i) one or more modifications that cause the engineered host cell to overexpress one or more endogenous genes; (ii) one or more modifications that cause the engineered host cell to underexpress one or more endogenous genes; (iii) one or more genetic modifications that cause the engineered host cell to express one or more non-native genes; and (iv) combinations thereof. In certain embodiments, the engineered host cell is cultured in a medium that includes a molecule selected from tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, and one or more of the selected molecules are chemical intermediates, including molecules in biosynthetic pathways or cofactors.In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from (i) a nucleic acid sequence encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequence encoding phenylalanine ammonia lyase activity; (iii) a nucleic acid sequence encoding cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence 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 comprising chalcone synthase and chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. coli. In certain embodiments, the one or more genetic modifications reduce fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamic acid-4-hydroxylase, where the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.In certain embodiments, the engineered host cell comprises: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, where the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, where the 4-coumarate-CoA ligase converts phenylalanine to trans-cinnamic acid; The exogenous nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms p-coumaroyl-CoA from coumaric acid; (b) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (c) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (d) a nucleic acid sequence encoding a flavanone-3-hydroxylase activity, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (e) any combination thereof. In certain embodiments, the flavonoid is catechin.

[0026] In yet another aspect, the present invention provides a method for increasing the production of flavonoids or anthocyanins, comprising providing an engineered host cell comprising one or more genetic modifications, the genetic modifications resulting in the engineered host cell producing flavonoids or anthocyanins via multiple chemical intermediates from a carbon source that may also be an energy source. In certain embodiments, the production of flavonoids or anthocyanins from a carbon source that may also be an energy source occurs by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a medium comprising a molecule selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors. In certain embodiments, the one or more genetic modifications cause the metabolic flux to flavonoid precursors to increase.In certain embodiments, the one or more genetic modifications cause the formation of by-products to be reduced.In certain embodiments, the one or more genetic modifications are selected from: (i) one or more modifications that allow the engineered host cell to overexpress one or more endogenous genes; (ii) one or more modifications that allow the engineered host cell to underexpress one or more endogenous genes; (iii) one or more genetic modifications that allow the engineered host cell to express one or more non-native genes; and (iv) combinations thereof.In certain embodiments, the engineered host cell is cultured in a medium containing a molecule selected from tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, where one or more of the selected molecules are chemical intermediates, including molecules in biosynthetic pathways or cofactors.In certain embodiments, the engineered host cell contains at least one or more nucleic acid sequences selected from (i) a nucleic acid sequence encoding tyrosine ammonia-lyase activity; (ii) a nucleic acid sequence encoding phenylalanine ammonia-lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof.In certain embodiments, the engineered host cell contains at least one or more peptides selected from (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprising chalcone synthase and chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is E. Coli. In certain embodiments, the one or more genetic modifications reduce fatty acid biosynthesis. In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) a nucleic acid sequence encoding tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding cinnamic acid-4-hydroxylase, wherein the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.In certain embodiments, the engineered host cell comprises: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, where the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, where the 4-coumarate-CoA ligase converts phenylalanine to trans-cinnamic acid; The exogenous nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms p-coumaroyl-CoA from coumaric acid; (b) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (c) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (d) a nucleic acid sequence encoding a flavanone-3-hydroxylase activity, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (e) any combination thereof. In certain embodiments, the flavonoid is catechin.

[0027] In yet another aspect, the present invention provides a plurality of engineered host cells, each of the plurality of engineered host cells comprising one or more genetic modifications that result in the production of flavonoids or anthocyanins via multiple chemical intermediates from a carbon source that can also be an energy source. In certain embodiments, the production of flavonoids or anthocyanins from a carbon source that can also be an energy source occurs by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass containing glycerol, and (vi) any combination thereof. In certain embodiments, the engineered host cells are cultured in a medium comprising a molecule selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass containing glycerol, and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors. In certain embodiments, the one or more genetic modifications lead to increasing the metabolic flux to flavonoid precursors or cofactors.In certain embodiments, the one or more genetic modifications cause the reduction of the formation of by-products.In certain embodiments, the one or more genetic modifications are selected from (i) one or more modifications that allow the engineered host cell to overexpress one or more endogenous genes; (ii) one or more modifications that allow the engineered host cell to underexpress one or more endogenous genes; (iii) one or more genetic modifications that allow the engineered host cell to express one or more non-native genes; and (iv) combinations thereof.In certain embodiments, at least one of the engineered cells from the plurality of engineered host cells is cultured in a medium containing a molecule selected from tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, where one or more of the selected molecules are chemical intermediates, including molecules or cofactors in biosynthetic pathways. In certain embodiments, at least one of the engineered cells from the plurality of engineered host cells contains at least one or more nucleic acid sequences selected from (i) a nucleic acid sequence encoding a tyrosine ammonia lyase activity; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase activity; (iii) a nucleic acid sequence encoding a cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cells from the plurality of engineered host cells comprises at least one or more peptides selected from (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprising chalcone synthase and chalcone isomerase; and (iv) any combination thereof.In certain embodiments, at least one of the engineered host cells is E. coli.In certain embodiments, the one or more genetic modifications reduce fatty acid biosynthesis.In certain embodiments, at least one of the engineered host cells from the plurality of engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamic acid-4-hydroxylase, where the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.In certain embodiments, the engineered host cell comprises: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, where the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, where the 4-coumarate-CoA ligase converts phenylalanine to trans-cinnamic acid; The exogenous nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms p-coumaroyl-CoA from coumaric acid; (b) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (c) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (d) a nucleic acid sequence encoding a flavanone-3-hydroxylase activity, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (e) any combination thereof. In certain embodiments, the flavonoid is catechin.

[0028] In yet another aspect, the present invention provides a method for increasing the production of flavonoids or anthocyanins, comprising providing a plurality of engineered host cells, each of the plurality of engineered host cells comprising one or more genetic modifications that result in the engineered host cell producing flavonoids or anthocyanins via a plurality of chemical intermediates from a carbon source that may also be an energy source. In certain embodiments, the production of flavonoids or anthocyanins from a carbon source that may also be an energy source occurs by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the engineered host cells are cultured in a medium comprising a molecule selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors. In certain embodiments, the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors. In certain embodiments, the one or more genetic modifications cause reduced formation of by-products. In certain embodiments, the one or more genetic modifications are selected from (i) one or more modifications that allow the engineered host cell to overexpress one or more endogenous genes; (ii) one or more modifications that allow the engineered host cell to underexpress one or more endogenous genes; (iii) one or more genetic modifications that allow the engineered host cell to express one or more non-native genes; and (iv) combinations thereof.In certain embodiments, at least one of the engineered cells from the plurality of engineered host cells is cultured in a medium containing a molecule selected from tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid, where one or more of the selected molecules are chemical intermediates, including molecules or cofactors in biosynthetic pathways. In certain embodiments, at least one of the engineered cells from the plurality of engineered host cells contains at least one or more nucleic acid sequences selected from (i) a nucleic acid sequence encoding a tyrosine ammonia lyase activity; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase activity; (iii) a nucleic acid sequence encoding a cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof. In certain embodiments, at least one of the engineered host cells from the plurality of engineered host cells comprises at least one or more peptides selected from (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprising chalcone synthase and chalcone isomerase; and (iv) any combination thereof.In certain embodiments, at least one of the engineered host cells is E. coli.In certain embodiments, the one or more genetic modifications reduce fatty acid biosynthesis.In certain embodiments, at least one of the engineered host cells from the plurality of engineered host cells comprises an exogenous nucleic acid sequence selected from: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase forms 4-coumaric acid using tyrosine as a substrate; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamic acid-4-hydroxylase, where the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.In certain embodiments, the engineered host cell comprises: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, where the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, where the 4-coumarate-CoA ligase converts phenylalanine to trans-cinnamic acid; The exogenous nucleic acid sequence includes a nucleic acid sequence selected from the group consisting of: (a) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms p-coumaroyl-CoA from coumaric acid; (b) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (c) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (d) a nucleic acid sequence encoding a flavanone-3-hydroxylase activity, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (e) any combination thereof. In certain embodiments, the flavonoid is catechin.

[0029] 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 converting 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 E. coli. In certain embodiments, the E. coli cell further comprises a gene from a fungus. In certain embodiments, the acetyl-CoA carboxylase is an ortholog of an acetyl-CoA carboxylase from Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and has at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, the one or more genetic modifications are deletion or attenuation of one or more fatty acid biosynthesis genes, resulting in the reduction of fatty acid biosynthesis.In certain embodiments, the one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS).In certain embodiments, the acetyl-CoA synthase is selected from the ortholog of the acetyl-CoA synthase gene of E.coli, the acetyl-CoA synthase gene of Salmonella typhimurium, and the acetyl-CoA synthase gene of any other species that has at least 50% amino acid identity with the acetyl-CoA synthase gene of E.coli and Salmonella typhimurium.In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), where the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), where PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from Streptomyces coelicolor malonyl-CoA synthetase, Rhodopseudomonas palustris malonyl-CoA synthetase, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications to reduce fatty acid biosynthesis are selected from: (i) mutation or downregulation of the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of the beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of the acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from: (i) an 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) a malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:16; (iv) a malonyl-CoA synthase having an amino acid sequence at least 80% identical to SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; (v) a 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) a 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 combination thereof.

[0030] In another aspect, the present invention provides a method for increasing the production of flavonoids, comprising an engineered host cell, wherein one or more engineered host cells comprise one or more genetic modifications for increasing the production and / or availability of malonyl-CoA. In certain embodiments, the production and / or availability of malonyl-CoA is increased by converting 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 E. coli. In certain embodiments, the E. coli cell further comprises a gene from a fungus. In certain embodiments, the acetyl-CoA carboxylase is from Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and the orthologue of the acetyl-CoA carboxylase has at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species.In certain embodiments, the one or more genetic modifications are deletion or attenuation of one or more fatty acid biosynthesis genes, resulting in a decrease in fatty acid biosynthesis.In certain embodiments, the one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS).In certain embodiments, the acetyl-CoA synthase is selected from the acetyl-CoA synthase gene of E. coli, the acetyl-CoA synthase gene of Salmonella typhimurium, and the orthologue of the acetyl-CoA synthase gene in any other species that has at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium.In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), where the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), where PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from Streptomyces coelicolor malonyl-CoA synthetase, Rhodopseudomonas palustris malonyl-CoA synthetase, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications to reduce fatty acid biosynthesis are selected from: (i) mutation or downregulation of the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of the beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of the acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from: (i) an 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) a malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:16; (iv) a malonyl-CoA synthase having an amino acid sequence at least 80% identical to SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; (v) a 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) a 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 combination thereof.

[0031] In another aspect, the present invention provides engineered host cells comprising one or more genetic modifications for increasing endogenous biosynthesis of tyrosine. In certain embodiments, the one or more genetic modifications comprise upregulation of 3-deoxy-D-arabino-heptulosonic acid synthase. In certain embodiments, the 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 combination thereof. In certain embodiments, the one or more genetic modifications comprise downregulation of the L-phenylalanine biosynthetic pathway. In certain embodiments, the one or more genetic modifications comprise expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, the one or more genetic modifications comprise expression of exogenous transketolase (tktA). In certain embodiments, the one or more genetic modifications comprise disruption of the tyrR gene. In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) expression or overexpression of the (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of a 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 the A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.

[0032] In another aspect, the present invention provides a method for increasing the endogenous biosynthesis of tyrosine, comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications for increasing the endogenous biosynthesis of tyrosine. In certain embodiments, the one or more genetic modifications comprise upregulation of 3-deoxy-D-arabino-heptulosonic acid synthase. In certain embodiments, the 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 combination thereof. In certain embodiments, the one or more genetic modifications comprise downregulation of the L-phenylalanine biosynthesis pathway. In certain embodiments, the one or more genetic modifications comprise expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, the one or more genetic modifications comprise expression of exogenous transketolase (tktA). In certain embodiments, the one or more genetic modifications include disruption of the tyrR gene. In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) expression or overexpression of the (D146N) variant of phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of the 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 the A354V variant of chorismate mutase (tyrA); (vi) and any combination thereof.

[0033] In another aspect, the present invention provides engineered host cells comprising one or more genetic modifications to increase the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from (i) Carica papaya anthocyanin synthase (SEQ ID NO: 13); (ii) an anthocyanin synthase having at least 80% identical amino acid sequence to SEQ ID NO: 66, SEQ ID NO: 67, SEQ ID NO: 68 or SEQ ID NO: 69; (iii) an anthocyanin synthase having at least 80% identical amino acid sequence to SEQ ID NO: 13; and (iv) any combination thereof. In certain embodiments, the one or more engineered host cells comprise flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase from Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof. In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications are selected from the group consisting of (i) anthocyanin synthase, (ii) flavonoid-3-glucosyltransferase (3GT), and (iii) a combination thereof.

[0034] In another aspect, the present 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 for increasing the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from (i) Carica papaya anthocyanin synthase (SEQ ID NO: 13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO: 13; and (iv) any combination thereof. In certain embodiments, the one or more engineered host cells comprise flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase from Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof. In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications are selected from the group consisting of (i) anthocyanin synthase, (ii) flavonoid-3-glucosyltransferase (3GT), and (iii) a combination thereof.

[0035] In another aspect, the present invention provides a method for increasing conversion 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 an anthocyanin synthase selected from (i) Carica papaya anthocyanin synthase (SEQ ID NO: 13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO: 13; and (iv) any combination thereof. In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications are selected from the group consisting of (i) anthocyanin synthase, (ii) flavonoid-3-glucosyltransferase (3GT), and (iii) combinations thereof.

[0036] In another aspect, the present invention provides a method for increasing conversion of cyanidin to cyanidin-3-glucoside (Cy3G), delphinidin to delphinidin-3-glucoside (De3G), or pelargonidin to pelagonidin-3-glucoside (Pe3G), comprising a flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase of Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof.

[0037] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications for increasing production of dihydroquercetin (DHQ), dihydromyricein (DHM), eriodictoyl (EDL), and / or pentahydroxyflayaone (PHF), the engineered host cell comprising a 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 precursors for increased production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflayanone (PHF) are naringenin and / or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises a peptide selected from the 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, the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, the flavonoid 3'-hydroxylase (F3'H) is fused to a cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3',5'-hydroxylase (F3'5'H) is fused to a cytochrome P450 reductase (CPR).In certain embodiments, the 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, the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:8. In certain embodiments, the 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, the flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to a polypeptide selected from the 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, the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:98. In certain embodiments, the flavanone-3-hydroxylase (F3H) has an amino acid sequence that is at least 80% identical to a polypeptide selected from the 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.

[0038] In another aspect, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF), comprising an engineered host cell, the engineered host cell comprising a 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 increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF) is naringenin and / or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises a peptide selected from the 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, the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, the flavonoid 3'-hydroxylase (F3'H) is fused to the cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3',5'-hydroxylase (F3'5'H) is fused to a cytochrome P450 reductase (CPR). In certain embodiments, the 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, the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:8. In certain embodiments, the 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, the flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to a polypeptide selected from the 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, the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:98. In certain embodiments, the flavanone-3-hydroxylase (F3H) has an amino acid sequence that is at least 80% identical to a polypeptide selected from the 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. A brief description of some aspects of the drawings [Brief description of the drawings]

[0039] [Figure 1] FIG. 1 shows the metabolic pathways of flavonoid and anthocyanin bioproduction in the engineered cells and methods of preparing anthocyanins described herein.

[0040] [Diagram 2] FIG. 2 shows the structures of flavonoid and anthocyanin molecules that can be produced using the engineered cells and methods of preparing anthocyanins described herein.

[0041] [Diagram 3] FIG. 3 shows an HPLC spectrum showing peaks corresponding to molecules prepared using the engineered cells and methods of preparing anthocyanins described herein.

[0042] [Figure 4-1] FIG. 4 shows the flavonoid and anthocyanin bioproduction pathways in the engineered cells and methods of preparing anthocyanins described herein. [Figure 4-2] Same as above. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0043] VII. DETAILED DESCRIPTION OF THE PRESENTINVENTION The present application provides engineered cells for producing one or more flavonoids, cultures containing engineered cells, and methods for producing one or more flavonoids or at least one anthocyanin. The terms "flavonoid", "flavonoid product", or "flavonoid compound" are used herein to refer to members of a diverse group of phytonutrients found in nearly all fruits and vegetables. As used herein, the terms "flavonoid", "flavonoid product", or "flavonoid compound" are used interchangeably to refer to molecules containing a general structure of a 15-carbon skeleton consisting 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), catechin, flavanone, and flavanonol. Flavonoid compounds of interest include, but are not limited to, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin, pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, and kaempferol.Anthocyanins are in the form of anthocyanidin glycosides and acylated anthocyanins.Anthocyanin compounds of interest include, but are not limited to, cyanidin glycosides, delphinidin glycosides, pelargonidin glycosides, peonidin glycosides, and petunidin glycosides.

[0044] The term "precursor" or "flavonoid precursor" as used herein may refer to any intermediate present in the biosynthetic pathway leading to the production of catechin or anthocyanin. Flavonoid precursors may include, but are not limited to, tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.

[0045] Engineered cells for the production of flavonoids or anthocyanins may have one or more modifications, including, but not limited to, downregulation, disruption or deletion of an endogenous gene, upregulation of an endogenous gene, and introduction of an exogenous gene.

[0046] The term "non-naturally occurring" when used in reference to an enzyme is intended to mean that the nucleic acid or polypeptide contains at least one genetic modification not normally found in naturally occurring polypeptides or nucleic acid sequences. Naturally occurring nucleic acids and polypeptides may be referred to as "wild type" or "original". Host cells, organisms or microorganisms that contain at least one genetic modification produced by human intervention may also be referred to as "non-naturally occurring", "engineered", "genetically engineered" or "recombinant".

[0047] A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide, is genetically engineered by recombinant DNA technology to contain a gene or nucleic acid sequence that does not naturally code for an enzyme or polypeptide, or to express an endogenous gene at a level that exceeds its expression level in unmodified cells. As a non-limiting example, a host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide, may have any modification that affects the coding sequence of the gene, the location of the gene on a chromosome, or the regulatory element associated with the gene. Overexpression of a gene may also be by increasing the copy number of the gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to underexpress or have a reduced expression of a gene, nucleic acid sequence, or to underexpress an enzyme or polypeptide, may have any modification that affects the coding sequence of the gene, the location of the gene on a chromosome, or the regulatory element associated with the gene. Particularly included are gene disruptions, including any insertion, deletion or sequence mutation in or of a gene or part of a gene that affects the expression or activity of the encoded polypeptide.Gene disruptions include "knock-out" mutations that eliminate the expression of a gene.Modifications to under-express a gene also include modifications of the regulatory region of the gene, which can reduce its expression.

[0048] The terms "exogenous" or "heterologous" are intended to mean that the referenced molecule or 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, for example, by integration into a host chromosome, or as non-chromosomal genetic material that can be introduced using a vehicle such as a plasmid. Thus, the term "endogenous" refers to a referenced molecule or activity that is naturally present in the host.

[0049] Genes or nucleic acid sequences can be stably or transiently introduced into host cells using techniques well known in the art, including but not limited to conjugation, electroporation, chemical conversion, 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 code targeting signals, such as N-terminal mitochondrial or other targeting signals, and these 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 proteins.

[0050] The percent identity (% identity) between two sequences is determined by aligning the sequences to maximize homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Omega, etc., compare and determine the similarity or identity of raw sequences, and also determine the presence or significance of gaps in the sequence that can be assigned weights or scores. Such algorithms are also known in the art and can be similarly applied to determine the similarity or identity of nucleotide or amino acid sequences, and can be useful in identifying orthologs of genes of interest. Additional sequences added to the polypeptide sequence, such as, but not limited to, immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity.

[0051] Homologs are genes or genes that have the same or identical function in different organisms. Orthologous genes may encode proteins that have sequence similarity of about 45%-100% amino acid sequence identity, more preferably about 60%-100% amino acid sequence identity. Genes may also be considered orthologs when they share a three-dimensional structure but do not necessarily share a sufficient amount of sequence similarity to indicate that they evolved from a common ancestor, to the extent that no primary sequence similarity is identifiable. Paralogs are genes related by duplication within a genome and can evolve new functions even if they are closely related to the original gene.

[0052] The engineered cells for producing flavonoids include an exogenous nucleic acid sequence encoding a tyrosine ammonia-lyase (TAL) activity (alternatively or in addition, an exogenous nucleic acid encoding a phenylalanine ammonia-lyase (PAL) activity, and an exogenous nucleic acid encoding a cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding a chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding a chalcone isomerase (CHI) activity. Optionally, the engineered cells may further comprise an exogenous nucleic acid sequence encoding a flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding a flavonoid 3'-hydroxlase (F3'H) activity or a flavonoid 3',5'-hydroxylase (F3'5'H), an exogenous nucleic acid sequence encoding a cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding a dihydroflavonol-4-reductase (DFR) activity, and / or an exogenous nucleic acid sequence encoding a leucoanthocyanidin reductase (LAR) activity.

[0053] A 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 Saccharothrix espanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). TALs having 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 NO: 1 that have tyrosine ammonia lyase activity to produce p-coumaric acid from tyrosine are also contemplated for use in the engineered cells provided herein. [Table 1]

[0054] Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the nonoxidative deamination of L-phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is Brevibacillus laterosporus phenylalanine ammonia-lyase (PAL; SEQ ID NO: 2). PAL having 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 phenylalanine ammonia lyase activity to produce trans-cinnamic acid from phenylalanine are also contemplated for use in the engineered cells provided herein.

[0055] Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and catalyzes the formation of p-coumaric acid from trans-cinnamic acid. C4H of Helianthus annuus L. (C4H; SEQ ID NO: 3), C4H having SEQ ID NOs: 30-32, and C4H homologues from other species, as well as variants of naturally occurring C4H 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: 3 (C4H, Helianthus annuus L.) that have activity of C4H are contemplated for use in the engineered cells provided herein.

[0056] 4-Coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester. 4CL from Petroselinum crispum (SEQ ID NO: 4), 4CL in Table 2, 4CL having SEQ ID NOs: 33-36, and 4CL homologues from other species, as well as naturally occurring variants of 4CL 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 4CL activity are contemplated for use in the engineered cells provided herein. [Table 2]

[0057] Chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaroyl-CoA to produce the naringenin precursor naringenin chalcone, or naringenin. An exemplary chalcone synthase is Petunia x hybrida chalcone synthase (CHS, SEQ ID NO: 5). Genes listed in Table 3, CHS having SEQ ID NOs: 37-40, as well as 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 chalcone synthase activity are also contemplated for use in the engineered cells provided herein. [Table 3-1] [Table 3-2]

[0058] Chalcone isomerase (CHI; also called chalcone flavonone isomerase) catalyzes the stereospecific intramolecular isomerization of naringenin chalcone to its corresponding (2S)-flavanone. CHI (SEQ ID NO: 6) of Medicago sativa, CHI of Table 4, CHI having SEQ ID NOs: 41-44, and CHI homologs of other species, as well as naturally occurring variants of 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 chalcone isomerase activity are contemplated for use in the engineered cells provided herein. [Table 4-1] [Table 4-2]

[0059] A nucleic acid sequence encoding CHI can, in some embodiments, be fused to a nucleic acid sequence encoding CHS in an engineered cell as provided herein, such that CHI activity is fused to chalcone synthase activity, i.e., a fusion protein having both condensation and cyclization activity is produced in the engineered cell.

[0060] Flavanone 3-hydroxylases (F3H) catalyze 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 exhibit catalytic function as flavonol synthases (EC:1.14.20.6). F3H of Rubus occidentalis (SEQ ID NO: 7), F3H having SEQ ID NOs: 45-48, the F3Hs listed in Table 5, as well as 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 an F3H are contemplated for use in the engineered cells provided herein. [Table 5]

[0061] Flavonoid 3'-hydroxylase (F3'H) belongs to the cytochrome P450 family and has the systematic name of flavonoids, NADPH:oxygen oxidoreductase (3'-hydroxylating). In the flavonoid biosynthetic pathway, F3'H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. The F3'H of Brassica napus (F3'H; SEQ ID NO: 8), F3'H having SEQ ID NOs: 49-52, those listed in Table 6, as well as 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 are contemplated for use in the engineered cells provided herein. The F3'H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. The cytochrome P450 reductase is a diflavin oxidoreductase that provides electrons to the F3'H. The P450 reductase may be from the same species as the F3'H or may be a different species than the F3'H. The Catharanthus roseus CPR (SEQ ID NO: 9), additional CPRs listed in Table 7, CPRs having SEQ ID NOs: 53-55, CPR homologs from 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 CPR activity are contemplated for use in the engineered cells provided herein. In various embodiments, the N-terminal nucleic acid sequences in the genes for the F3'H and / or CPRs originating from eukaryotic cells can encode targeting leader peptides, which can be removed, if desired, prior to introduction into the prokaryotic host cell.In some embodiments, the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin). [Table 6]

[0062] [Table 7]

[0063] A nucleic acid sequence encoding an 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.

[0064] In cells engineered to produce dihydromyricetin, 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 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 Delphinium grandiflorum F3'5'H (SEQ ID NO: 10). Also contemplated for use in the engineered cells provided herein include the F3'5'H having SEQ ID NOs: 56-57, F3'5'H homologs from 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 NO: 10, which have the activity of an F3'5'H.

[0065] Dihydroflavonol-4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (taxifolin, DHQ), or dihydromyricetin (DHM) to reduce these compounds to the corresponding cis-flavan-3,4-diols (DHK to leucopelargonidin; taxifolin to leucocyanidin; DHM to leucodelphinidin). An exemplary DFR is the Anthurium andraeanum DFR (SEQ ID NO: 11). Also contemplated for use in the engineered cells provided herein include the DFRs in Table 8, the DFRs having SEQ ID NOs:58-61, and DFR homologues from other species, as well as variants of naturally occurring DFRs 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 8-1] [Table 8-2]

[0066] Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin. LAR (SEQ ID NO: 12) from Desmodium uncinatum, LAR having SEQ ID NOs: 62-65, and LAR homologs from other species, as well as naturally occurring variants of 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 LAR, are contemplated for use in the engineered cells provided herein.

[0067] Optionally, the cells are further engineered to contain anthocyanin synthase (ANS) that catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin, or leucodelphinidin to definidin. ANS from Carica papaya (SEQ ID NO: 13), ANS having SEQ ID NOs: 66-69, and ANS homologs from other species, as well as naturally occurring variants of 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 ANS activity are contemplated for use in the engineered cells provided herein.

[0068] Optionally, the cells are further engineered to contain flavonoid-3-glucosyltransferase (3GT) to generate anthocyanins by the transfer of a sugar moiety, such as, but not limited to, UDP-α-D-glucose, to anthocyanidins to form glycosylated anthocyanins. Naturally occurring variants of 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 3GT activity are contemplated for use in the engineered cells provided herein.

[0069] In various embodiments, host cells can be engineered to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce by-product formation, or improve cell fitness by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or down-regulate competing pathways to enhance flavonoid or anthocyanin production.Enhancing or improving flavonoid or anthocyanin production can be increasing yield, increasing titer, or increasing production rate.

[0070] Thus, the host cell engineered for the production of flavonoids or anthocyanins can be engineered to include any of the following, or any combination: overexpression of acetyl-CoA carboxylase (ACC) or ACC variant; expression or overexpression of at least one enzyme for increasing the malonyl-CoA supply of cells independent of the ACC stage; expression or overexpression of at least one enzyme for increasing the tyrosine supply; expression or overexpression of at least one enzyme for increasing the CoA available for synthesis of the precursor malonyl-CoA or p-coumaroyl-CoA; expression or overexpression of at least one enzyme for increasing heme biosynthesis; deletion or downregulation of at least one fatty acid synthesis enzyme; at least one alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, phosphate acetyltransferase, or acetate kinase; at least one enzyme of the fatty acid degradation pathway, at least one thioesterase, or at least one TCA gene.The above list of modifications is non-limiting.

[0071] Malonyl-CoA is the direct precursor of chalcone synthase for sequential condensation with p-coumaroyl-CoA. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized in a multi-step reaction by ATP-dependent carboxylation of acetyl-CoA by acetyl-CoA carboxylase (ACC). First, a biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as the CO2 donor. In the second reaction, a carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes, including fungi, both reactions are catalyzed by large single-chain proteins, but in E. coli and other bacteria, the activities are catalyzed by multisubunit enzymes. Host cells can be engineered to express exogenous acetyl-CoA carboxylase or variant ACC to increase malonyl-CoA synthesis from acetyl-CoA, for example. For example, Mucor circinelloides (SEQ ID NO: 15) acetyl-CoA carboxylase can be introduced into the host cell. Further examples of ACC genes that can be used in the engineered cells provided herein include, but are not limited to, the genes listed in Table 9, genes having 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 the reference gene. Additionally, 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]

[0072] Further 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 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 provide the function of increasing acetyl-CoA supply when cells are supplied with acetate as a co-feed or when acetate is produced as a by-product. Other acyl-CoA ligases with their primary 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 ACS expressed in the host cell can be prokaryotic or eukaryotic. Culture of engineered host cells that overexpress the nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthases that can be expressed in engineered host cells to produce flavonoids or anthocyanins include, but are not limited to, the ACS gene of E. coli, the ACS of Salmonella typhimurium (SEQ ID NO: 16), and orthologs of these ACS in other species that 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% amino acid identity with these ACS.

[0073] Alternatively or in addition, engineered host cells 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 requires control by its substrates and products. Nicotinamide adenine dinucleotide hydrogen (NADH), the product of the PDH reaction, is a competitive inhibitor of the PDH complex. It has been demonstrated that the NADH sensitivity of the PDH complex is present in LPD, an enzyme that interacts with NAD+ as a substrate. Thus, variants of the Lpd subunit of PDH can be expressed that contain one or more mutations that reduce the inhibition of PDH by NADH. Such an example is an LPD variant in E. coli that contains the E354K mutation, and the mutated enzyme is less sensitive to NADH inhibition than the native LPD.

[0074] Alternatively, or in addition to the strategies for increasing ACC activity and for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms independent of the activity of ACC can be used.For example, the engineered cell for producing flavonoids or anthocyanins as provided herein can include an exogenous nucleic acid sequence encoding malonyl-CoA synthetase (EC 6.2.1.14) that produces malonyl-CoA from malonate. Acyl-CoA synthetase catalyzes the conversion of carboxylic acid to its acyl-CoA thioester through an ATP-dependent two-step reaction. In the first step, free fatty acid is converted to acyl-AMP intermediate and pyrophosphate is released. In the second step, the activated acyl group is coupled to the thiol group of CoA, resulting in the release of AMP and acyl-CoA product. Non-limiting examples of malonyl-CoA synthetases include malonyl-CoA synthetase from Streptomyces coelicolor (SEQ ID NO: 17), matB from Rhodopseudomonas palustris (SEQ ID NO: 77), matB from Rhizobium sp., BUS003 (SEQ ID NO: 78), matB from 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 reference sequence. Malonate can be added to the culture medium of a culture containing cells engineered to express malonyl-CoA synthetase, if desired. In Rhizobium trifolii, the matB gene is part of the matABC operon, matA encodes malonyl-CoA decarboxylase, and matC encodes a putative dicarboxylate carrier protein or malonate transporter.The engineered cell comprising an exogenous gene encoding a malonyl-CoA synthetase can further comprise a malonate transporter, e.g., a malonate transporter encoded by a matC gene, e.g., a malonate transporter encoded by the matC gene of Streptomyces coelicolor (SEQ ID NO: 18), a malonate transporter encoded by the matC gene of Rhizobiales bacterium (SEQ ID NO: 80), a malonate transporter encoded by the matC gene of Rhizobium leguminosarum (SEQ ID NO: 81), a malonate transporter encoded by the matC gene of Agrobacterium vitis (SEQ ID NO: 82), a malonate transporter encoded by the matC gene of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by the matC gene of Sinorhizobium sp. (SEQ ID NO: 84). The exogenous nucleic acid sequence may also include an exogenous nucleic acid sequence encoding a malonate transporter encoded by DctPQM of D. 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. The cell culture of the host cell engineered to express malonyl-CoA synthetase and the malonate transporter may include a culture medium containing malonate.

[0075] In a further embodiment, the cells engineered to produce flavonoids or anthocyanins are further engineered to contain an exogenous nucleic acid sequence encoding malonate CoA-transferase (EC:2.8.3.3; also called alpha subunit of malonate decarboxylase), which generates malonyl-CoA by direct transfer of CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has malonate CoA-transferase activity. The α subunit, which is the product of the mdcA gene, is a malonate CoA-transferase, and the β subunit, which is the product of the mdcD gene, is a malonyl-CoA decarboxylase. The γ subunit, which is the product of the mdcE gene, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The δ subunit, which is the product of the mdcC gene, was an acyl-carrier protein with 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 portion of the prosthetic group binds onto the α subunit and exchanges the acetyl group for a malonyl group. Because the transfer of the thioester should be thermodynamically favorable, engineered cells can contain nucleic acids encoding malonate CoA-transferase to increase malonyl-CoA supply. Examples of mdcA that can be expressed in engineered cells as provided herein include, but are not limited to, mdcA of Acinetobacter calcoaceticus (SEQ ID NO: 19), mdcA in Table 10, mdcA having SEQ ID NOs: 84-87, or transferases 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

[0076] In some embodiments, the cells engineered to produce flavonoids or anthocyanins are 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 the CoA biosynthetic pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It consumes an adenosine triphosphate (ATP) molecule to phosphorylate pantothenic acid (vitamin B5) to form 4'-phosphopantothenic acid. This 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 (found primarily in eukaryotes, but also in 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 enzyme CoaX is not subject to feedback inhibition by CoA. In some embodiments, host cells can be engineered to contain a nucleic acid sequence encoding a type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, but not limited to, the Pseudomonas aeruginosa CoaX gene (EC:2.7.1.33, SEQ ID NO:20), Streptomyces sp. CLI2509 CoaX (SEQ ID NO:88), Streptomyces cinereus CoaX (SEQ ID NO:89), or Kitasatospora kifunensis CoaX (SEQ ID NO:90). Cultures of engineered cells for the production of flavonoids or anthocyanins may, in some embodiments, comprise a medium that contains pantothenate, a precursor for CoA biosynthesis, and may also optionally contain cysteine, which is used in CoA biosynthesis.

[0077] Further strategies for increasing malonyl-CoA flux into 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, thus limiting flavonoid formation. Without limiting the embodiments to any particular mechanism, by limiting fatty acid biosynthesis, the malonyl-CoA supply available for flavonoid biosynthesis can be increased. 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 can be mutated or downregulated is the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of engineered host cells that can be downregulated include the beta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and / or acyl carrier protein (E. coli acpP).

[0078] Additional genetic modifications that may be present in engineered host cells to produce flavonoids or anthocyanins include downregulation, disruption or deletion of gene targets that divert carbon flux to form by-products such as ethanol, acetate and lactate. These include genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In E. coli host cells, genes that are downregulated, disrupted or deleted may include adhE, ldhA, poxB, and ackA-pta.

[0079] Additionally, engineered cells for the production of flavonoids or anthocyanins may have one or more genes encoding thioesterases that are downregulated, disrupted or deleted to prevent hydrolysis of the precursors malonyl-CoA, acetyl-CoA, and / or p-coumaroyl-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short, medium, long and very long chain), bile acid-CoAs, and methyl-branched-CoAs to free fatty acids 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.

[0080] In further embodiments, engineered cells for the production of flavonoids or anthocyanins can have one or more of the 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, the adhesin A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and / or the gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted or deleted.

[0081] Alternatively or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase the supply of alpha-ketoglutarate, which functions as a cofactor for flavonoid and anthocyanin pathway enzymes.Other TCA enzymes that can be downregulated include citrate synthase, which converts acetyl-CoA to citrate.

[0082] In further embodiments, engineered host cells for the production of flavonoids or anthocyanins are also contemplated to upregulate the endogenous biosynthesis of the amino acid tyrosine. Tyrosine is one of the precursors of flavonoid biosynthesis, and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from raw materials 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 shikimic acid pathway. The shikimic acid pathway is the central metabolic route that leads to the formation of tryptophan (TRP), tyrosine (TYR) and phenylalanine (PHE), and this pathway exists exclusively in plants and microorganisms. It begins with the condensation of intermediates of glycolysis and the pentose phosphate pathway, phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), respectively, which enter the pathway by a series of condensations and redox reactions to shikimate via 3-deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP), 3-dehydroquinate (DHQ), and 3-dehydroshikimate (DHS). From there, the central branch point metabolite chorismate is obtained via shikimate-3-phosphate following ATP hydrolysis and introduction of a second PEP. The first step of the shikimate pathway is catalyzed by the DAHP synthase isozyme and is regulated by feedback inhibition. In E. coli, there are three DAHP synthase isozymes (aroF, aroG, aroH), each of which is feedback inhibited by one of three aromatic amino acids (TYR, PHE, TRP), in contrast to the two DAHP synthases in plants, which are not feedback inhibited. In plants and bacteria, the subsequent five steps are catalyzed by a single enzyme. From the central intermediate chorismate, the pathway branches to anthranilate and prephenate, leading to the synthesis of the aromatic amino acids para-hydroxybenzoate (pHBA) and para-aminobenzoate (pABA), the last of which is a precursor for folate metabolism.Strategies for increasing L-tyrosine production can include, but are not limited to, transcriptional deregulation, removal of feedback inhibition, overexpression of rate-limiting enzymes, and / or deletion of the L-phenylalanine branch of the aromatic acid biosynthesis pathway.For example, in E. coli host, the tyrR gene can be disrupted, feedback inhibition-resistant versions of DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and / or the rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN) / shikimate dehydrogenase (ydiB) can be overexpressed.In addition, ppsA, aroG, and / or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.

[0083] In further embodiments, engineered host cells for the production of flavonoids or anthocyanins are also contemplated, which are further engineered to upregulate the endogenous biosynthesis of the cofactor heme. One of the exogenous genes in the engineered cells provided herein, cytochrome P450 (CYP), contains heme as a cofactor. Improving heme supply can be an effective strategy for increasing flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There are two known alternative routes by which this committed intermediate is produced. One route is the C4 pathway (the shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA synthase (ALAS). The C4 pathway is limited to mammals, fungi, and purple non-sulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions that result in the biosynthesis of ALA from the five-carbon skeleton of glutamic acid. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions are required to convert ALA to heme, including the assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chain, and incorporation of reduced iron into the molecule. 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 heme precursor ALA production. Non-limiting examples of hemA genes that can be overexpressed include, but are not limited to, a mutant hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO:21), and hemA having SEQ ID NO:91-93. Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway.Non-limiting examples of heterologous ALAS that can be expressed in E. coli include Rhodobacter capsulatus ALAS (SEQ ID NO: 22), ALAS having 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. Additionally, one or more of the downstream genes that catalyze the synthesis of heme from ALA (E. coli hemB, hemC, hemD, hemE, hemF, hemG, heMI, or hemH) can be overexpressed to direct flux from ALA towards heme production. Cultures of engineered cells for the production of flavonoids or anthocyanins may, in some embodiments, contain media that includes succinate and glycine, which are precursors for heme biosynthesis via the C4 pathway.

[0084] The engineered cells that produce flavonoids can be engineered to contain multiple pathways to enhance flavonoid production. Those skilled in the art will recognize that the embodiments described herein can be combined in multiple ways. The examples of engineered cells with multiple genetic modifications are merely illustrative and do not limit the scope of the present invention.

[0085] Enzymes to be expressed or overexpressed in engineered cells according to the present invention are shown in Table 11.

[0086] host cell The host cell as provided herein can be a prokaryotic or eukaryotic cell. The eukaryotic cell can be a microbial eukaryotic cell, such as a fungal cell or a yeast cell. Prokaryotic cells that can be engineered as provided herein include bacterial and cyanobacterial cells.

[0087] Hosts can be selected based on their ability to take up and utilize particular carbon sources, nitrogen sources or precursor molecules, or can be engineered to take up and utilize molecules that can be added to the culture medium.

[0088] Non-limiting examples of microbial hosts suitable for the bioproduction of flavonoids include, but are not limited to, any Gram-negative organism, more particularly, members of the Enterobacteriaceae family, such as E. coli, any Gram-positive microorganism, such as Bacillus subtilis, Lactobacillus sp. or Lactococcus sp.; yeasts, such as Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species. More specifically, suitable microbial hosts for the bioproduction of flavonoids 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.

[0089] Culture medium In yet another aspect, a method for producing a flavonoid or anthocyanin, comprising incubating a culture of an engineered host cell as provided herein to produce the flavonoid or anthocyanin. The method may further comprise recovering the flavonoid or anthocyanin from the culture medium, the whole culture, or the cells.

[0090] The culture comprises engineered cells for the production of flavonoids or anthocyanins in the culture medium. In various embodiments, the engineered cells can be prokaryotic or eukaryotic. The culture medium comprises at least one carbon source that is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose and xylose. Such carbon sources can be refined or crude, including glycerol-containing biomass, e.g., crude glycerol, which occurs as a by-product of biodiesel production from corn waste. In addition, the culture medium can comprise one or more other carbon sources or compounds to increase precursor production or cofactor supply, such as, but not limited to, 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.

[0091] The culture conditions may include aerobic growth conditions, microaerobic growth conditions, or any combination of alternating aerobic / microaerobic growth conditions. Additionally, the culture conditions may include shake flasks, fermentation, and other large-scale culture procedures. Exemplary growth conditions for obtaining flavonoid products include aerobic or microaerobic fermentation conditions. The culture conditions can be continuously scaled up and expanded to produce flavonoid products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation protocol, cells are grown in a bioreactor with well-controlled growth temperature, oxygen, pH, carbon source, and other compounds. The desired temperature may be, for example, 20-37° C., depending on the growth characteristics of the producing cells and the desired conditions for the fermentation product. The pH of the bioreactor may be controlled in the range of 5-8 or may be left unattended in some cases. The batch fermentation period may range from a few hours to several days, for example, 8-96 hours. Upon completion of the culture period, the contents of the fermenter can be passed through a cell separation unit to remove cells and cell debris. Cells can be enzymatically or chemically lysed or destroyed, as desired, before or after separation of the cells from the fermentation broth to release additional products. To purify the flavonoids and / or anthocyanins to homogeneity, the solution containing the flavonoids and / or anthocyanins was concentrated and the product was purified by ion exchange or silica-based chromatography. The resulting solution was either freeze-dried to obtain the product in solid form or concentrated into a solution.

[0092] In some embodiments, the method for producing flavonoids or anthocyanins comprises culturing the engineered cells disclosed herein in a culture medium to produce flavonoids or anthocyanins. In some embodiments, glycerol is used as a carbon source. 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, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can 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, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 50% to about 99%, e.g., about 50% to about 95% (e.g., about 50%, 55%, 60%, 65%, 70%, 75%, 80% to about 85%, 90%, 95%, 97.5%, 99% or 99.9%).In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 50%, to about 55%, to about 60%, to about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, or to about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 55%, to about 60%, to about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, or to about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 60%, to about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, or to about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 65%, to about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, or to about 99%.In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 70%, to about 75%, to about 80%, to about 85%, to about 90%, to about 95%, or to about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 75% to about 80%, about 85%, about 90%, about 95%, or about 99%, about 80% to about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of from about 85%, to about 90%, to about 95%, or to about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated at a purity of about 90%, about 95% or about 99%, or about 95% to about 99%, or greater. EXAMPLES

[0093] VIII. Examples Use of modified cells to produce products

[0094] Example 1 Production of naringenin in E. coli E. coli cells derived from MG1655 were 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 the substrates tyrosine and coumaric acid were fed to the culture medium. ACC was expressed on a medium copy number plasmid (15-20 copies), while TAL, 4CL, CHS, and CHI were expressed chromosomally. Cells at OD2.5 were grown in 48-well plates in minimal medium supplemented with trace elements, vitamins, 1 mM tyrosine, 1 mM coumaric acid, and 2% glycerol at 30 degrees for 24 hours with a shaking speed of 600 RPM. The cell culture was extracted with DMSO in a 1:1 ratio and centrifuged for 15 minutes. The supernatant was analyzed for naringenin by HPLC. The cells produced 232 μM naringenin.

[0095] 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) can be used together with one or more homologues 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 a combination of two or more of these, to prepare variants of the host cells described above, where 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.

[0096] Example 2 Production of dihydrokaempferol in E. coli. E. coli cells derived from MG1655 were engineered to overexpress F3H (SEQ ID NO: 7) on the chromosome to produce dihydrokaempferol when the substrate naringenin was fed to the culture medium. Cells at OD 0.5-0.7 were grown in 24-well plates in minimal medium supplemented with 2% glycerol, trace elements, 0.8 mM naringenin, 65 mg / L 5-aminolevulinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid at 30 degrees for 18 hours with a shaking speed of 200 RPM. The cell culture was extracted with DMSO and centrifuged for 15 minutes. The supernatant was analyzed for dihydrokaempferol by HPLC. The cells produced 315 μM dihydrokaempferol.

[0097] Variants of the above-mentioned host cells can be prepared using a homologue of F3H (SEQ ID NO: 7), where 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.

[0098] 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 fed to the culture medium. F3H was overexpressed on the chromosome, while F3'H and CPR were overexpressed on a medium copy number plasmid. Cells at OD 0.5-0.7 were grown in 24-well plates in minimal medium supplemented with 2% glucose, 0.8 mM naringenin, 65 mg / L 5-aminolevulinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate and 2.5 mM ascorbic acid at 30 degrees for 18 hours with a shaking speed of 200 RPM. The cell culture was extracted with 50% DMSO and centrifuged for 15 minutes. The supernatant was analyzed for taxifolin by HPLC. The cells produced 500 μM taxifolin.

[0099] One or more of F3H (SEQ ID NO:7), F3'H (SEQ ID NO:8) and CPR (SEQ ID NO:9) can be used together with one or more homologues of F3H (SEQ ID NO:7), F3'H (SEQ ID NO:8) and CPR (SEQ ID NO:9), or a combination of two or more of these, to prepare variants of the host cells described above, where 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.

[0100] Example 4 Anthocyanidin and anthocyanin production 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 fed to the culture medium. ANS and 3GT were overexpressed on the chromosome. Cells at OD 0.5-0.7 were grown in 24-well plates in minimal medium fed with 1.0% glucose, 2.0 mM (+)-catechin, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid at 30 degrees for 18 hours with a shaking speed of 200 RPM. The cell culture was 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 of cyanidin-3-O-glucoside.

[0101] Either or both of ANS (SEQ ID NO:13) and 3GT (SEQ ID NO:14) can be used together with homologs of ANS (SEQ ID NO:13), 3GT (SEQ ID NO:14) and / or both to prepare variants of the host cells described above, where 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.

[0102] Analysis method Example 5 Flavonoid Precursors and Flavonoids Extraction of total flavonoids from E. coli for sampling of naringenin, eriodictyol, dihydrokaempferol and taxifolin was performed using whole cell broth. 500 μL of whole cell broth was vortexed with 500 μL of DMSO (dimethyl sulfoxide) for 30 seconds and centrifuged for 15 minutes. 50 μL of the supernatant was transferred to an HPLC vial for HPLC analysis.

[0103] The HPLC method was as follows: An Agilent 1200 HPLC was equipped with an Ascentis C18 column 150 mm×4.6 mm, 3 μm with an R-18 (3 μm) guard column. The column was heated to 30° C. and the sample block was 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 the following gradient: 0.1% phosphoric acid in water (solvent A), acetonitrile (solvent B) and methanol (solvent C): [Table 13]

[0104] The run time was 15 min in total, with naringenin, eriodictyol, dihydrokaempferol and taxifolin eluting at 12.50, 11.56, 10.20 and 8.85 min, respectively. A diode array detector (DAD) was used for detection of the molecules of interest at 288 nm.

[0105] Example 6 Anthocyanidins and Anthocyanins For sampling of (+)-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 with a LiChrospher 100 RP-8 (5 μm) LiChroCART 4-4 guard column. The column was heated to 25° C. and the sample block was maintained at 25° C. For each sample, 10 μL was injected and the products were eluted at a flow rate of 1.0 ml / min using the following gradient: 90% A to 10% A in 12 min; 90% A, 0.5 min; and 90% A, 3.5 min for column equilibration. The run time was 16 min in total, with cyanidin-3-glycoside eluting at 6.95 min and cyanidin eluting at 8.9 min. A diode array detector (DAD) was used for detection of the molecules of interest at either 280 nm or 530 nm.

[0106] Example 7 Flavonoid production This example provides a combination of modifications to the E. coli host genome, including deletion and overexpression of enzymes from other organisms to reproduce the bioproduction pathway described in Figure 4. Thus, the present invention provides engineered host cells that contain one or more genetic modifications (as shown in Figure 4 and as described in this Example 7, and as described above herein), which result in the engineered host cell producing flavonoids or anthocyanins via multiple chemical intermediates from a carbon source that can also be an energy source. In certain embodiments, the production of flavonoids or anthocyanins from a carbon source that can also be an energy source occurs by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, and (v) any combination thereof. In certain embodiments, the engineered host cells are cultured in a medium that contains molecules selected from the group consisting of (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, and (v) any combination thereof. As shown in Figure 4, in certain embodiments, the one or more genetic modifications lead to an increase in metabolic flux to flavonoid precursors or cofactors. As shown in Figure 4, in certain embodiments, the one or more genetic modifications cause a reduction in the formation of by-products. As shown in Figure 4, in certain embodiments, the one or more genetic modifications are selected from (i) one or more modifications that cause the engineered host cell to overexpress one or more endogenous genes; (ii) one or more modifications that cause the engineered host cell to underexpress one or more endogenous genes; (iii) one or more genetic modifications that cause the engineered host cell to express one or more non-native genes; and (iv) combinations thereof.

[0107] As shown in FIG. 4, in certain embodiments, the engineered host cells are cultured in a medium comprising a molecule selected from tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

[0108] As shown in Figure 4, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from (i) a nucleic acid sequence encoding tyrosine ammonia lyase activity; (ii) a nucleic acid sequence encoding phenylalanine ammonia lyase activity; (iii) cinnamate 4-hydroxylase; and (iv) any combination thereof. As shown in Figure 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 comprising chalcone synthase and chalcone isomerase; and (iv) any combination thereof.

[0109] As shown in Figure 4, in certain embodiments, one or more genetic modifications reduce fatty acid biosynthesis. As shown in Figure 4, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from: (i) a nucleic acid sequence encoding tyrosine ammonia lyase, where the encoded tyrosine ammonia lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding phenylalanine ammonia lyase, where the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding cinnamic acid-4-hydroxylase, where the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.

[0110] 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 sequence encoding a tyrosine ammonia-lyase activity; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase activity; (iii) a nucleic acid sequence encoding a cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof.

[0111] As shown in FIG. 4, in certain embodiments, the engineered host cell comprises: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase, where the encoded tyrosine ammonia-lyase uses tyrosine as a substrate to form 4-coumaric acid; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, where the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, where the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, where the 4- (v) a nucleic acid sequence encoding a chalcone synthase activity, where the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, where the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof.

[0112] The above compositions can be used in the methods described herein for increasing the production of flavonoids or anthocyanins. Such methods involve providing any of the above compositions that result in the enzymatic conversion of glycerol to flavonoids or anthocyanins via multiple chemical intermediates (e.g., as shown in part or in whole in Figure 4) by engineered host cells.

[0113] In yet another aspect, it is envisioned that the pathway described in Figure 4 can be performed using multiple engineered host cells rather than the single host cell described above. In such an embodiment, the multiple engineered host cells have one or more genetic modifications that result in the enzymatic conversion of glycerol to flavonoids or anthocyanins (as shown in Figure 4) via multiple chemical intermediates by the engineered host cells.

[0114] An embodiment of the present invention will now be described with reference to FIG.

[0115] Step 1: Conversion of pyruvate to acetate. Deletion of poxB reduces carbon loss and eliminates by-products.

[0116] Step 2: Conversion of pyruvate to lactate. ldhA is deleted to reduce carbon loss and eliminate by-products.

[0117] Step 3: Conversion of acetyl-CoA to acetate. Deletion of ackA-pta reduces carbon loss and eliminates by-products.

[0118] Step 4: Conversion of acetyl-CoA to ethanol (EtOH). adhE is deleted to reduce carbon loss and eliminate by-products.

[0119] Step 5: Conversion of acetyl-CoA to a substrate for the tricarboxylic acid cycle (TCA).

[0120] Step 6: Conversion of acetyl-CoA to mal-CoA. Heterologous ACC is expressed to increase the concentration of available mal-CoA. Heterologous ACC can be obtained from a fungal species. Thus, an embodiment of the present invention provides an engineered host cell comprising 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 E. coli. In certain embodiments, the acetyl-CoA carboxylase is from Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and is an ortholog of an acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, the one or more genetic modifications are deletion or attenuation of one or more fatty acid biosynthesis genes, resulting in the reduction of fatty acid biosynthesis.In certain embodiments, the one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS).In certain embodiments, the acetyl-CoA synthase is selected from the ortholog of the acetyl-CoA synthase gene of E.coli, the acetyl-CoA synthase gene of Salmonella typhimurium, and the acetyl-CoA synthase gene of any other species that has at least 50% amino acid identity with the acetyl-CoA synthase gene of E.coli and Salmonella typhimurium.In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), where the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), where PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from Streptomyces coelicolor malonyl-CoA synthetase, Rhodopseudomonas palustris malonyl-CoA synthetase, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications to reduce fatty acid biosynthesis are selected from: (i) mutation or downregulation of the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of the beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of the acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from: (i) an 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) a malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:16; (iv) a malonyl-CoA synthase having an amino acid sequence at least 80% identical to SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; (v) a 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) a 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 combination thereof.

[0121] In another aspect, the present invention provides a method for increasing the production of flavonoids, comprising an engineered host cell, wherein one or more engineered host cells comprise one or more genetic modifications for increasing 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 E. coli. In certain embodiments, the acetyl-CoA carboxylase is from Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and is an ortholog of the acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, the one or more genetic modifications are deletion or attenuation of one or more fatty acid biosynthesis genes, resulting in a decrease in fatty acid biosynthesis. In certain embodiments, the one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS).In certain embodiments, the acetyl-CoA synthase is selected from the orthologs of the acetyl-CoA synthase gene of E. coli, the acetyl-CoA synthase gene of Salmonella typhimurium, and the acetyl-CoA synthase gene of any other species that has at least 50% amino acid identity with the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium.In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), where the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), where PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from Streptomyces coelicolor malonyl-CoA synthetase, Rhodopseudomonas palustris malonyl-CoA synthetase, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications to reduce fatty acid biosynthesis are selected from: (i) mutation or downregulation of the gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of the beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of the acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from: (i) an 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) a malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:16; (iv) a malonyl-CoA synthase having an amino acid sequence at least 80% identical to SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:79; (v) a 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) a 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 combination thereof.

[0122] Step 7: Conversion of mal-CoA to malonyl-ACP (acyl carrier protein). Malonyl-coA-ACP transacylase (fabD) is downregulated to increase carbon flux.

[0123] Step 8: Conversion of malonyl-ACP to 3-ketyoacyl-ACP. Beta-ketoacyl-ACP synthase II (fabF) is downregulated to increase carbon flux.

[0124] Step 9: Conversion of mal-CoA to naringenin chalcone; Conversion of coumaryl-CoA to naringenin chalcone. Heterologous CHS is overexpressed.

[0125] Step 10: Conversion of naringenin chalcone to naringenin. Heterologous CHI is overexpressed.

[0126] 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 pentahydroxyflavone (PHF) to dihydromyricetin (DHM). Overexpressing heterologous F3'5'H, F3H, F3H and / or CPR. Thus, as shown in Figure 4, in another aspect, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF), comprising an engineered host cell, the engineered host cell comprising a cytochrome P450 reductase (CPR) and at least one of a flavanone-3'-hydroxylase (F3'H) or a flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursors for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF) are naringenin and / or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises a peptide selected from the 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, the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence.In certain embodiments, the flavonoid 3'-hydroxylase (F3'H) is fused to a cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3',5'-hydroxylase (F3'5'H) is fused to a cytochrome P450 reductase (CPR). In certain embodiments, the flavanone-3'-hydroxylase (F3H) has an amino acid sequence that is at least 80% identical to the polypeptide set forth in SEQ ID NO:7. In certain embodiments, the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence that is at least 80% identical to the polypeptide set forth in SEQ ID NO:8. In certain embodiments, the cytochrome P450 reductase (CPR) has an amino acid sequence that is at least 80% identical to the polypeptide set forth in SEQ ID NO:9. In certain embodiments, the flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to a polypeptide selected from the 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, the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 98.

[0127] As shown in Figure 4, in another aspect, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF), comprising an engineered host cell, the engineered host cell comprising a cytochrome P450 reductase (CPR) and at least one of a flavanone-3'-hydroxylase (F3'H) or a flavonoid 3',5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF) is naringenin and / or dihydrokaempferol (DHK). In certain embodiments, the engineered host cell further comprises a peptide selected from the 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, the flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, the cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, the flavonoid 3'-hydroxylase (F3'H) is fused to the cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3',5'-hydroxylase (F3'5'H) is fused to a cytochrome P450 reductase (CPR). In certain embodiments, the flavanone-3-hydroxylase (F3H) has an amino acid sequence that is at least 80% identical to the polypeptide set forth in SEQ ID NO:7.In certain embodiments, the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:8. In certain embodiments, the 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, the flavonoid 3',5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to a polypeptide selected from the 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, the cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:98.

[0128] Step 14: Conversion of dihydroquercetin (DHQ) to leucocyanidin (LC); dihydrokaempferol (DHK) to leucopelargonidin (LP); and dihydromyricetin (DHM) to leucodelphinidin (LD). Heterologous DFRs are overexpressed.

[0129] Step 15: Conversion of leucocyanidin (LC) to catechin; leucodelphinidin (LD) to gallocatechin; and leucopelargonidin (LP) to afzelechin. Heterologous LAR is overexpressed.

[0130] Step 16: converting catechin to cyanidin; converting leucocyanidin (LC) to catechin; converting leucodelphinidin (LD) to delphinidin; converting gallocatechin to delphinidin; converting leucopelargonidin (LP) to pelargonidin; or converting afzelechin to pelargonidin. Overexpressing heterologous ANS. Step 16 can be performed in vivo or in cell-free medium. Thus, as shown in Figure 4, in another aspect, the present invention provides engineered host cells comprising one or more genetic modifications to increase the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from (i) an anthocyanin synthase of Carica papaya (SEQ ID NO: 13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO: 13; and (iv) any combination thereof. In certain embodiments, the one or more engineered host cells comprise a flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase of Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof. In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT).In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications are selected from the group consisting of (i) anthocyanin synthase, (ii) flavonoid-3-glucosyltransferase (3GT), and (iii) combinations thereof.

[0131] In another aspect, the present 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 for increasing the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from (i) Carica papaya anthocyanin synthase (SEQ ID NO: 13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO: 13; and (iv) any combination thereof. In certain embodiments, the one or more engineered host cells comprise flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase from Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof. In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications include overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications are selected from the group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyltransferase (3GT), and (iii) combinations thereof.

[0132] In another aspect, the present invention provides a method for increasing conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), comprising an anthocyanin synthase selected from: (i) anthocyanin synthase of Carica papaya (SEQ ID NO:13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO:13; and (iv) any combination thereof.

[0133] In another aspect, the present invention provides a method for increasing the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), comprising a flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyltransferase is selected from (i) a flavonoid-3-glucosyltransferase of Vitis labrusca (SEQ ID NO: 14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof.

[0134] Step 17: Conversion of pelargonidin to callistephin; conversion of delphinidin to mirtilin (De3G); conversion of cyanidin to Cy3G. Heterologous 3GT was overexpressed in E. coli. Step 17 can be performed in vivo or as a cell-free reaction.

[0135] Step 18: Conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine.

[0136] Step 19: Conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA is overexpressed to upregulate tyrosine.

[0137] Step 20: Conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). Overexpression of aroG variants results in upregulation of tyrosine.

[0138] Step 21: Conversion of deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) to dehydroquinic acid (DHQ); Conversion of erythrose-4-phosphate (E4P) to dehydroquinic acid (DHQ).

[0139] Step 22: Conversion of dehydroquinic acid (DHQ) to 3-dehydroshikimic acid (DHS).

[0140] Step 23: Conversion of 3-dehydroshikimate (DHS) to shikimate (SHK). aroE is overexpressed and tyrosine is upregulated.

[0141] Step 24: Conversion of shikimic acid (SHK) to shikimic acid-3-phosphate (S3P).

[0142] Step 25: Conversion of shikimate-3-phosphate (S3P) to 5-enolpyruvylshikimate-3-phosphate (EPSP).

[0143] Step 26: Conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate (CHA).

[0144] Step 27: Conversion of chorismate (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variants are overexpressed.

[0145] Stage 28: Conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine; Conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Thus, as shown in FIG. 4, an embodiment of the present invention provides an engineered host cell comprising one or more genetic modifications to increase the endogenous biosynthesis of tyrosine. In certain embodiments, the one or more genetic modifications comprise upregulation of 3-deoxy-D-arabino-heptulosonic acid synthase. In certain embodiments, the 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 combination thereof. In certain embodiments, the one or more genetic modifications comprise downregulation of the L-phenylalanine biosynthetic pathway. In certain embodiments, the one or more genetic modifications comprise expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, the one or more genetic modifications include expression of an exogenous transketolase (tktA). In certain embodiments, the one or more genetic modifications include disruption of the tyrR gene.

[0146] As shown in Figure 4, in another aspect, the present invention provides a method for increasing the endogenous biosynthesis of tyrosine, comprising an engineered cell, wherein the engineered host cell comprises one or more genetic modifications for increasing the endogenous biosynthesis of tyrosine. In certain embodiments, the one or more genetic modifications comprise upregulation of 3-deoxy-D-arabino-heptulosonic acid synthase. In certain embodiments, the 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 combination thereof. In certain embodiments, the one or more genetic modifications comprise downregulation of the L-phenylalanine biosynthesis pathway. In certain embodiments, the one or more genetic modifications comprise expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, the one or more genetic modifications comprise expression of exogenous transketolase (tktA). In certain embodiments, the one or more genetic modifications include a disruption of the tyrR gene.

[0147] Step 29: Conversion of tyrosine to coumarate. Heterologous TAL is overexpressed.

[0148] Step 30: Conversion of courmaric acid to coumaryl-CoA. Heterologous 4CL is overexpressed.

[0149] Step 31: Conversion of glutamic acid (Glut) to glutamyl-tRNA.

[0150] Step 32: Conversion of glutamyl-tRNA to glutamic acid semialdehyde (GSA). hemA is overexpressed to upregulate ALA.

[0151] Step 33: Conversion of glutamic semialdehyde (GSA) to delta-aminolevulinic acid (ALA). ALA is upregulated by overexpressing hemL.

[0152] Step 34: Conversion of delta-aminolevulinic acid (ALA) to porphobilinogen (PBG).

[0153] Step 35: Conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB).

[0154] Step 36: Conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III (UPPIII).

[0155] Step 37: Conversion of uroporphyrinogen III (UPPIII) to coproporphyrinogen III (CPPIII).

[0156] Step 38: Conversion of coproporphyrinogen III (CPPIII) to protoporphyrinogen IX (PPPIX).

[0157] Step 39: Conversion of protoporphyrinogen IX (PPPIX) to protoporphyrin IX, which is then covered with heme.

[0158] Step 40: Conversion of prephenic acid (PPA) to phenylpyruvic acid (POPP).

[0159] Step 41: Conversion of phenylalanine (Phe) to cinnamic acid. Heterologous PAL and / or TAL are overexpressed.

[0160] Step 42: Conversion of cinnamic acid to coumaric acid. Heterologous C4H / CPR is overexpressed. [Table 11-1] [Table 11-2] [Table 11-3] [Table 11-4]

Table 11-5

Table 11-6

Table 11-7

Table 11-8

Table 11-9

Table 11-10

Table 11-11

Table 11-12

Table 11-13

Table 11-14

Table 11-15

Table 11-16

Table 11-17

Table 11-18

Table 11-19

Table 11-20

Table 11-21

Table 11-22

Table 11-23

Table 11-24

Table 11-25

Table 11-26

Table 11-27

Table 11-28

Table 11-29

Table 11-30

Table 11-31

Table 11-32

Table 11-33

Table 11-34

Table 11-35

Table 11-36

Table 11-37

Table 11-38

Table 11-39

Table 11-40

Table 11-41

Table 11-42

Table 11-43

Table 11-44

Table 11-45

Table 11-46

Table 11-47

Table 11-48

Table 11-49

Table 11-50

[0161]

Table 12-1

Table 12-2

Table 12-3

Table 12-4

Claims

1. An engineered host cell comprising one or more genetic modifications that result in the production by the engineered host cell of a flavonoid or anthocyanin by enzymatic conversion from a carbon source, which may also be an energy source, via multiple chemical intermediates, wherein the engineered host cell is E. coli, and the one or more genetic modifications comprise the expression of a heterologous malonyl-CoA synthetase.

2. 2. The engineered host cell of claim 1, wherein the carbon source is selected from the group consisting of: (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof.

3. 2. The engineered host cell of claim 1 , cultured in a medium comprising molecules selected from the group consisting of: (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, (v) biomass comprising glycerol, and (vi) any combination thereof.

4. The engineered host cell of claim 1, wherein the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors, cause reduced formation of by-products, or reduce fatty acid biosynthesis, compared to a corresponding host cell not having the one or more genetic modifications.

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

6. (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase activity; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase activity; (iii) a nucleic acid sequence encoding a cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof, (vi) chalcone isomerase; (vii) chalcone synthase; (viii) a fusion protein comprising chalcone synthase and chalcone isomerase; and (ix) any combination thereof; and / or (x) a nucleic acid sequence encoding a tyrosine ammonia-lyase, wherein the encoded tyrosine ammonia-lyase forms 4-coumaric acid using tyrosine as a substrate; (xi) a nucleic acid sequence encoding a phenylalanine ammonia-lyase, wherein the encoded phenylalanine ammonia-lyase converts phenylalanine to trans-cinnamic acid; (xii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (xiii) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, wherein the 4-coumarate-CoA ligase converts cinnamic acid to trans-cinnamic acid; (xiv) a nucleic acid sequence encoding a chalcone synthase activity, where the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (xv) a nucleic acid sequence encoding a chalcone isomerase activity, where the chalcone isomerase forms naringenin from naringenin chalcone; (xvi) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (xvii) any combination thereof. The engineered host cell of claim 1.

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

8. 10. A method for increasing production of a flavonoid or anthocyanin, comprising the step of providing an engineered host cell comprising one or more genetic modifications that result in the production by the engineered host cell of a flavonoid or anthocyanin by enzymatic conversion from a carbon source, which may also be an energy source, via multiple chemical intermediates, wherein the engineered host cell is an engineered host cell according to any one of claims 1, 4-7.

9. A plurality of engineered host cells, each of said plurality of said engineered host cells comprising one or more genetic modifications that result in the production by said engineered host cell of a flavonoid or anthocyanin by enzymatic conversion from a carbon source, which may also be an energy source, via a plurality of chemical intermediates, wherein at least one of said plurality of said engineered host cells is E. coli, and said one or more genetic modifications comprise expression of a heterologous malonyl-CoA synthetase.

10. The plurality of engineered host cells described in claim 9, wherein the one or more genetic modifications lead to increased metabolic flux to flavonoid precursors or cofactors, cause reduced formation of by-products, or reduce fatty acid biosynthesis, compared to a corresponding host cell not having the one or more genetic modifications.

11. 10. The plurality of engineered host cells of claim 9, wherein at least one of the engineered host cells is cultured in a medium comprising a molecule selected from the group consisting of tyrosine, phenylalanine, malonate, p-coumarate, bicarbonate, acetate, pantothenate, biotin, thiamine, alpha-ketoglutarate, ascorbate, and 5-aminolevulinic acid.

12. At least one of the engineered host cells comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a tyrosine ammonia-lyase activity; (ii) a nucleic acid sequence encoding a phenylalanine ammonia-lyase activity; (iii) a nucleic acid sequence encoding a cinnamate 4-hydroxylase activity; (iv) a nucleic acid sequence encoding a 4-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof. At least one of the engineered host cells comprises at least one or more peptides selected from the group consisting of: (vi) chalcone isomerase; (vii) chalcone synthase; (viii) a fusion protein comprising chalcone synthase and chalcone isomerase; and (ix) any combination thereof; and / or At least one of the engineered host cells is capable of detecting a nucleic acid sequence encoding: (x) a nucleic acid sequence encoding a tyrosine ammonia lyase, wherein the encoded tyrosine ammonia lyase uses tyrosine as a substrate to form 4-coumaric acid; (xi) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (xii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (xiii) a nucleic acid sequence encoding a 4-coumarate-CoA ligase activity, wherein the 4-coumarate- (xiv) a nucleic acid sequence encoding a chalcone synthase activity, where the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (xv) a nucleic acid sequence encoding a chalcone isomerase activity, where the chalcone isomerase forms naringenin from naringenin chalcone; (xvi) a nucleic acid sequence encoding a flavanone-3-hydroxylase, where the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (xvii) any combination thereof.

10. A plurality of engineered host cells according to claim 9.

13. 10. The plurality of engineered host cells of claim 9, wherein the flavonoid is a catechin.

14. 10. A method for increasing production of a flavonoid or anthocyanin, comprising the step of providing a plurality of engineered host cells, each of said plurality of engineered host cells comprising one or more genetic modifications that result in the production by said engineered host cell of a flavonoid or anthocyanin from a carbon source, which may also be an energy source, via a plurality of chemical intermediates, said engineered host cell being an engineered host cell according to any one of claims 1, 4-7.

15. The engineered host cell of claim 1, wherein the malonyl-CoA synthetase comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:

17.

16. The engineered host cell of claim 1, wherein the malonyl-CoA synthetase comprises an amino acid sequence having at least 95% identity to the amino acid sequence of SEQ ID NO:77, SEQ ID NO:78 or SEQ ID NO:

79.

17. An engineered host cell comprising one or more genetic modifications to an endogenous gene to increase production and / or availability of malonyl-CoA compared to a wild-type host cell, wherein the engineered host cell expresses an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 85% identical to the polypeptide set forth in SEQ ID NO:

15.

18. 18. The engineered host cell of claim 17, wherein the production and / or availability of malonyl-CoA is increased by conversion of acetyl-CoA to malonyl-CoA.

19. The engineered host cell of claim 17, comprising one or more genetic modifications that increase expression of acetyl-CoA carboxylase (ACC) compared to a wild-type host.

20. 18. The engineered host cell of claim 17, which is E. coli.

21. 21. The engineered host cell of claim 20, wherein the E. coli further comprises a gene from a fungus.

22. 20. The engineered host cell of claim 19, 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 carboxylases having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species.

23. The engineered host cell of claim 17, wherein the one or more genetic modifications are a deletion or attenuation of one or more fatty acid biosynthetic genes resulting in reduced fatty acid biosynthesis compared to a wild-type host.

24. The engineered host cell of claim 17, further comprising a peptide selected from the group consisting of: (i) a malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:19; (ii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO:16; (iii) a malonyl-CoA synthase having an amino acid sequence at least 80% identical to SEQ ID NO:77, SEQ ID NO:78, or SEQ ID NO:79; (iv) a 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; (v) a 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 (vi) any combination thereof.

25. The engineered host cell of claim 17, wherein the engineered host cell expresses an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 90% identical to SEQ ID NO:

15.

26. The engineered host cell of claim 17, wherein the engineered host cell expresses an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 95% identical to SEQ ID NO:

15.

27. 27. A method for increasing production of flavonoids, comprising an engineered host cell, wherein one or more of the engineered host cells comprises one or more genetic modifications to increase production and / or availability of malonyl-CoA, and wherein the one or more engineered host cells are the engineered host cells of any one of claims 17-26.

28. An engineered host cell comprising one or more genetic modifications to increase endogenous biosynthesis of tyrosine, said one or more genetic modifications comprising downregulation or deletion of pyruvate oxidase, and upregulation of chorismate mutase.

29. 30. The engineered host cell of claim 28, wherein the one or more genetic modifications further comprise: (i) upregulation of 3-deoxy-D-arabino-heptulosonate synthase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; or (v) any combination thereof.

30. 30. The engineered host cell of claim 28, wherein the one or more genetic modifications further comprise downregulation of the L-phenylalanine biosynthetic pathway, expression of an exogenous phosphoenolpyruvate synthase (ppsA), expression of an exogenous transketolase (tktA), or disruption of a tyrR gene.

31. 30. The engineered host cell of claim 28, wherein the one or more genetic modifications further comprise: (i) expression or overexpression of a feedback inhibition resistant phospho-2-dehydro-3-deoxyheptonate aldolase; (ii) expression or overexpression of a variant of 3-dehydroquinate synthase (aroB); (iii) overexpression of transketolase tktA; (iv) deletion of tyrR; (v) expression or overexpression of a feedback inhibition resistant chorismate mutase (tyrA); or (vi) any combination thereof.

32. 32. A method for increasing endogenous biosynthesis of tyrosine, the method comprising the step of preparing an engineered host cell comprising one or more genetic modifications for increasing endogenous biosynthesis of tyrosine, the one or more genetic modifications being the genetic modification of any one of claims 28 to 31.

33. The engineered host cell of claim 28, wherein the one or more genetic modifications further comprise downregulation or deletion of lactate dehydrogenase.

34. The method described in claim 32, wherein the one or more genetic modifications further include down-regulation or deletion of lactate dehydrogenase.

35. An engineered host cell comprising one or more genetic modifications to increase the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G).

36. 36. The engineered host cell of claim 35, wherein the one or more genetic modifications comprise overexpression of an anthocyanin synthase, the anthocyanin synthase being selected from the group consisting of: (i) an anthocyanin synthase of Carica papaya (SEQ ID NO: 13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO: 13; and (iv) any combination thereof.

37. 37. The engineered host cell of claim 36, further comprising a flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyltransferase is selected from the group consisting of: (i) a flavonoid-3-glucosyltransferase of Vitis labrusca (SEQ ID NO:14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof.

38. 38. A method for increasing production of a flavonoid, comprising an engineered host cell, the engineered host cell comprising one or more genetic modifications to increase conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), the one or more genetic modifications being the genetic modification of claim 37.

39. 1. A method for increasing the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), comprising an anthocyanin synthase selected from the group consisting of: (i) an anthocyanin synthase of Carica papaya (SEQ ID NO:13); (ii) an anthocyanin synthase having 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) an anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ ID NO:13; and (iv) any combination thereof.

40. 1. A method for increasing the conversion of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G), comprising: labrusca flavonoid-3-glucosyltransferase (SEQ ID NO:14); (ii) a flavonoid-3-glucosyltransferase having 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 combination thereof.

41. An engineered host cell comprising one or more genetic modifications for increasing production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF) compared to a wild-type cell, wherein the engineered host cell expresses a cytochrome P450 reductase (CPR) having an amino acid sequence at least 80% identical to SEQ ID NO:9, cytochrome b5, and at least one of flavanone-3-hydroxylase (F3H), flavanone-3'-hydroxylase (F3'H), or flavonoid 3',5'-hydroxylase (F3'5'H).

42. 42. The engineered host cell of claim 41, wherein the precursors for increased production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF) are naringenin and / or dihydrokaempferol (DHK).

43. 42. The engineered host cell of claim 41, wherein the flavonoid 3',5'-hydroxylase (F3'5'H) is truncated to remove an N-terminal leader sequence, fused to a cytochrome P450 reductase (CPR), or has an amino acid sequence at least 80% identical to SEQ ID NO:10, SEQ ID NO:56, or SEQ ID NO:

57.

44. a flavonoid 3'-hydroxylase (F3'H) fused to a cytochrome P450 reductase (CPR) or having an amino acid sequence at least 80% identical to SEQ ID NO:8; and / or The flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to SEQ ID NO:7, SEQ ID NO:45, SEQ ID NO:46, SEQ ID NO:47, or SEQ ID NO:

48.

42. The engineered host cell of claim 41.

45. 42. The engineered host cell of claim 41, wherein the cytochrome P450 reductase (CPR) has an amino acid sequence that is at least 95% identical to the polypeptide set forth in SEQ ID NO:

9.

46. The cytochrome b ５ has an amino acid sequence that is at least 80% identical to SEQ ID NO:

98.

47. 50. A method for increasing production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL), and / or pentahydroxyflavone (PHF), comprising an engineered host cell, the engineered host cell comprising a 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), the engineered host cell being the engineered host cell of any one of claims 41-46.

48. The engineered host cell of claim 41, wherein the engineered host cell is E. coli.

49. The method of claim 47, wherein the engineered host cell is E. coli.

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