Biological production of flavonoids using microbial hosts

Abstract

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

Description

Technical Field

[0001] The present invention relates to materials (including engineered cells and cell lines) and methods involved in the production of flavonoids, anthocyanins and other organic compounds. Background Art

[0002] Flavonoids and anthocyanins are natural products produced in plants that have a variety of effects, 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 are able to scavenge free radicals and can act as enzyme inhibitors and anti-inflammatory agents. With these recognized health and color benefits, much research has gone into understanding how these compounds are made in nature.

[0003] Myricetin is a common plant-derived flavonoid widely recognized for its nutritional value. It is one of the key ingredients in various foods and beverages. The compound exhibits a wide range of activities, including strong antioxidant, anticancer, antidiabetic, and anti-inflammatory activities. Kaempferol is a polyphenolic antioxidant found in fruits and vegetables. Many studies have described the beneficial effects of dietary kaempferol in reducing the risk of chronic diseases, especially cancer. Quercetin is a plant pigment and a potent antioxidant flavonoid found primarily in onions, grapes, berries, cherries, broccoli, and citrus fruits. It is a versatile antioxidant known to possess protective capabilities against tissue damage induced by various drug toxicities.

[0004] Flavonoids and anthocyanins are synthesized from phenylpropanoid starting units and malonyl-cofactor-A (malonyl-CoA) extenders, which are then modified to produce many polyphenolic compounds such as taxifolin, naringenin, and (+)-catechins. However, in most cases, these compounds are extracted or chemically manufactured. Summary of the invention

[0005] To move away from agricultural and chemical-derived products, we created engineered cells for the bioproduction of flavonoids and anthocyanins. This approach provides a viable pathway for the rapid, safe, economical, and sustainable production of multiple important flavonoids, including myricetin, kaempferol, and quercetin.

[0006] Herein, a series of flavonoids and anthocyanins, including naringenin, eriodictyol, taxifolin, dihydrokaempferol, (+)-catechin, cyanidin, cyanidin-3-glucoside, myricetin, kaempferol and quercetin, are produced using modified microbial host organisms. Herein, the engineered cells include 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 caused by the production of by-products.

[0007] In a first aspect, the present invention provides engineered host cells, wherein these engineered host cells include one or more genetic modifications, to increase the production of flavonols by one or more enzyme conversion carbon sources through the engineered host cells. In one embodiment, the flavonols are selected from the group consisting of kaempferol, myricetin and quercetin. In another embodiment, the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL). In another embodiment, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in these engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in these engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in these engineered host cells; and (iv) combinations thereof. In another embodiment, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homologue thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homologue thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3', 5'-hydroxylase (F3'5'H) or a homologue thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homologue thereof, and (v) any combination thereof. In another embodiment, the engineered host cell is Escherichia coli. In another embodiment, the production of flavonols by enzymatic conversion of a carbon source includes one or more chemical intermediates. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3', 5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, 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 another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, flavonol synthase (FLS) has an amino acid sequence at least 80% identical to any one of the polypeptides listed in SEQ ID NOs.99-122.In another embodiment, the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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. In another embodiment, the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H). In another embodiment, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in (i) SEQ ID NO:8, (ii) SEQ ID NO.49, (iii) SEQ ID NO.50, (iv) SEQ ID NO.51, and (v) SEQ ID NO.52. In another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), naringenin, or a combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), or (iii) any combination thereof. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), dihydrokaempferol (DHK), eriodictyol (EDL), naringenin, kaempferol, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), or (iv) any combination thereof. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), dihydroquercetin (DHQ), dihydrokaempferol (DHK), eriodictyol (EDL), naringenin, quercetin, kaempferol or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H) or (v) any combination thereof.

[0008] On the other hand, the present invention provides a method for increasing the production of flavonols, comprising an engineered host cell, wherein the engineered host cells comprise one or more genetic modifications to increase the production of flavonols by converting carbon sources by one or more enzymes through the engineered host cell. In one embodiment, the flavonol is selected from the group consisting of kaempferol, myricetin and quercetin. In another embodiment, the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL). In another embodiment, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in these engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in these engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in these engineered host cells; and (iv) combinations thereof. In another embodiment, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homologue thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homologue thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3', 5'-hydroxylase (F3'5'H) or a homologue thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homologue thereof, and (v) any combination thereof. In another embodiment, the engineered host cell is Escherichia coli. In another embodiment, the production of flavonols by enzymatic conversion of a carbon source includes one or more chemical intermediates. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3', 5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, 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 another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthase (FLS).In another embodiment, the flavonol synthase (FLS) has an amino acid sequence at least 80% identical to any one of the polypeptides listed in SEQ ID NO.99-122. In another embodiment, the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS) and (iii) any combination thereof. In another embodiment, the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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. In another embodiment, the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H). In another embodiment, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in (i) SEQ ID NO: 8, (ii) SEQ ID NO.49, (iii) SEQ ID NO.50, (iv) SEQ ID NO.51, and (v) SEQ ID NO.52. In another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), naringenin, or a combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), or (iii) any combination thereof. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), dihydrokaempferol (DHK), eriodictyol (EDL), naringenin, kaempferol, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), or (iv) any combination thereof.In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), dihydroquercetin (DHQ), dihydrokaempferol (DHK), eriodictyol (EDL), naringenin, quercetin, kaempferol or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H) or (v) any combination thereof. BRIEF DESCRIPTION OF THE DRAWINGS

[0009] Figure 1 Metabolic pathways for bioproduction of flavonoids and anthocyanins in the engineered cells and methods for making anthocyanins described herein are shown.

[0010] Figure 2 Shown are the structures of flavonoid and anthocyanin molecules that can be produced using the engineered cells and methods of making anthocyanins described herein.

[0011] Figure 3 Shown is an HPLC profile showing peaks corresponding to molecules produced using the engineered cells and methods of producing anthocyanins described herein.

[0012] Figure 4 Pathways for bioproduction of flavonoids and anthocyanins in the engineered cells and methods for making anthocyanins described herein are shown.

[0013] Figure 5 The production pathways of flavonols including myricetin, kaempferol, and quercetin are shown.

[0014] FIG6 shows specific carbon sources, intermediates, and enzyme combinations for the production of flavonols including kaempferol, quercetin, and myricetin.

[0015] Figure 7 Data are provided for the bioproduction of quercetin using the methods of the invention. DETAILED DESCRIPTION

[0016] The present application provides engineered cells for producing one or more flavonoids, cultures comprising 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 variety of phytonutrients found in almost all fruits and vegetables. As used herein, the terms "flavonoid", "flavonoid product" or "flavonoid compound" are used interchangeably and refer to molecules with a general structure containing a 15-carbon skeleton consisting of two benzene rings (A and B) and a heterocycle. Flavonoids may include, but are not limited to, isoflavone types (e.g., genistein), flavonoid types (e.g., apigenin), flavonol types (e.g., kaempferol), flavanone types (e.g., naringenin), chalcone types (e.g., phloretin), anthocyanidin types (e.g., cyanidin), catechins, flavanones, and dihydroflavonols. The target flavonoid compounds include, but are not limited to, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphinidin, pentahedralidin, cyanidin, catechin, delphinidin, pelargonidin and kaempferol. Anthocyanins exist in the form of anthocyanidin glycosides and acylated anthocyanins. The target anthocyanin compounds include, but are not limited to, cyanidin glycosides, delphinidin glycosides, pelargonidin glycosides, peony pigment glycosides and morning glory pigment glycosides.

[0017] As used herein, the term "precursor" or "flavonoid precursor" may refer to any intermediate present in the biosynthetic pathway leading to the production of catechins or anthocyanins. Flavonoid precursors may include, but are not limited to, tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvic acid, acetyl-CoA, and naringenin.

[0018] Cells engineered for the production of flavonoids or anthocyanins may have one or more modifications including, but not limited to, down-regulation, disruption or deletion of endogenous genes, up-regulation of endogenous genes, and introduction of exogenous genes.

[0019] The term "non-naturally occurring" when applied to enzymes is intended to mean that a nucleic acid or polypeptide includes at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids and polypeptides may be referred to as "wild type" or "original". A host cell, organism, or microorganism that includes at least one genetic modification produced by human intervention may also be referred to as "non-naturally occurring," "engineered," "genetically engineered," or "recombinant."

[0020] Host cells, organisms or microorganisms engineered to express or overexpress genes or nucleic acid sequences or overexpress enzymes or polypeptides have been genetically engineered by recombinant DNA technology to include genes or nucleic acid sequences that do not naturally encode enzymes or polypeptides or to express endogenous genes at a level exceeding their expression level in unaltered cells. As a non-limiting example, host cells, organisms or microorganisms engineered to express or overexpress genes or nucleic acid sequences, or overexpress enzymes or polypeptides may have any modification of the coding sequence of the gene, the position of the gene on the chromosome, or the regulatory elements associated with the gene that affect the gene. Overexpression of a gene may also be achieved by increasing the copy number of the gene in a cell or organism. Similarly, host cells, organisms or microorganisms engineered to underexpress genes, nucleic acid sequences, or to have reduced expression of genes, nucleic acid sequences, or to underexpress enzymes or polypeptides may have any modification of the coding sequence of the gene, the position of the gene on the chromosome, or the regulatory elements associated with the gene that affect the gene. Specifically include gene disruption, which includes any insertion, deletion or sequence mutation that affects the activity of its expression or the encoded polypeptide of a gene or a part of a gene. Gene disruption includes a "knockout" mutation that eliminates gene expression. Modification of low-expression genes also includes modification of gene regulatory regions that can reduce their expression.

[0021] The term "exogenous" or "heterologous" is intended to mean that the reference molecule or reference activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introducing the encoding nucleic acid into the host genetic material, such as by integrating the encoding nucleic acid into the host chromosome or as non-chromosomal genetic material that can be introduced onto a vector such as a plasmid. Thus, the term "endogenous" means that the reference molecule or activity is naturally present in the host.

[0022] 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 transformation, transduction, and transfection. Optionally, for exogenous expression in Escherichia coli or other prokaryotic cells, some nucleic acid sequences in the gene or cDNA of the eukaryotic nucleic acid can encode targeting signals (e.g., N-terminal mitochondrial targeting signals or other targeting signals), which can be removed before being transformed into prokaryotic host cells if necessary. In addition, genes can be codon optimized using techniques well known in the art to achieve optimized expression of proteins.

[0023] When the sequence is aligned to obtain maximum homology, the identity percentage (identity %) between the two sequences is determined. Algorithms well known to those skilled in the art (such as Align, BLAST, Clustal Omega, etc.) are relatively and determine rough sequence similarity or identity, and determine the existence or significance of the gap that can be assigned weight or score in the sequence. Such algorithms are also known in the art, and are similarly applicable to determining nucleotide or amino acid sequence similarity or identity, and can be used to identify the ortholog of the target gene. The other sequence added to the polypeptide sequence, such as but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect identity %.

[0024] Homologs are one or more genes that have the same or equivalent function in different organisms. Orthologous genes can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, more preferably about 60% to 100% amino acid sequence identity. Genes can also be considered orthologs if they have three-dimensional structural similarity but not necessarily sequence similarity, or if there is sufficient amount to indicate that they evolved from a common ancestor to the extent that primary sequence similarity cannot be identified. Paralogs are genes that are related by duplication within a genome and can evolve new functions, even if these functions are related to the original function.

[0025] Provided herein are cells engineered for producing flavonoids, anthocyanins and other organic compounds, wherein these engineered cells include one or more genetic modifications, which increase the production of flavonoids by increasing the metabolic flux leading to flavonoid precursors and / or reducing the carbon loss caused by the production of by-products. As a non-limiting example, the genetic modification can be a modification for overexpressing or underexpressing one or more endogenous genes in an engineered host cell, or can be a modification for expressing one or more non-natural genes in an engineered host cell. Engineered cells as provided herein can include a variety of genetic modifications.

[0026] Also provided is a cell culture for producing one or more flavonoids or anthocyanins. The cell culture includes an engineered cell as disclosed herein in a culture medium, the culture medium includes a carbon source, which can also be an energy source, such as glycerol, sugar or organic acid. In various embodiments, the culture medium can include at least one feed molecule, such as but not limited to one or more organic acids or amino acids that can be converted into flavonoid precursors (such as tyrosine, p-coumaroyl-CoA or malonyl-CoA). Examples of feed molecules include but are not limited to acetic acid, malonic acid, tyrosine, phenylalanine, pantothenic acid, coumaric acid, etc. In some embodiments, the feed molecule can have a reduced or low purity. For example, glycerol as a feed molecule can be crude glycerol, including biomass containing glycerol, such as glycerol obtained as a by-product of biodiesel processing. Alternatively or in addition, the culture medium can include a supplementary compound, which can be a cofactor or a precursor of a cofactor used by an enzyme that works in the flavonoid pathway, such as bicarbonate, biotin, thiamine, pantothenic acid, α-ketoglutaric acid, ascorbic acid or 5-aminolevulinic acid.

[0027] Also provided are methods for producing flavonoids and anthocyanins, comprising culturing cells engineered to produce flavonoids or anthocyanins as provided herein under conditions where the cells produce flavonoids or anthocyanins. In some examples, these methods include culturing the engineered cells in a culture medium that includes at least one feed molecule or supplement, such as, but not limited to, tyrosine, phenylalanine, malonic acid, p-coumaric acid, bicarbonate, acetic acid, pantothenic acid, biotin, thiamine, α-ketoglutaric acid, ascorbic acid, and 5-aminolevulinic acid. These methods may further include recovering at least one of the flavonoids from the culture medium, the whole culture, or the cells.

[0028] In a first aspect, provided herein are cells engineered to produce one or more flavonoids or anthocyanins, wherein the cells include, in addition to nucleic acid sequences encoding 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, colorless anthocyanidin reductase activity and dihydroflavonol-4-reductase activity, one or more genetic modifications for improving the production of flavonoids or anthocyanins.As described herein, cells engineered to produce one or more flavonoids are engineered to include: an exogenous nucleic acid sequence encoding a tyrosine ammonia lyase activity that can use tyrosine as a substrate to form 4-coumaric acid (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 that can convert phenylalanine to trans-cinnamic acid, and an exogenous nucleic acid sequence encoding a cinnamate-4-hydroxylase activity that forms 4-coumaric acid from trans-cinnamic acid; an exogenous nucleic acid sequence encoding a CoA ligase activity that forms p-coumaroyl-CoA from coumaric acid nucleic acid sequence (e.g., 4-coumarate-CoA ligase, 4CL, EC:6.2.1.12); an exogenous nucleic acid sequence encoding a polyketide synthase activity that uses malonyl-CoA and p-coumaryl-CoA as substrates to form naringenin chalcone (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 through its cyclase activity (e.g., chalcone-flavanone isomerase, CHI, EC:5.5.1.6); an exogenous nucleic acid sequence encoding a flavanone that forms dihydrokaempferol from naringenin or a flavanone from eriodictyol An exogenous nucleic acid sequence encoding an alkane-3-hydroxylase activity (e.g., naringenin-3-dioxygenase, F3H, EC: 1.14.11.9); an exogenous nucleic acid sequence encoding a flavonoid 3'-hydroxylase or a flavonoid 3'5'-hydroxylase activity coupled to an exogenous nucleic acid sequence encoding a cytochrome P450 reductase activity to form taxifolin or dihydromyricetin from dihydrokaempferol or to form eriodictyol or pentahydroxyflavone from naringenin (e.g., flavonoid 3'-monooxygenase, F3'H, EC: 1.14.13.21, EC: 1.14.14.82; cytochrome P450 / NA DPH--P450 reductase, EC: 1.14.14.1; F3'5'H, EC: 1.14.14.81); an exogenous nucleic acid sequence encoding a dihydroflavonol-4-reductase activity that forms leucocyanidin from taxaflavin, leucodelphinidin from dihydromyricetin, or leucopelargonidin from dihydrokaempferol (e.g., dihydroflavonol-4-reductase, EC: 1.1.1); and an exogenous nucleic acid sequence encoding a leucoanthocyanidin reductase activity that forms catechins from leucocyanidin (e.g., leucoanthocyanidin reductase, LAR, EC: 1.17.1.3).Optionally, the cell engineered to produce anthocyanins is further engineered to include an exogenous nucleic acid sequence encoding an anthocyanin synthase activity that forms cyanidin from catechin or leucocyanidin, delphinidin from leucodelphinidin, or pelargonidin from leucopelargonidin (e.g., anthocyanin synthase, ANS, EC: 1.14.20.4), and an exogenous nucleic acid sequence encoding a glucosyltransferase activity that forms cyanidin-3-O-β-D-glucoside from cyanidin, delphinidin-3-O-β-D-glucoside from delphinidin, or pelargonidin-3-O-β-D-glucoside from pelargonidin (e.g., anthocyanin 3-O-glucosyltransferase, 3GT, EC: 2.4.1.115). The cells engineered to produce flavonoids or anthocyanins provided herein 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 reducing the formation of by-products.

[0029] The cell that is engineered to produce flavonoids is further engineered to increase the supply of precursor malonyl-CoA. A strategy to increase malonyl-CoA includes increasing acetyl-CoA carboxylase (ACC) activity. In various embodiments, the ACC enzyme is overexpressed in a host strain, and the ACC enzyme is a large single-chain polypeptide in most eukaryotic organisms (including fungi), and is a multi-subunit enzyme in plants and bacteria (such as Escherichia coli). The example of acetyl-CoA carboxylase that can be expressed in a host cell engineered to produce flavonoids or anthocyanins includes but is not limited to the ACC gene of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, Ustilago maydis, and the orthologs of these ACCs in other species with at least 50% amino acid identity with these ACCs.

[0030] Other strategies to increase malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). In some embodiments, an acetyl-CoA synthase (ACS) that converts acetate and CoA into acetyl-CoA is overexpressed in a host cell. A culture of an engineered host cell comprising an overexpressed nucleic acid sequence encoding an ACS may optionally include acetic acid in the culture medium. Examples of acetyl-CoA synthases that can be expressed in a host cell engineered to produce flavonoids or anthocyanins include, but are not limited to, ACS genes of Escherichia coli, ACS of Salmonella typhimurium, and orthologs of these ACS in other species with at least 50% amino acid identity to these ACS.

[0031] In other embodiments, engineered host cells that overexpress a gene encoding pyruvate dehydrogenase (PDH) that converts pyruvate into acetyl-CoA are also contemplated. In addition, in E. coli, variants of the Lpd subunit of PDH can be expressed that include a mutation (E354K) that reduces the inhibition of PDH by NADH.

[0032] Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that are not dependent on ACC activity can be adopted. In some embodiments, cells engineered to produce flavonoids or anthocyanins are further engineered to increase the malonyl-CoA supply of cells, and the cells include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase that produces malonyl-CoA from malonate. Examples of malonyl-CoA synthetase include malonyl-CoA synthetase of Streptomyces coelicolor, Rhodopseudomonas palustris, or malonyl-CoA synthetase with at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetase. Malonic acid can optionally be added to a culture medium comprising a culture of cells engineered to express malonyl-CoA synthetase. The engineered cells comprising an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as the malonate transporter encoded by the matC gene of, for example, Streptomyces coelicolor, or the malonate transporter encoded by DctPQM of Sinorhizobium medicae.

[0033] In additional embodiments, the cells engineered to produce flavonoids or anthocyanins are further engineered to include an exogenous nucleic acid sequence encoding a malonate CoA transferase that prepares malonyl-CoA by directly transferring CoA from acetyl-CoA. Examples of malonate CoA transferases that can be expressed in engineered cells as provided herein include, but are not limited to, the alpha subunit (mdcA) of malonate decarboxylase from Acinetobacter calcoaceticus, Geobacillus sp, or a transferase with at least 50% identity to any of these or other naturally occurring malonate CoA transferases.

[0034] 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 the production of acetyl-CoA, malonyl-CoA and / or p-coumaroyl-CoA. Strategies to increase the supply of CoA include upregulating endogenous pantothenate kinase (PanK) (EC: 2.7.1.33) that produces CoA from pantothenate. Alternatively or in addition, the host cell can be engineered to include a nucleic acid sequence encoding a type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as the CoaX gene (EC: 2.7.1.33) of Pseudomonas aeruginosa. In some embodiments, the culture of the cells engineered to produce flavonoids or anthocyanins can include a culture medium that includes pantothenate (a precursor for CoA biosynthesis) and can optionally also include cysteine ​​for CoA biosynthesis.

[0035] Other strategies that increase the malonyl-CoA flux leading to flavonoid pathway include sudden change or down-regulation of one or more genes that work in fatty acid biosynthesis. Without limiting the embodiment to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply that can be used for flavonoid biosynthesis. In some embodiments, gene β-ketoacyl-ACP synthase II (Escherichia coli fabF) can be destroyed to reduce fatty acid biosynthesis. Another example of the fatty acid biosynthetic gene of the host cell that can be suddenly changed or down-regulated is the gene of encoding malonyl-CoA-ACP transacylase (Escherichia coli fabD). Other fatty acid biosynthetic genes of the through engineering host cell that can be down-regulated include β-ketoacyl-ACP synthase I enzyme (Escherichia coli fabB) and acyl carrier protein (Escherichia coli acpP).

[0036] Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption or deletion of genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetylphosphotransferase and acetate kinase. In an E. coli host cell, downregulated, disrupted or deleted genes may include aldehyde-alcohol dehydrogenase (adhE), lactate dehydrogenase (ldhA), pyruvate oxidase (poxB) and the enzyme acetate kinase phosphate acetyltransferase (ackA-pta).

[0037] In addition, cells engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted or deleted to prevent hydrolysis of the precursors malonyl-CoA, acetyl-CoA and / or p-coumaryl-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.

[0038] Alternatively or additionally, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or down-regulated to increase the supply of α-ketoglutarate, which serves as a cofactor for one or more of the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be down-regulated include citrate synthase, which converts acetyl-CoA into citric acid.

[0039] In other embodiments, engineered host cells for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of the amino acid tyrosine are also contemplated. Tyrosine is one of the precursors of flavonoid biosynthesis, and its conversion into coumaric acid is the first step of the pathway. L-Tyrosine is one of the three aromatic amino acids derived from the shikimic acid pathway. The initial step of the shikimic acid pathway is catalyzed by the DAHP synthase isozyme and regulated by feedback inhibition. The strategy for increasing tyrosine production may include, but is not limited to, transcriptional dysregulation, removal of feedback inhibition, overexpression of the rate-limiting enzyme, and / or the absence of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host, the tyrR gene may be destroyed, feedback inhibition-resistant forms of DAHP synthase (aroG) and chorismate mutase (tyrA) may be introduced, and / or the rate-limiting enzyme shikimate kinase (aroK or aroL) and quinic acid (QUIN) / shikimate dehydrogenase (ydiB) may be overexpressed. In addition, phosphoenolpyruvate synthase (ppsA) and transketolase (tktA) can be exogenously introduced to enhance tyrosine production.

[0040] In other embodiments, it is also contemplated that an engineered host cell for producing flavonoids or anthocyanins is further engineered to upregulate the endogenous biosynthesis of the cofactor heme. Cytochrome P450 (CYP) is one of the exogenous genes in the engineered cells provided herein, containing heme as a cofactor. Improving the heme supply may be an effective strategy to increase the biosynthesis of flavonoids. 5-aminolevulinic acid (ALA) is the first precursor of the heme pathway. The strategy to increase the heme supply includes the overexpression of the gene of the synthetic precursor ALA. In the Escherichia coli host, ALA is formed by the 5-carbon skeleton of glutamic acid (C5 pathway). The three enzymes involved in the biosynthesis of ALA are glutamyl-tRNA synthetase (gltX), glutamyl-tRNA reductase (hemA) and glutamate-1-semialdehyde aminotransferase (hemL). In the E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and / or HemL to increase the production of heme precursor ALA. Non-limiting examples of hemA genes that can be overexpressed include mutant hemA (two lysine residues are inserted 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 (ALS is synthesized by the condensation of glycine and succinyl-CoA). Non-limiting examples of heterologous ALAS that can be expressed in E. coli include Bradyrhizobium japonicum (Bradyrhizobium japonicum) (EC: 2.3.1.37), ALAS from Rhodobacter capsulatus, or an ALAS having at least 50% sequence identity to a naturally occurring ALAS. In addition, one or more of the downstream genes that catalyze the synthesis of heme from ALA (e.g., in E. coli, hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) can be overexpressed to drive flux from ALA to heme production. In some embodiments, a culture of cells engineered for the production of flavonoids or anthocyanins can include a culture medium that includes succinate and / or glycine, which are precursors for the biosynthesis of heme via the C4 pathway.

[0041] On the other hand, provided herein is a cell culture comprising an engineered cell as provided herein in a culture medium, wherein the culture medium comprises a carbon source that is also an energy source for the cell, wherein as a non-limiting example, the carbon source can be, for example, glycerol, a sugar, or an organic acid. The culture medium can further comprise a feed molecule for producing flavonoids or anthocyanins. The feed molecule can be, for example, acetic acid, malonic acid, tyrosine, pantothenic acid, coumaric acid, biotin, α-ketoglutaric acid, ascorbic acid, 5-aminolevulinic acid, succinic acid, or glycine. In some embodiments, the culture comprises a culture medium comprising a carbon source and at least one supplement, and the at least one supplement is a cofactor of an enzyme or a precursor of an enzyme cofactor.

[0042] In yet another aspect, methods for producing flavonoids and anthocyanins include incubating a culture of an engineered host cell as provided herein to produce flavonoids or anthocyanins. These methods may further include recovering at least one flavonoid from the cells, the culture medium, or the entire culture.

[0043] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications that cause the engineered host cell to produce flavonoids or anthocyanins from a carbon source, which may also be an energy source, through a variety of chemical intermediates. In certain embodiments, flavonoids or anthocyanins are produced from glycerol by enzymatic conversion. In certain embodiments, flavonoids or anthocyanins are produced from a carbon source that may also be an energy source by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a culture medium comprising a molecule selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications result in an increase in metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications result in a reduction in by-product formation. In certain embodiments, the one or more genetic modifications are selected from: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications for expressing one or more non-native genes in the engineered host cell; and (iv) combinations thereof. In certain embodiments, the engineered host cell is cultured in a culture medium comprising a molecule selected from the group consisting of tyrosine, phenylalanine, malonic acid, p-coumaric acid, bicarbonate, acetic acid, pantothenic acid, biotin, thiamine, α-ketoglutarate, ascorbic acid, and 5-aminolevulinic acid, wherein one or more of the selected molecules are chemical intermediates, including molecules or cofactors in a biosynthetic pathway. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the following: (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-coumarate-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 the following: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprising a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is Escherichia coli. In certain embodiments, one or more genetic modifications reduce fatty acid biosynthesis.In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 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 an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumaric acid-CoA (v) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof. In certain embodiments, the flavonoid is a catechin.

[0044] In yet another aspect, the present invention provides a method for increasing the production of flavonoids or anthocyanins, the method comprising: providing an engineered host cell comprising one or more genetic modifications, the one or more genetic modifications causing the engineered host cell to produce flavonoids or anthocyanins from a carbon source through a variety of chemical intermediates, the carbon source may also be an energy source. In certain embodiments, flavonoids or anthocyanins are produced from a carbon source that may also be an energy source by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a culture medium comprising a molecule selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the one or more genetic modifications result in an increase in metabolic flux to a flavonoid precursor or cofactor. In certain embodiments, the one or more genetic modifications cause an increase in metabolic flux to a flavonoid precursor. In certain embodiments, one or more genetic modifications result in a reduction in byproduct formation. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for overexpressing one or more endogenous genes in engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in engineered host cells; and (iv) combinations thereof. In certain embodiments, engineered host cells are cultured in a culture medium comprising a molecule selected from the following: tyrosine, phenylalanine, malonic acid, p-coumaric acid, bicarbonate, acetic acid, pantothenic acid, biotin, thiamine, α-ketoglutaric acid, ascorbic acid, and 5-aminolevulinic acid, wherein one or more of the selected molecules are chemical intermediates, including molecules or cofactors in a biosynthetic pathway. In certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the following: (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 comprises at least one or more peptides selected from: (i) chalcone isomerase; (ii) chalcone synthase; (iii) a fusion protein comprising a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, the engineered cell is an Escherichia coli. In certain embodiments, one or more genetic modifications reduce fatty acid biosynthesis.In certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 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 an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumaric acid-CoA (v) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof. In certain embodiments, the flavonoid is a catechin.

[0045] In yet another aspect, the present invention provides a variety of engineered host cells, wherein each of the plurality of engineered host cells comprises one or more genetic modifications, which result in the production of flavonoids or anthocyanins from a carbon source, which may also be an energy source, through a variety of chemical intermediates. In certain embodiments, flavonoids or anthocyanins are produced from a carbon source that may also be an energy source by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cell is cultured in a culture medium comprising a molecule selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications result in an increase in metabolic flux to a flavonoid precursor or cofactor. In certain embodiments, one or more genetic modifications result in an increase in metabolic flux to a flavonoid precursor or cofactor. In certain embodiments, one or more genetic modifications result in a reduction in byproduct formation. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for overexpressing one or more endogenous genes in engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in engineered host cells; and (iv) combinations thereof. In certain embodiments, at least one of the engineered cells from the various engineered host cells is cultured in a culture medium comprising a molecule selected from the group consisting of tyrosine, phenylalanine, malonic acid, p-coumaric acid, bicarbonate, acetic acid, pantothenic acid, biotin, thiamine, α-ketoglutaric acid, ascorbic acid, and 5-aminolevulinic acid, wherein one or more of the selected molecules are chemical intermediates, including molecules or cofactors in a biosynthetic pathway. In certain embodiments, at least one of the engineered cells from the plurality of 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. 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 the group consisting of: (i) a chalcone isomerase; (ii) a chalcone synthase; (iii) a fusion protein comprising a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof. In certain embodiments, at least one of the engineered host cells is an Escherichia coli.In certain embodiments, 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 the following: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine into trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 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 an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumaric acid-CoA (v) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof. In certain embodiments, the flavonoid is a catechin.

[0046] In another aspect, the present invention provides a method for increasing the production of flavonoids or anthocyanins, the method comprising: providing a plurality of engineered host cells, wherein each of the plurality of engineered host cells comprises one or more genetic modifications, the one or more genetic modifications causing the engineered host cells to produce flavonoids or anthocyanins from a carbon source through a plurality of chemical intermediates, the carbon source may also be an energy source. In certain embodiments, flavonoids or anthocyanins are produced from a carbon source that may also be an energy source by enzymatic conversion. In certain embodiments, the carbon source is selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, the engineered host cells are cultured in a culture medium comprising a molecule selected from the group consisting of: (i) glycerol, (ii) sugar, (iii) organic acid, (iv) amino acid, (v) biomass comprising glycerol; and (vi) any combination thereof. In certain embodiments, one or more genetic modifications result in an increase in metabolic flux to flavonoid precursors or cofactors. In certain embodiments, one or more genetic modifications result in an increase in metabolic flux to a flavonoid precursor or cofactor. In certain embodiments, one or more genetic modifications result in a reduction in byproduct formation. In certain embodiments, one or more genetic modifications are selected from: (i) one or more modifications for overexpressing one or more endogenous genes in an engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in an engineered host cell; (iii) one or more genetic modifications for expressing one or more non-natural genes in an engineered host cell; and (iv) a combination thereof. In certain embodiments, at least one of the engineered cells from the various engineered host cells is cultured in a culture medium comprising a molecule selected from the group consisting of: tyrosine, phenylalanine, malonic acid, p-coumaric acid, bicarbonate, acetic acid, pantothenic acid, biotin, thiamine, α-ketoglutaric acid, ascorbic acid, and 5-aminolevulinic acid, wherein one or more of the selected molecules are chemical intermediates, including molecules or cofactors in a biosynthetic pathway. In certain embodiments, at least one of the engineered cells from the plurality of 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. 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 the group consisting of: (i) a chalcone isomerase; (ii) a chalcone synthase; (iii) a fusion protein comprising a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.In certain embodiments, at least one engineered host cell is Escherichia coli. In certain embodiments, one or more genetic modifications reduce fatty acid biosynthesis. In certain embodiments, at least one of the engineered host cells from the multiple engineered host cells comprises an exogenous nucleic acid sequence selected from the following: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine into trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamic acid-4-hydroxylase, wherein the cinnamic acid-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 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 an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine to trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumaric acid-CoA (v) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof. In certain embodiments, the flavonoid is a catechin.

[0047] On the other hand, 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 into malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from the following: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is Escherichia coli. In certain embodiments, the Escherichia coli cell further comprises a gene from a fungus. In certain embodiments, acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces stargensis, Ustilago maydis, and an ortholog of an acetyl-CoA carboxylase with at least 50% amino acid identity with the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modifications are the deletion or weakening of one or more fat biosynthetic genes that cause a reduction in fatty acid biosynthesis. In certain embodiments, one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from the group consisting of: an acetyl-CoA synthase gene of Escherichia coli, an acetyl-CoA synthase gene of Salmonella typhimurium, and an ortholog of an acetyl-CoA synthase gene in any other species having at least 50% amino acid identity with the acetyl-CoA synthase gene of Escherichia coli and Salmonella typhimurium. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from the malonyl-CoA synthetase of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications that reduce fatty acid biosynthesis are selected from: (i) mutation or down-regulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene β-ketoacyl-ACP synthase II (E. coli fabF); (iii) down-regulation of β-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) down-regulation of acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from the group consisting of: (i) an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide shown 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 shown in SEQ ID NO: 19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide shown 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; 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.

[0048] On the other hand, the present invention provides a method for increasing the production of flavonoids, comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and / or availability of malonyl-CoA. In certain embodiments, the production and / or availability of malonyl-CoA is increased by converting acetyl-CoA into malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from the following: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is Escherichia coli. In certain embodiments, the Escherichia coli cell further comprises a gene from a fungus. In certain embodiments, acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces stargensis, Ustilago maydis, and an ortholog of an acetyl-CoA carboxylase having at least 50% amino acid identity with the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modifications are the deletion or weakening of one or more fat biosynthetic genes that cause a reduction in fatty acid biosynthesis. In certain embodiments, one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS). In certain embodiments, acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of Escherichia coli, acetyl-CoA synthase gene of Salmonella typhimurium, and acetyl-CoA synthase gene of any other species with at least 50% amino acid identity with acetyl-CoA synthase gene of Escherichia coli and Salmonella typhimurium. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), wherein PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from the malonyl-CoA synthetase of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications that reduce fatty acid biosynthesis are selected from: (i) mutation or down-regulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene β-ketoacyl-ACP synthase II (E. coli fabF); (iii) down-regulation of β-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) down-regulation of acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from the group consisting of: (i) an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide shown 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 shown in SEQ ID NO: 19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide shown 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; 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.

[0049] On the other hand, the invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprise the upregulation of 3-deoxy-D-arabino-heptulose synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combination thereof. In certain embodiments, one or more genetic modifications comprise the downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprise the expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprise the expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprise the destruction of 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 2-dehydro-3-deoxyheptonic acid phosphoaldolase; (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.

[0050] On the other hand, 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 to increase the endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprise the upregulation of 3-deoxy-D-arabino-heptulose synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combination thereof. In certain embodiments, one or more genetic modifications comprise the downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprise the expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprise the expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprise the destruction of 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 2-dehydro-3-deoxyheptonic acid phosphoaldolase; (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.

[0051] In another aspect, the present invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the conversion of cyanidin 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) anthocyanin synthase of Carica papaya (SEQ.ID NO: 13); (ii) 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) anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ.ID NO: 13; and (iv) any combination thereof. In certain embodiments, 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 in 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 comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise 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.

[0052] 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 to increase the conversion of cyanidin 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) anthocyanin synthase of papaya (SEQ.ID NO: 13); (ii) 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) anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ.ID NO: 13; and (iv) any combination thereof. In certain embodiments, 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 in Vitis vinifera (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 comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise 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.

[0053] In another aspect, the present invention provides a method for increasing the conversion of cyanidin or catechin to cyanidin-3-glucoside (Cy3G), delphinidin or gallocatechin to delphinidin-3-glucoside (De3G), or avercatechin or pelargonidin to pelargonidin-3-glucoside (Pe3G), comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO: 13); (ii) 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) 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 comprise 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.

[0054] In another aspect, the present invention provides a method for increasing the conversion of cyanidin to cyanidin-3-glucoside (Cy3G), delphinidin to delphinidin-3-glucoside (De3G) or pelargonidin to pelargonidin-3-glucoside (Pe3G), comprising a flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyltransferase is selected from: (i) a flavonoid-3-glucosyltransferase in Vitis vinifera (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.

[0055] In another aspect, the present invention provides an engineered host cell comprising one or more genetic modifications to increase the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or pentahedralone (PHF), wherein the engineered host cell comprises 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 pentahedralone (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 a flavonoid 3', 5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused to cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) is fused to cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.9. In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to 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 that is at least 80% identical to the polypeptide shown 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.

[0056] In another aspect, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or quinahydroxyflavone (PHF), comprising an engineered host cell, wherein the engineered host cell comprises 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 quinahydroxyflavone (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 a flavonoid 3', 5'-hydroxylase (F3'5'H). In certain embodiments, the engineered host cell produces pentahydroxyflavone or dihydromyricetin. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is truncated to remove the N-terminal leader sequence. In certain embodiments, cytochrome P450 reductase (CPR) is truncated to remove the N-terminal leader sequence. In certain embodiments, flavonoid 3'-hydroxylase (F3'H) is fused to cytochrome P450 reductase (CPR). In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) is fused to cytochrome P450 reductase (CPR). In certain embodiments, flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.9. In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to 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 that is at least 80% identical to the polypeptide shown 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.

[0057] Engineered cells for producing flavonoids include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase (TAL) activity (alternatively or additionally, an exogenous nucleic acid encoding phenylalanine ammonia lyase (PAL) activity and an exogenous nucleic acid encoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding chalcone isomerase (CHI) activity. Optionally, the engineered cell may further comprise an exogenous nucleic acid sequence encoding a flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding a flavonoid 3'-hydroxylase (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 colorless anthocyanidin reductase (LAR) activity.

[0058] 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 produce p-coumaric acid. An exemplary tyrosine ammonia lyase is Saccharothrixespanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). It is also contemplated that TALs with SEQ ID NOs: 23-26, TALs listed in Table 1, TALs with 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 with TAL homologs and variants having tyrosine ammonia lyase activity that produces p-coumaric acid from tyrosine are used in the engineered cells provided herein.

[0059] Table 1. Tyrosine ammonia lyase

[0060] organism GenBank Accession Number Rhodotorula glutini AGZ04575.1 Flavobacterium johnsoniae WP_012023194.1 Herpetosiphon aurantiacus ABX02653.1 Rhodobacter capsulatus ADE83766.1 Saccharothrix hispanica AKE50820.1 Trichosporon cutaneum AKE50834.1

[0061] Similar to tyrosine ammonia lyase, phenylalanine ammonia lyase (PAL) can be a member of the aromatic amino acid deaminase family, which catalyzes the non-oxidative 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). It is also contemplated that PALs with SEQ ID NOs: 27-29, PALs with 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 with phenylalanine ammonia lyase activity producing trans-cinnamic acid from phenylalanine are used in the engineered cells provided herein.

[0062] 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 sunflower (Helianthus annuus L.) (C4H; SEQ ID NO: 3), C4H with SEQ ID NOs: 30-32 and C4H homologs of other species, and variants of naturally occurring C4H with C4H activity 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 with SEQ ID NO: 3 (C4H, sunflower) are contemplated for use in the engineered cells provided herein.

[0063] 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 with SEQ ID NO: 33-36 and 4CL homologs from other species, and variants of naturally occurring 4CL with 4CL activity 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 with SEQ ID NO: 4 (4CL, parsley) are contemplated for use in the engineered cells provided herein.

[0064] Table 2. 4-Coumarate-CoA ligase

[0065] organism GenBank Accession Number Parsley CAA31697.1 Tea Tree (Camellia sinensis) ASU87409.1 Chili pepper (Capsicum annuum) KAF3620173.1 Chestnut (Castanea mollissima) KAF3954751.1 Carrot (Daucus carota) AIT52344.1 Gynura bicolor BAJ17664.1 Ipomoea purpurea AHJ60263.1 Honeysuckle (Lonicera japonica) AGE10594.1 Wolfberry (Lycium chinense) QDL52638.1 Lotus (Nelumbo nucifera) XP_010265453.1 Blueberry Tree (Nyssa sinensis) KAA8540582.1 tomato NP_001333770.1 Striga asiatica GER48539.1

[0066] Chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three malonyl-CoA molecules with one p-coumaroyl-CoA molecule to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase (CHS, SEQ ID NO: 5) of Petunia x hybrida. It is also contemplated that the genes listed in Table 3, CHS with SEQ ID: 37-40, and CHS homologs and variants with SEQ ID NO: 5 (CHS, Petunia x hybrida) 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 with chalcone synthase activity are used in the engineered cells provided herein.

[0067] Table 3. Chalcone synthase

[0068]

[0069]

[0070] Chalcone isomerase (CHI, also known as chalcone flavonoid isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone to its corresponding (2S)-flavanone. CHI of Medicago sativa (SEQ ID NO: 6), CHI of Table 4, CHI with SEQ ID NO: 41-44 and CHI homologs of other species, and variants of naturally occurring CHI with chalcone isomerase activity 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 with SEQ ID NO: 6 (CHI, Medicago sativa) are contemplated for use in the engineered cells provided herein.

[0071] Table 4. Chalcone isomerase

[0072]

[0073]

[0074] In some embodiments, a nucleic acid sequence encoding CHI can be fused with a nucleic acid sequence encoding CHS in an engineered cell as provided herein, such that CHI activity is fused with chalcone synthase activity, i.e., a fusion protein having both condensation and cyclization activities is produced in the engineered cell.

[0075] Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substrates include (2S)-eriodictyol, (2S)-dihydroquercetin, and (2S)-pinoretin. Some F3H enzymes are bifunctional, also catalyzing as flavonol synthases (EC: 1.14.20.6). F3H of Rubus occidentalis (SEQ ID NO: 7), F3H having SEQ ID NOs: 45-48, F3H listed in Table 5, and other F3H homologs and variants having F3H activity 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 with SEQ ID NO: 7 (F3H, Rubus occidentalis) are contemplated for use in the engineered cells provided herein.

[0076] Table 5. Flavanone 3-hydroxylase

[0077]

[0078]

[0079] Flavonoid 3'-hydroxylase (F3'H) belongs to the cytochrome P450 family and its systematic name is flavonoid, NADPH: oxygen oxidoreductase (3'-hydroxylating). In the flavonoid biosynthetic pathway, F3'H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. In cells engineered for enhanced quercetin production, flavonoid 3'-hydroxylase (F3'H) can be used to convert kaempferol to quercetin. F3'H of Brassica napus (F3'H; SEQ ID NO: 8), F3'H with SEQ ID NO: 49-52, those listed in Table 6, and homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid identity with these F3'H are contemplated for use in the engineered cells provided herein. F3'H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. Cytochrome P450 reductase is a diflavin oxidoreductase that donates electrons to F3'H. The P450 reductase can be from the same species as F3'H or from a different species than F3'H. The CPR of Catharanthus roseus (SEQ ID NO: 9), other CPRs listed in Table 7, CPRs having SEQ ID NOs: 53-55, CPR homologs of other species, and variants of naturally occurring CPRs having CPR activity 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 CPRs are contemplated for use in the engineered cells provided herein. In various embodiments, the N-terminal nucleic acid sequence in the F3'H and / or CPR genes derived from eukaryotic cells can encode a targeting leader peptide that can be removed prior to introduction into a prokaryotic host cell if desired. In some embodiments, the hydroxylase complex HpaBC from E. coli is used to hydroxylate naringenin to eriodictyol or to hydroxylate dihydrokaempferol to dihydroquercetin (taxifolin).

[0080] Table 6. Flavonoid 3'-hydroxylase

[0081]

[0082]

[0083] Table 7. Cytochrome P450 Reductase

[0084] organism GenBank Accession Number Catharanthus roseus CAA49446.1 Brassica napus XP_013706600.1 pitcherwort GAV59576.1 tea tree XP_028084858.1

[0085] In some embodiments, a nucleic acid sequence encoding F3'H can be fused to a nucleic acid sequence encoding a CPR in an engineered cell as provided herein, such that F3'H activity is fused to CPR activity.

[0086] In cells engineered to produce dihydromyricetin, flavonoid 3',5'-hydroxylase (F3'5'H) can be used to convert dihydrokaempferol to dihydromyricetin or to convert naringenin to pentahydroxyflavone, which is further converted to dihydromyricetin by F3H. F3'5'H, whose systematic name is flavanone, NADPH:oxygen oxidoreductase, catalyzes the formation of 3',5'-dihydroxyflavanone from flavanone.

[0087] In cells engineered to enhance myricetin production, flavonoid 3', 5'-hydroxylase (F3'5'H) can be used to convert kaempferol and quercetin into myricetin. An exemplary F3'5'H is Delphinium grandiflorum F3'5'H (SEQ ID NO: 10). It is also contemplated that F3'5'H with SEQ ID NO: 56-57, F3'5'H homologs of other species, and naturally occurring F3'5'H variants with F3'5'H activity 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 with SEQ ID NO: 10 are used in the engineered cells provided herein.

[0088] Flavonol synthase (FLS) catalyzes the oxidation of dihydroflavonols to generate flavonols. In cells engineered to enhance the production of myricetin, kaempferol and / or quercetin, flavonol synthase (FLS) can be used to catalyze the conversion of dihydromyricetin (DHM) to myricetin, dihydrokaempferol (DHK) to kaempferol, and dihydroquercetin (DHQ) to quercetin. An exemplary flavonol synthase (FLS) is FLS from parsley (EC: 1.14.11.23). FLS of parsley (SEQ ID NO:99), as well as other FLS homologs and variants having FLS activity having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% amino acid identity to SEQ ID NOs:99-122 are contemplated for use in the engineered cells provided herein.

[0089] Dihydroflavonol 4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (taxifolin, DHQ) or dihydromyricetin (DHM), reducing these compounds to the corresponding cis-flavan-3,4-diols (DHK is reduced to leucopelargonidin; taxifolin is reduced to leucocyanidin; DHM is reduced to leucodelphinidin). An exemplary DFR is Anthurium andraeanum DFR (SEQ ID NO: 11). Also contemplated for use in the engineered cells provided herein are the DFRs in Table 8, DFRs having SEQ ID NOs: 58-61, DFR homologs from other species, and 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.

[0090] Table 8. Dihydroflavonol 4-reductase

[0091] organism GenBank Accession Number Eustoma grandiflorum BAD34461.1 Anthurium AAP20866.1 tea tree AAT66505.1 Bayberry KAB1203810.1 Dendrobium moniliforme AEB96144.1 Strawberry (Fragaria x ananassa) AHL46451.1 Rose XP_024167119.1 Acer palmatum AWN08247.1 Blueberry Tree KAA8531902.1 mountain grape I82380.1 Acacia XP_027329642.1 Angelonia angustifolia AHM27144.1 Pyrus pyrifolia Q84KP0.1 cocoa XP_017985307 cocoa XP_007051597.2 Cabbage (Brassica oleracea var. capitata) QKO29328.1 Raspberry (Rubus idaeus) AXK92786.1 Sweet Orange (Citrus sinensis) AAY87035.1 Gerbera hybrida P51105.1 pitcherwort GAV76940.1 Ginkgo biloba AGR34043.1 Dryopteris erythrosora QFQ61498.1 Dryopteris erythrina QFQ61499.1 pitcherwort GAV76942.1

[0092] Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechins from 3,4-cis leucocyanidin. LAR also synthesizes affcatechin and gallatechin. LAR (SEQ ID NO: 12) of Desmodium uncinatum, LAR with SEQ ID NO: 62-65 and LAR homologs of other species, and variants of naturally occurring LAR with LAR activity 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 with SEQ ID NO: 12 (LAR, Desmodium uncinatum) are contemplated for use in the engineered cells provided herein.

[0093] Optionally, the cell is further engineered to include anthocyanin synthase (ANS), which catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin or leucodelphinidin to delphinidin. ANS of papaya (SEQ ID NO: 13), ANS with SEQ ID NOs: 66-69 and ANS homologs of other species, and naturally occurring ANS variants having ANS activity 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 with SEQ ID NO: 13 (ANS, papaya) are contemplated for use in the engineered cells provided herein.

[0094] Optionally, the cell is further engineered to include a flavonoid-3-glucosyltransferase (3GT) to produce anthocyanins by transferring a sugar moiety (e.g., but not limited to, UDP-α-D-glucose) to anthocyanidins to form glycosylated anthocyanidins. 3GT of Vitis vinifera (SEQ ID NO: 14), 3GTs having SEQ ID NOs: 70-73 and 3GT homologs from other species, and naturally occurring 3GT variants having 3GT activity 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 vinifera) are contemplated for use in the engineered cells provided herein.

[0095] In various aspects, host cells can be engineered to enhance the production of flavonoids or anthocyanins by introducing additional exogenous pathways and / or modifying endogenous metabolic pathways to remove or downregulate competing pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving the production of flavonoids or anthocyanins can be to increase yield, titer, or production rate.

[0096] Thus, the host cell engineered for the production of flavonoids or anthocyanins can be engineered to include any one or any combination of the following: overexpression of acetyl-CoA carboxylase (ACC) or ACC variants; expression or overexpression of at least one enzyme that is independent of the ACC step for increasing the cellular malonyl-CoA supply; expression or overexpression of at least one enzyme to increase tyrosine supply; expression or overexpression of at least one enzyme to increase CoA availability for the synthesis of precursor malonyl-CoA or p-coumaryl-CoA; expression or overexpression of at least one enzyme to increase heme biosynthesis; deletion or downregulation of at least one fatty acid synthase; 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 foregoing list of modifications is non-limiting.

[0097] Malonyl-CoA is the direct precursor of chalcone synthase and p-coumaryl-CoA for sequential condensation. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation of acetyl-CoA in a multistep reaction. First, the biotin carboxylase domain uses bicarbonate as CO2 donor to catalyze the ATP-dependent carboxylation of biotin. In the second reaction, the carboxyl group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotic organisms including fungi, both reactions are catalyzed by large single-chain proteins, but in Escherichia coli and other bacteria, activity is catalyzed by multi-subunit enzymes. Host cells can be engineered to, for example, express exogenous acetyl-CoA carboxylase or variant ACC to increase the synthesis of malonyl-CoA by acetyl-CoA. For example, Mucor circinelloides (SEQ ID NO:15) acetyl-CoA carboxylase can be introduced into host cells. Other examples of ACC genes that can be used in engineered cells provided herein include, but are not limited to, genes listed in Table 9, genes with SEQ ID NO: 74-76, naturally occurring orthologs of these ACC, or variants with 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 the reference gene. In addition, 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.

[0098] Table 9. Acetyl-CoA carboxylase

[0099]

[0100] Other strategies to increase malonyl-CoA include increasing acetyl-CoA, which is converted into malonyl-CoA by acetyl-CoA carboxylase (ACC). Acetyl-CoA can be synthesized by acetic acid by acyl-CoA ligase in ATP-dependent reactions. The formation of new chemical bonds between acetyl-CoA synthetase (ACS) or acetic acid-CoA ligase (EC 6.2.1.1.) catalyzes acetic acid and CoA coenzyme A (CoA). When acetic acid is supplied to cells as a co-supplement or acetic acid is produced as a byproduct, the ACS with natural activity to acetic acid will provide the function of increasing the supply of acetyl-CoA. Other acyl-CoA ligases with its main activity to other acid substrates can also have significant activity to acetic acid, and are feasible candidates for providing acetic acid-CoA ligase activity in the engineered cells provided herein. The ACS expressed in the host cell can be prokaryotic or eukaryotic. The culture of the engineered host cell expressing the nucleic acid sequence encoding ACS can optionally include acetic acid in the culture medium. Examples of acetyl-CoA synthases that can be expressed in a host cell engineered to produce flavonoids or anthocyanins include, but are not limited to, the ACS genes of Escherichia 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.

[0101] Alternatively or in addition, the engineered host cell can overexpress a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate into acetyl-CoA to increase the supply of acetyl-CoA. PDH catalyzes an irreversible metabolic step, and the control of its activity is complex, involving the control of its substrates and products. Nicotinamide adenine dinucleotide hydrogen (NADH), the product of the PDH reaction, is a competitive inhibitor of the PDH complex. The NADH sensitivity of the PDH complex has been shown to exist in LPD, which is an enzyme that interacts with NAD+ as a substrate. Therefore, variants of the Lpd subunit of PDH can be expressed, which include one or more mutations that reduce the inhibition of PDH by NADH. Such an example is an LPD variant containing an E354K mutation in Escherichia coli, and the mutated enzyme is less sensitive to NADH inhibition than native LPD.

[0102] Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that are not dependent on ACC activity can be adopted. For example, the cells engineered to produce flavonoids or anthocyanins as provided herein can include an exogenous nucleic acid sequence encoding malonyl-CoA synthetase (EC 6.2.1.14), which produces malonyl-CoA from malonic acid. Acyl-CoA synthetase catalyzes carboxylic acid to convert into its acyl-CoA thioester by an ATP-dependent two-step reaction. In the first step, free fatty acids are converted into acyl-AMP intermediates, releasing pyrophosphate. In the second step, activated acyl is coupled to the thiol group of CoA, releasing AMP and acyl-CoA products. Non-limiting examples of malonyl-CoA synthetase include malonyl-CoA synthetase of Streptomyces coelicolor (SEQ ID NO: 17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the reference sequence. Malonic acid may optionally be added to the culture medium comprising a culture of cells engineered to express malonyl-CoA synthetase. In Rhizobium trifolii, the matB gene is part of the matABC operon, matA encodes a malonyl-CoA decarboxylase and matC encodes a putative dicarboxylate carrier protein or malonate transporter.The engineered cells comprising an exogenous gene encoding a malonyl-CoA synthetase may also comprise an exogenous nucleic acid sequence encoding a malonate transporter, such as from, for example, Streptomyces coelicolor (SEQ ID NO: 18), Rhizobiales bacterium (SEQ ID NO: 80), Rhizobiales bacterium (SEQ ID NO: 81), Agrobacterium vitis (SEQ ID NO: 82), Neorhizobium sp. (SEQ ID NO: 83), NO:83), or a malonate transporter encoded by the matC gene of Sinorhizobium meliloti, or a malonate transporter encoded by the DctPQM of Sinorhizobium meliloti, or 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. A cell culture of a host cell engineered to express malonyl-CoA synthetase and malonate transporter may include a culture medium comprising malonate.

[0103] In other embodiments, the cell engineered to produce flavonoids or anthocyanins is further engineered to include an exogenous nucleic acid sequence encoding a malonate CoA transferase (EC: 2.8.3.3; also known as the alpha subunit of a malonate decarboxylase), which prepares malonyl-CoA by directly transferring CoA from acetyl-CoA. For example, the alpha subunit of the malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has malonate CoA transferase activity. The mdcA gene product alpha subunit is a malonate CoA transferase, and the mdcD gene product beta subunit is a malonyl-CoA decarboxylase. The mdcE gene product gamma subunit can play a role in subunit interactions to form a stable complex or as a cocarboxylase. The mdcC gene product delta subunit is an acyl carrier protein with a unique CoA-like cofactor. When the α subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA portion of the prosthetic group is bound to the α subunit to exchange the acetyl group for a malonyl group. Since thioester transfer should be thermodynamically favorable, the engineered cell can include a nucleic acid encoding a malonate CoA transferase to increase the malonyl-CoA supply. Examples of mdcA that can be expressed in engineered cells as provided herein include, but are not limited to, mdcA (SEQ ID NO: 19) of Acinetobacter calcoaceticus, mdcA of Table 10, mdcA with SEQ ID NO: 84-87, or a transferase with 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.

[0104] Table 10. Malonate CoA transferase (malonate decarboxylase subunit α)

[0105]

[0106] In some embodiments, cells engineered to produce flavonoids or anthocyanins are further engineered to increase the supply of coenzyme A (CoA) to increase its availability for the production of acetyl-CoA, malonyl-CoA and / or p-coumaryl-CoA. Strategies to increase the supply of CoA include expressing or overexpressing at least one enzyme of the CoA biosynthetic pathway. Pantothenate kinase (EC2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4'-phosphopantothenate at the expense of adenosine triphosphate (ATP) molecules. It is the rate-limiting step in CoA biosynthesis. Three different types of PanK have been identified—PanK-I (found in bacteria), PanK-II (primarily found in eukaryotes, but also found 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 some CoA esters. Type III enzyme CoaX is not subject to feedback inhibition by CoA. In some embodiments, the host cell can be engineered to include 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 CoaX gene of Pseudomonas aeruginosa (EC: 2.7.1.33, SEQ ID NO: 20), the CoaX of Streptomyces CLI2509 (SEQ ID NO: 88), the CoaX of Streptomyces cinereus (SEQ ID NO: 89), or the CoaX of Kitasatospora kifunensis (SEQ ID NO: 90). In some embodiments, the culture of cells engineered for the production of flavonoids or anthocyanins can include a culture medium that includes pantothenate (a precursor for CoA biosynthesis), and can optionally also include cysteine ​​for CoA biosynthesis.

[0107] Other strategies that increase the malonyl-CoA flux leading to the flavonoid pathway include mutation or downregulation of one or more genes that work in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavonoid biosynthesis for the precursor malonyl-CoA, therefore limiting the formation of flavonoids. Without limiting the embodiment to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply that can be used for flavonoid biosynthesis. In some embodiments, gene β-ketoacyl-ACP synthase II (Escherichia coli fabF) can be destroyed, weakened or lack to reduce fatty acid biosynthesis. Another example of the fatty acid biosynthesis gene of the host cell that can mutate or downregulate is the gene of encoding malonyl-CoA-ACP transacylase (Escherichia coli fabD). Other fatty acid biosynthesis genes of the through engineering host cell that can downregulate include β-ketoacyl-ACP synthase I enzyme (Escherichia coli fabB) and / or acyl carrier protein (Escherichia coli acpP).

[0108] Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption or deletion of gene targets that divert carbon flux to form byproducts such as ethanol, acetate and lactate. They include genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetylphosphotransferase and acetate kinase. In E. coli host cells, downregulated, disrupted or deleted genes may include adhE, ldhA, poxB and ackA-pta.

[0109] In addition, cells engineered for the production of flavonoids or anthocyanins may have one or more genes encoding thioesterases downregulated, disrupted or deleted to prevent hydrolysis of the precursors malonyl-CoA, acetyl-CoA and / or p-coumaryl-CoA. Acyl-CoA thioesterases (ACOTs) catalyze the hydrolysis of acyl-CoAs (short chain, medium chain, long chain and very long chain), bile acid-CoAs and methyl branched chain acyl-CoAs into 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 may be downregulated, disrupted or deleted.

[0110] In other embodiments, cells engineered for the production of flavonoids or anthocyanins may have one or more of the fatty acid degradation genes downregulated, disrupted or deleted to improve the supply of precursors to the flavonoid pathway. For example, in E. coli, the acyl-CoA dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, the adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and / or the gene encoding fatty acid oxidation complex subunit alpha (fadB) may be downregulated, disrupted or deleted.

[0111] Alternatively or additionally, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase the supply of α-ketoglutarate, which serves 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.

[0112] In other embodiments, engineered host cells for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine are also contemplated. Tyrosine is one of the precursors of flavonoid biosynthesis, and its conversion into 4-coumaric acid is the first step of the pathway. Effective biosynthesis of L-tyrosine from raw materials such as glucose or glycerol is necessary to make bioproduction economically feasible. L-tyrosine is one of the three aromatic amino acids derived from the shikimic acid pathway. The shikimic acid pathway is a central metabolic route that leads to the formation of tryptophan (TRP), tyrosine (TYR) and phenylalanine (PHE), which is only present in plants and microorganisms. It begins with the condensation of phosphoenolpyruvate (PEP) and erythrose-4-phosphate (E4P), an intermediate of glycolysis and pentose phosphate pathway, which enter shikimic acid via 3-deoxy-d-arabino-heptulose acid-7-phosphate (DAHP), 3-dehydroquinic acid (DHQ), and 3-dehydroshikimic acid (DHS) through a series of condensation and redox reactions. From there, the central branch point metabolite chorismate is obtained by shikimate-3-phosphate under ATP hydrolysis and introduction of the second PEP. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated by feedback inhibition. There are three DAHP synthase isozymes (aroF, aroG, aroH) in Escherichia coli, each of which is feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), while the two DAHP synthases of plants are not subject to feedback inhibition. In plants and bacteria, the subsequent five steps are catalyzed by a single enzyme. Starting from the central intermediate chorismate, the pathway branches into anthranilic acid and prephenic acid, resulting in the synthesis of aromatic amino acids, p-hydroxybenzoic acid (pHBA) and p-aminobenzoic acid (pABA), which is a precursor of folic acid metabolism. The strategy to increase L-tyrosine production can include but is not limited to the deletion of the L-phenylalanine branch of the transcriptional dysregulation, removal of feedback inhibition, overexpression of the rate-limiting enzyme and / or the aromatic acid biosynthetic pathway. For example, in an E. coli host, the tyrR gene can be destroyed, feedback inhibition resistant forms 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.

[0113] In other embodiments, engineered host cells for producing flavonoids or anthocyanins are also contemplated, which are further engineered to upregulate the endogenous biosynthesis of the cofactor heme. Cytochrome P450 (CYP) is one of the exogenous genes in the engineered cells provided herein, containing heme as a cofactor. Improving heme supply may be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first precursor of the heme pathway. There are two known alternative pathways to produce this key intermediate. One route is the C4 pathway (Shemin pathway), which involves succinyl-CoA and glycine condensed into 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, resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions are required to convert ALA into heme, including assembling eight ALA molecules into cyclic tetrapyrroles, modifying side chains, and incorporating reduced iron into molecules. In E. coli hosts, 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, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and / or HemL to increase the production of heme precursor ALA. Non-limiting examples of hemA genes that can be overexpressed include, but are not limited to, mutant hemA genes (EC:1.1.1.70, SEQ ID NO:21) from Salmonella typhimurium and hemA with SEQ ID NO:91-93. Alternatively or in addition, heterologous ALAS genes can be introduced to produce ALA via C4 pathways. Non-limiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Rhodobacter capsulatus (SEQ ID: 22), ALAS having SEQ ID NOs: 94-97, or an ALAS that is 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% identical to any of these or other naturally occurring ALAS. In addition, one or more of the downstream genes that catalyze the synthesis of heme from ALA (hemB, hemC, hemD, hemE, hemF, hemG, heml, or hemH in E. coli) can be overexpressed to drive flux from ALA to heme production.In some embodiments, a culture of cells engineered for the production of flavonoids or anthocyanins can include a culture medium that includes succinate and / or glycine, which are precursors for the biosynthesis of heme via the C4 pathway.

[0114] The engineered cells producing flavonoids can be engineered to include multiple approaches to enhance the production of flavonoids. Those skilled in the art will recognize that the embodiments described herein can be combined in various ways. The examples of engineered cells with multiple genetic modifications are only exemplary and do not limit the scope of the invention.

[0115] The enzymes expressed or overexpressed in the engineered cells according to the present invention are listed in Table 11.

[0116] Host cells

[0117] Host cells provided herein can be prokaryotic cells or eukaryotic cells. Eukaryotic cells can be microbial eukaryotic cells, such as fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanobacterial cells.

[0118] Hosts can be selected based on their ability to take up and utilize specific 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.

[0119] Non-limiting examples of suitable microbial hosts for bioproduction of flavonoids include, but are not limited to, any Gram-negative organism, more particularly a member of the Enterobacteriaceae family, such as Escherichia coli; any Gram-positive microorganism, such as Bacillus subtilis, Lactobacillus sp., or Lactococcus sp.; yeast, such as Saccharomyces cerevisiae, Pichia pastoris, or Pichia stipitis; and other groups or microbial species. More specifically, suitable microbial hosts for 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.

[0120] Culture medium

[0121] In yet another aspect, methods for producing flavonoids or anthocyanins include incubating a culture of an engineered host cell as provided herein to produce flavonoids or anthocyanins. These methods may further include recovering the flavonoids or anthocyanins from the culture medium, the whole culture or the cells.

[0122] The culture comprises cells engineered to produce flavonoids or anthocyanins in the culture medium. In various embodiments, the engineered cells can be prokaryotic cells or eukaryotic cells. The culture medium includes at least one carbon source, which is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose and xylose. Such carbon sources can be purified or crude, including biomass containing glycerol, such as crude glycerol produced as a byproduct of biodiesel produced from corn waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply, such as but not limited to tyrosine, phenylalanine, coumaric acid, acetic acid, malonic acid, succinic acid, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenic acid, α-ketoglutaric acid and ascorbic acid. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, α-ketoglutaric acid, 5-aminolevulinic acid and ascorbic acid are provided in the culture medium.

[0123] The culture conditions may include any combination of aerobic growth conditions, microaerobic growth conditions, or alternating aerobic / microaerobic growth conditions. In addition, 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 may be amplified and continuously grown to produce flavonoid products. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation scheme, cells grow in a bioreactor where growth temperature, oxygen, pH, carbon source, and other compounds are well controlled. The desired temperature may be, for example, 20-37° C., depending on the growth characteristics of the production cells and the desired conditions of the fermented product. The pH of the bioreactor may be controlled within the range of 5-8, or may be uncontrolled in some cases. The batch fermentation period may last from several hours to several days, for example, 8 to 96 hours. At the end of the culture period, the fermentor contents may be passed through a cell separation unit to remove cells and cell debris. As required, enzymatic or chemical lysis or destruction of cells may be performed before or after separation of cells from the fermented broth to release additional products. To purify the flavonoids and / or anthocyanins to homogeneity, the solution containing the flavonoids and / or anthocyanins is concentrated and the product is purified by ion exchange or silica-based chromatography. The resulting solution is lyophilized to produce the product in solid form or concentrated to a liquid solution.

[0124] In some embodiments, the method for producing flavonoids or anthocyanins includes culturing the engineered cells disclosed herein in a culture medium to produce flavonoids or anthocyanins. In some embodiments, glycerol is used as a carbon raw material. In some embodiments, glycerol is crude glycerol. In some embodiments, the method includes separating 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, leucocyanin, 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, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside can be isolated with a purity of from about 50% to about 99%, such as from about 50% to about 95% (e.g., from: 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, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 50% to about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 55% to about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%.In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 60% to about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 65% to about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 70% to about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 75% to about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80% to about 85%, about 90%, about 95%, or about 99%. In some embodiments, naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside, or pelargonidin glucoside can be isolated with a purity of from about 85% to 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 with a purity of from about 90% to about 95%, or about 99%, or from about 95% to about 99% or more.

[0125] I. Embodiment

[0126] Using modified cells to produce products

[0127] Example 1 - Production of naringenin in E. coli

[0128] 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 provided in the culture medium. ACC was expressed on a medium copy plasmid (15-20 copies), while TAL, 4CL, CHS and CHI were expressed on the chromosome. Cells at OD 2.5 were cultured in a 48-well plate at a shaking speed of 600 RPM at 30 degrees in minimal medium supplied with trace elements, vitamins, 1 mM tyrosine, 1 mM coumaric acid and 2% glycerol for 24 hours. The cell culture was extracted with DMSO at a ratio of 1: 1 and centrifuged for 15 minutes. The supernatant was analyzed for naringenin by HPLC. The cells produced 232 μM naringenin.

[0129] Variants of the foregoing host cells can be prepared using one or more of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) and one or more homologs of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), or CHI (SEQ ID NO: 6), or a combination of two or more thereof, wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the reference enzyme.

[0130] Example 2 - Production of dihydrokaempferol in E. coli

[0131] 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 provided in the culture medium. Cells at OD 0.5-0.7 were cultured in 24-well plates in minimal medium supplied with 2% glycerol, trace elements, 0.8mM naringenin, 65mg / L 5-aminolevulinic acid, 0.1mM ferrous sulfate, 0.1mM 2-oxoglutarate, and 2.5mM ascorbic acid at 30 degrees with a shaking speed of 200RPM for 18 hours. The cell culture was extracted with DMSO and centrifuged for 15 minutes. Dihydrokaempferol in the supernatant was analyzed by HPLC. The cells produced 315μM dihydrokaempferol.

[0132] Variants of the aforementioned host cells can be prepared using homologs of F3H (SEQ ID NO: 7), wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the reference enzyme.

[0133] Example 3 - Production of Taxifolin in E. coli

[0134] The 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 provided in the culture medium. F3H was overexpressed on the chromosome, while F3'H and CPR were overexpressed on medium copy plasmids. Cells at OD 0.5-0.7 were cultured in 24-well plates in minimal medium supplied with 2% glucose, 0.8mM naringenin, 65mg / L 5-aminolevulinic acid, 0.1mM ferrous sulfate, 0.1mM 2-oxoglutarate and 2.5mM ascorbic acid at 30 degrees with a shaking speed of 200RPM for 18 hours. The cell culture was extracted with 50% DMSO and centrifuged for 15 minutes. Taxifolin in the supernatant was analyzed by HPLC. The cells produced 500μM Taxifolin.

[0135] Variants of the aforementioned host cells can be prepared using one or more of F3H (SEQ ID NO: 7), F3'H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9), and one or more homologs 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 thereof, wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity with the reference enzyme.

[0136] Example 4 - Production of anthocyanidins and anthocyanins

[0137] The 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 is provided in the culture medium. ANS and 3GT were overexpressed on the chromosome. Cells with OD 0.5-0.7 were cultured in 24-well plates at 30°C with a shaking speed of 200RPM for 18 hours in minimal medium supplied with 1.0% glucose, 2.0mM (+)-catechin, 0.1mM 2-oxoglutarate and 2.5mM ascorbic acid. 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 50mg / L cyanidin-3-O-glucoside.

[0138] Variants of the aforementioned host cells can be prepared using one or both of ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) and homologs of ANS (SEQ ID NO: 13), 3GT (SEQ ID NO: 14) or both, wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the reference enzyme.

[0139] Example 5 - Production of Quercetin

[0140] 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), CHI (SEQ ID NO: 6), F3H (SEQ ID NO: 7), F3'H (SEQ ID NO: 8), CPR (SEQ ID NO: 9) and FLS (SEQ ID NO: 99) to produce quercetin when the substrate glycerol is provided in the culture medium. ACC, TAL, 4CL, CHS, CHI, F3H, F3'H, CPR and FLS are expressed on the chromosome.

[0141] Cells at OD 2.0 were cultured in 48-well plates in minimal medium supplied with trace elements, vitamins, 2% glycerol, 2.0 mM 5-aminolevulinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid at 30°C with a shaking speed of 600 RPM for 24 hours. The cell culture was extracted with methanol at a ratio of 5:1 (MeOH: cells) and centrifuged for 15 minutes. The supernatant was analyzed for quercetin by HPLC. The cells produced 256 μM quercetin.

[0142] Figure 7 Data demonstrating the production of quercetin by the methods described herein are provided. Panel (A) provides a profile of a quercetin analytical standard, while panel (B) provides a profile from a quercetin producing strain. Figure 7 As is evident from the data presented in, the methods provided herein demonstrate the production of quercetin in engineered cells.

[0143] One or more of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), CHI (SEQ ID NO: 6), F3H (SEQ ID NO: 7), F3'H (SEQ ID NO: 8), CPR (SEQ ID NO: 9), and FLS (SEQ ID NO: 99) and ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), CHI (SEQ ID NO: 6), F3H (SEQ ID NO: 7), F3'H (SEQ ID NO: 8), CPR (SEQ ID NO: 9), and FLS (SEQ ID NO: 99) can be used. NO:99) or a combination of two or more thereof to prepare variants of the aforementioned host cells, wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98% or at least 99% identity with the reference enzyme.

[0144] Analytical methods

[0145] Example 6 - Flavonoid Precursors and Flavonoids

[0146] To sample naringenin, eriodictyol, dihydrokaempferol and taxifolin, total flavonoids were extracted from E. coli on whole cell culture fluid. 500 μL of whole cell culture fluid was vortexed with 500 μL of DMSO (dimethyl sulfoxide) for 30 seconds and centrifuged for 15 minutes. For HPLC analysis, 50 μL of supernatant was transferred to HPLC vials.

[0147] The HPLC method is as follows: Agilent 1200HPLC was equipped with an Ascentis C18 column 150 mm × 4.6 mm, 3 μm, equipped 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 0.1% aqueous phosphoric acid (solvent A), acetonitrile (solvent B) and methanol (solvent C) with the following gradient:

[0148]

[0149] The total run time was 15 minutes, with naringenin, eriodictyol, dihydrokaempferol, and taxifolin eluting at 12.50, 11.56, 10.20, and 8.85 minutes, respectively. A diode array detector (DAD) was used to detect the target molecules at 288 nm.

[0150] Example 7 - Anthocyanidins and Anthocyanins

[0151] In order to sample (+)-catechin, cyanidin and cyanidin-3-glucoside, the reaction solution was acidified with 13M HCl (1:40v / v), extracted with 100% ethanol, then mixed, centrifuged and filtered through a 0.45μm filter.HPLC method is as follows: Agilent 1200HPLC was equipped with LiChrospher RP-8 column 250mm×4.6mm, 5μm, equipped with LiChrospher100RP-8 (5μm) LiChroCART 4-4 guard column. The column was heated to 25°C and the sample block was maintained at 25°C. For each sample, 10μL was injected and the product was eluted at a flow rate of 1.0ml / min using 0.1% phosphoric acid aqueous solution (solvent A) and acetonitrile (solvent B) with the following gradient: 90% A to 10% A for 12 minutes, 90% A for 0.5 minutes, and 90% A for 3.5 minutes for column balance. The total run time was 16 minutes with cyanidin-3-glycoside eluting at 6.95 minutes and cyanidin eluting at 8.9 minutes. A diode array detector (DAD) was used to detect the molecules of interest at either 280 nm or 530 nm.

[0152] Example 8 - Flavonoid Production

[0153] This example provides a combination of modifications to the E. coli host genome, including deletion and overexpression of enzymes from other organisms, to reproduce Figure 4 Therefore, the present invention provides an engineered host cell comprising one or more genetic modifications (such as Figure 4 As shown in and in this Example 8 and described above in this application), the one or more genetic modifications cause the engineered host cell to produce flavonoids or anthocyanins from a carbon source, which may also be an energy source, through a variety of chemical intermediates. In certain embodiments, flavonoids or anthocyanins are produced from a carbon source, which may also be an energy source, 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 cell is cultured in a culture medium comprising molecules selected from the group consisting of: (i) glycerol, (ii) sugars, (iii) organic acids, (iv) amino acids, and (v) any combination thereof. As Figure 4 As shown, in certain embodiments, one or more genetic modifications result in increased metabolic flux to flavonoid precursors or cofactors. Figure 4 As shown, in certain embodiments, one or more genetic modifications result in a reduction in by-product formation. Figure 4 As shown, in certain embodiments, the one or more genetic modifications are selected from: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications for expressing one or more non-native genes in the engineered host cell; and (iv) combinations thereof.

[0154] like Figure 4 As shown, in certain embodiments, the engineered host cells are cultured in a medium comprising a molecule selected from the group consisting of tyrosine, phenylalanine, malonate, p-coumaric acid, bicarbonate, acetate, pantothenic acid, biotin, thiamine, α-ketoglutarate, ascorbic acid, and 5-aminolevulinic acid.

[0155] like Figure 4 As shown, 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. Figure 4As shown, 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 a chalcone synthase and a chalcone isomerase; and (iv) any combination thereof.

[0156] like Figure 4 As shown, in certain embodiments, one or more genetic modifications reduce fatty acid biosynthesis. Figure 4 As shown, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine into trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (v) any combination thereof.

[0157] like Figure 4 As shown, in certain embodiments, the engineered host cell comprises at least one or more nucleic acid sequences selected from the following: (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-coumarate-CoA ligase (4CL) activity; and (v) any combination thereof.

[0158] like Figure 4As shown, in certain embodiments, the engineered host cell comprises an exogenous nucleic acid sequence selected from the group consisting of: (i) 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; (ii) a nucleic acid sequence encoding a phenylalanine ammonia lyase, wherein the encoded phenylalanine ammonia lyase converts phenylalanine into trans-cinnamic acid; (iii) a nucleic acid sequence encoding a cinnamate-4-hydroxylase, wherein the cinnamate-4-hydroxylase produces 4-coumaric acid from trans-cinnamic acid; (iv) a nucleic acid sequence encoding a 4-coumaric acid-C (v) a nucleic acid sequence encoding a chalcone synthase activity, wherein the chalcone synthase forms naringenin chalcone from malonyl-CoA and p-coumaroyl-CoA; (vi) a nucleic acid sequence encoding a chalcone isomerase activity, wherein the chalcone isomerase forms naringenin from naringenin chalcone; (vii) a nucleic acid sequence encoding a flavanone-3-hydroxylase, wherein the flavanone-3-hydroxylase forms dihydrokaempferol from naringenin; and (viii) any combination thereof.

[0159] The compositions described above can be used in the methods described herein to increase the production of flavonoids or anthocyanins. Such methods involve providing any of the above compositions to cause an engineered host cell to enzymatically convert glycerol to flavonoids or anthocyanins (e.g., Figure 4 part or all of it).

[0160] On the other hand, imagine Figure 4 The pathways shown in can be performed using multiple engineered host cells, rather than a single host cell as described above. In such embodiments, the multiple engineered host cells have one or more genetic modifications that cause the engineered host cells to enzymatically convert glycerol to flavonoids or anthocyanins (e.g., Figure 4 shown).

[0161] Reference now Figure 4 Aspects of the invention are described.

[0162] Step 1: Conversion of pyruvate to acetate. poxB was deleted to reduce carbon loss and eliminate byproducts.

[0163] Step 2: Conversion of pyruvate to lactate. ldhA is deleted to reduce carbon loss and eliminate byproducts.

[0164] Step 3: Conversion of acetyl-CoA to acetate. ackA-pta is deleted to reduce carbon loss and eliminate byproducts.

[0165] Step 4: Conversion of acetyl-CoA to ethanol (EtOH). adhE was deleted to reduce carbon loss and eliminate byproducts.

[0166] Step 5: Acetyl-CoA is converted to substrates for the tricarboxylic acid cycle (TCA).

[0167] Step 6: Acetyl-CoA is converted to mal-CoA. Heterologous ACC is expressed to increase the concentration of available mal-CoA. Heterologous ACC can be obtained from fungal species. Therefore, embodiments of the present invention provide 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 the following: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is Escherichia coli. In certain embodiments, acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces stargensis, Ustilago maydis, and orthologs of acetyl-CoA carboxylases having at least 50% amino acid identity with the acetyl-CoA carboxylases of these aforementioned species. In certain embodiments, one or more genetic modifications are deletions or attenuations of one or more fat biosynthetic genes that result in reduced fatty acid biosynthesis. In certain embodiments, one or more genetic modifications are overexpressions of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from the group consisting of: an acetyl-CoA synthase gene of Escherichia coli, an acetyl-CoA synthase gene of Salmonella typhimurium, and an ortholog of an acetyl-CoA synthase gene in any other species having at least 50% amino acid identity with the acetyl-CoA synthase gene of Escherichia coli and Salmonella typhimurium. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from the malonyl-CoA synthetase of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications that reduce fatty acid biosynthesis are selected from: (i) mutation or down-regulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene β-ketoacyl-ACP synthase II (E. coli fabF); (iii) down-regulation of β-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) down-regulation of acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from the group consisting of: (i) an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide shown 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 shown in SEQ ID NO: 19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide shown 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; 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.

[0168] On the other hand, the present invention provides a method for increasing the production of flavonoids, comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase the production and / or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from the following: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is Escherichia coli. In certain embodiments, acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces stargensis, Ustilago maydis, and an ortholog of acetyl-CoA carboxylase with at least 50% amino acid identity with the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modifications are the deletion or weakening of one or more fat biosynthetic genes that cause fatty acid biosynthesis to decrease. In certain embodiments, one or more genetic modifications are overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from the group consisting of: an acetyl-CoA synthase gene of Escherichia coli, an acetyl-CoA synthase gene of Salmonella typhimurium, and an ortholog of an acetyl-CoA synthase gene in any other species having at least 50% amino acid identity with the acetyl-CoA synthase gene of Escherichia coli and Salmonella typhimurium. In certain embodiments, one or more genetic modifications are selected from the group consisting of: (i) overexpression of a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include an E354K mutation; (ii) an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) an exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combination thereof. In certain embodiments, the malonyl-CoA synthetase is selected from the malonyl-CoA synthetase of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, the one or more genetic modifications that reduce fatty acid biosynthesis are selected from: (i) mutation or down-regulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modification of the gene β-ketoacyl-ACP synthase II (E. coli fabF); (iii) down-regulation of β-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) down-regulation of acyl carrier protein (E. coli acpP); and (v) any combination thereof.In certain embodiments, the engineered host cell comprises a peptide selected from the group consisting of: (i) an acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide shown 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 shown in SEQ ID NO: 19; (iii) an acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide shown 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. 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.

[0169] Step 7: Conversion of mal-CoA to malonyl-ACP (acyl carrier protein). Malonyl-CoA-ACP transacylase (fabD) is downregulated to increase carbon flux.

[0170] Step 8: Conversion of malonyl-ACP to 3-ketoacyl-ACP. β-ketoacyl-ACP synthase II (fabF) is downregulated to increase carbon flux.

[0171] Step 9: Conversion of mal-CoA to naringenin chalcone; conversion of coumaroyl-CoA to naringenin chalcone. Overexpression of heterologous CHS.

[0172] Step 10: Conversion of naringenin to naringenin chalcone. Heterologous CHI overexpression.

[0173] 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). Heterologous F3'5'H, F3H, F3H and / or CPR are overexpressed. Therefore, if Figure 4As shown, on the other hand, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or pentahedral flavonoids (PHF), comprising an engineered host cell, wherein the engineered host cell comprises a cytochrome P450 reductase (CPR), and at least one of flavanone-3'-hydroxylase (F3'H) or flavonoid 3', 5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or pentahedral flavonoids (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 a 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 with the cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3', 5'-hydroxylase (F3'5'H) is fused with the cytochrome P450 reductase (CPR). In certain embodiments, the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.9. In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to 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, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.98.

[0174] like Figure 4As shown, on the other hand, the present invention provides a method for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or pentahedral flavonoids (PHF), comprising an engineered host cell, wherein the engineered host cell comprises a cytochrome P450 reductase (CPR), and at least one of flavanone-3'-hydroxylase (F3'H) or flavonoid 3', 5'-hydroxylase (F3'5'H). In certain embodiments, the precursor for increasing the production of dihydroquercetin (DHQ), dihydromyricetin (DHM), eriodictyol (EDL) and / or pentahedral flavonoids (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 a 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 with the cytochrome P450 reductase (CPR). In certain embodiments, the flavonoid 3', 5'-hydroxylase (F3'5'H) is fused with the cytochrome P450 reductase (CPR). In certain embodiments, the flavanone-3-hydroxylase (F3H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.7. In certain embodiments, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In certain embodiments, cytochrome P450 reductase (CPR) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.9. In certain embodiments, flavonoid 3', 5'-hydroxylase (F3'5'H) has an amino acid sequence at least 80% identical to 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, cytochrome b5 has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.98.

[0175] Step 14: Conversion of dihydroquercetin (DHQ) to leucocyanidin (LC); conversion of dihydrokaempferol (DHK) to leucopelargonidin (LP); and conversion of dihydromyricetin (DHM) to leucodelphinidin (LD). Heterologous DFR overexpression.

[0176] Step 15: Conversion of leucocyanidin (LC) to catechins; leucodelphinidin (LD) to gallocatechins; and leucopelargonidin (LP) to afrucatechins. Heterologous LAR Overexpression.

[0177] Step 16: conversion of catechins to cyanidins; conversion of leucocyanidins (LC) to catechins; conversion of leucodelphinidin (LD) to delphinidin; conversion of gallocatechin to delphinidin; conversion of leucopelargonidin (LP) to pelargonidin; or conversion of avercatechin to pelargonidin. Heterologous ANS overexpression. Step 16 can be performed in vivo or in a cell-free culture medium. Thus, if Figure 4As shown, on the other hand, the present invention provides an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the conversion of cyanidin 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) anthocyanin synthase of papaya (SEQ.ID NO: 13); (ii) 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) anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ.ID NO: 13; and (iv) any combination thereof. In certain embodiments, 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 in Vitis vinifera (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 comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise 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.

[0178] 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 to increase the conversion of cyanidin 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) anthocyanin synthase of papaya (SEQ.ID NO: 13); (ii) 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) anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ.ID NO: 13; and (iv) any combination thereof. In certain embodiments, 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 in Vitis vinifera (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 comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise overexpression of anthocyanin synthase and flavonoid-3-glucosyltransferase (3GT). In certain embodiments, the one or more genetic modifications comprise 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.

[0179] In another aspect, the present invention provides a method for increasing the conversion of cyanidin or catechin into anthocyanin-3-glucoside (Cy3G), which comprises anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of papaya (SEQ.ID NO:13); (ii) 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) anthocyanin synthase having an amino acid sequence at least 80% identical to SEQ.ID NO:13; and (iv) any combination thereof.

[0180] In another aspect, the present invention provides a method for increasing the conversion of cyanidin or catechin to cyanidin-3-glucoside (Cy3G), which comprises a flavonoid-3-glucosyltransferase (3GT), wherein the flavonoid-3-glucosyltransferase is selected from: (i) a flavonoid-3-glucosyltransferase in Vitis vinifera (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.

[0181] Step 17: Pelargonin is converted to asterin; delphinidin is converted to myrtlein (De3G); cyanidin is converted to Cy3G. Heterologous 3GT is overexpressed in E. coli. Step 17 can be performed in vivo or as a cell-free reaction.

[0182] Step 18: Conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine.

[0183] Step 19: Conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA is overexpressed to upregulate tyrosine.

[0184] Step 20: Conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonic acid-7-phosphate (DAHP) aroG variants are overexpressed to upregulate tyrosine.

[0185] Step 21: deoxy-d-arabinoheptulose-7-phosphate (DAHP) is converted into dehydroquinic acid (DHQ); erythrose-4-phosphate (E4P) is converted into dehydroquinic acid (DHQ).

[0186] Step 22: Dehydroquinic acid (DHQ) is converted to 3-dehydroshikimic acid (DHS).

[0187] Step 23: Conversion of 3-dehydroshikimate (DHS) to shikimate (SHK) aroE is overexpressed to upregulate tyrosine.

[0188] Step 24: Conversion of shikimate (SHK) to shikimate-3-phosphate (S3P).

[0189] Step 25: Shikimate-3-phosphate (S3P) is converted to 5-enolpyruvylshikimate-3-phosphate (EPSP).

[0190] Step 26: Conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismate (CHA).

[0191] Step 27: Conversion of chorismate (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variants are overexpressed.

[0192] Step 28: 4-hydroxy-phenylpyruvate (HPP) is converted to tyrosine; phenylpyruvate (POPP) is converted to phenylalanine (Phe). Figure 4 As shown, embodiments of the present invention provide an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprise the upregulation of 3-deoxy-D-arabino-heptulose synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combination thereof. In certain embodiments, one or more genetic modifications comprise the downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprise the expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprise the expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprise the destruction of tyrR gene.

[0193] like Figure 4 As shown, on the other hand, the present invention provides a method for increasing the endogenous biosynthesis of tyrosine, which comprises an engineered cell, wherein the engineered host cell comprises one or more genetic modifications to increase the endogenous biosynthesis of tyrosine. In certain embodiments, one or more genetic modifications comprise the upregulation of 3-deoxy-D-arabino-heptulose synthase. In certain embodiments, one or more genetic modifications are selected from: (i) upregulation of chorismate mutase; (ii) upregulation of prephenate dehydrogenase; (iii) overexpression of shikimate kinase; (iv) overexpression of shikimate dehydrogenase; and (v) any combination thereof. In certain embodiments, one or more genetic modifications comprise the downregulation of L-phenylalanine biosynthetic pathway. In certain embodiments, one or more genetic modifications comprise the expression of exogenous phosphoenolpyruvate synthase (ppsA). In certain embodiments, one or more genetic modifications comprise the expression of exogenous transketolase (tktA). In certain embodiments, wherein the one or more genetic modifications comprise the destruction of tyrR gene.

[0194] Step 29: Conversion of tyrosine to coumaric acid. Heterologous TAL overexpression.

[0195] Step 30: Conversion of coumaric acid to coumaroyl-CoA. Heterologous 4CL overexpression.

[0196] Step 31: Glutamate (Glut) is converted into glutamyl-tRNA.

[0197] Step 32: Glutamyl-tRNA is converted to glutamate semialdehyde (GSA). hemA is overexpressed to upregulate ALA.

[0198] Step 33: Conversion of glutamate semialdehyde (GSA) to delta-aminolevulinic acid (ALA). Overexpression of hemL upregulates ALA.

[0199] Step 34: Conversion of delta-aminolevulinic acid (ALA) to porphobilinogen (PBG).

[0200] Step 35: Conversion of porphobilinogen (PBG) to hydroxymethylbilane (HMB).

[0201] Step 36: Conversion of hydroxymethylbilane (HMB) to uroporphyrinogen III (UPPIII).

[0202] Step 37: Uroporphyrinogen III (UPPIII) is converted to coproporphyrinogen III (CPPIII).

[0203] Step 38: Conversion of coproporphyrinogen III (CPPIII) to protoporphyrinogen IX (PPPIX).

[0204] Step 39: Protoporphyrinogen IX (PPPIX) is converted to protoporphyrin IX, which is subsequently capped with heme.

[0205] Step 40: Conversion of prephenate (PPA) to phenylpyruvate (POPP).

[0206] Step 41: Conversion of phenylalanine (Phe) to cinnamic acid. Heterologous PAL and / or TAL are overexpressed.

[0207] Step 42: Conversion of cinnamic acid to coumaric acid. Heterologous C4H / CPR overexpression.

[0208] like Figure 5 As shown, in another aspect, the present invention provides engineered cells for increasing the production of kaempferol, myricetin and quercetin by engineering host cells through one or more enzyme conversions of a carbon source. Figure 5 It can be clearly seen that Figure 5 The enzymatic transformation described in provides Figure 4 Additional transformations to those provided in . Figure 4The enzymatic conversion in is also discussed in U.S. Application No. 17 / 720,020, which is incorporated herein by reference in its entirety. In one embodiment, the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL). In another embodiment, the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in these engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in these engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in these engineered host cells; and (iv) combinations thereof. In another embodiment, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homologue thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homologue thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3', 5'-hydroxylase (F3'5'H) or a homologue thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homologue thereof, and (v) any combination thereof. In another embodiment, the engineered host cell is Escherichia coli. In another embodiment, the production of flavonols by enzymatic conversion of a carbon source includes one or more chemical intermediates. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3', 5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, 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 another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, flavonol synthase (FLS) has an amino acid sequence at least 80% identical to any one of the polypeptides listed in SEQ ID NOs.99-122.In another embodiment, the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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. In another embodiment, the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H). In another embodiment, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), naringenin, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavanone-3'-hydroxylase (F3'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), dihydrokaempferol, eriodictyol (EDL), naringenin, kaempferol, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), and (iv) any combination thereof. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), dihydroquercetin (DHQ), dihydrokaempferol, eriodictyol (EDL), naringenin, quercetin, kaempferol or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H) and (v) any combination thereof.

[0209] On the other hand, the present invention provides a method for increasing the production of flavonols, comprising an engineered host cell, wherein the engineered host cells comprise one or more genetic modifications to increase the production of flavonols by converting carbon sources by one or more enzymes through the engineered host cell. In one embodiment, the flavonol is selected from the group consisting of kaempferol, myricetin and quercetin. In another embodiment, the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL). In another embodiment, one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in these engineered host cells; (ii) one or more modifications for underexpressing one or more endogenous genes in these engineered host cells; (iii) one or more genetic modifications for expressing one or more non-natural genes in these engineered host cells; and (iv) combinations thereof. In another embodiment, the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homologue thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homologue thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3', 5'-hydroxylase (F3'5'H) or a homologue thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homologue thereof, and (v) any combination thereof. In another embodiment, the engineered host cell is Escherichia coli. In another embodiment, the production of flavonols by enzymatic conversion of a carbon source includes one or more chemical intermediates. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3', 5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, 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 another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthase (FLS).In another embodiment, the flavonoid synthase (FLS) has an amino acid sequence at least 80% identical to any one of the polypeptides selected from the group consisting of SEQ ID NOs: 99-122. In another embodiment, the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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. In another embodiment, the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthase (FLS). In another embodiment, the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H). In another embodiment, flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptide shown in SEQ ID NO.8. In another embodiment, the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), naringenin, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavanone-3'-hydroxylase (F3'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof. In another embodiment, the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), dihydrokaempferol, eriodictyol (EDL), naringenin, kaempferol, or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), and (iv) any combination thereof. In another embodiment, the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), dihydroquercetin (DHQ), dihydrokaempferol, eriodictyol (EDL), naringenin, quercetin, kaempferol or any combination thereof, and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H) and (v) any combination thereof.

[0210] Figure 5It was shown that dihydromyricetin (DHM) is enzymatically converted to myricetin by flavonol synthase (FLS). Figure 5 It was also shown that kaempferol and quercetin are enzymatically converted to myricetin by flavonoid 3',5'-hydroxylase (F3'5'H). Figure 5 It was further shown that dihydrokaempferol (DHK) was enzymatically converted to kaempferol by flavonol synthase (FLS). Figure 5 It was shown that kaempferol is enzymatically converted to quercetin by flavanone-3'-hydroxylase (F3'H) and dihydroquercetin (DHQ) is enzymatically converted to quercetin by flavonol synthase (FLS).

[0211] Fig. 6A and 6B Several enzymatic pathways for the bioproduction of kaempferol, quercetin, and myricetin are shown. Fig. 6A and 6B As can be clearly seen, the present invention provides several pathways involving the conversion of various precursors to kaempferol, quercetin and myricetin.

[0212] Table 11: Enzyme sequences:

[0213]

[0214]

[0215]

[0216]

[0217]

[0218]

[0219]

[0220]

[0221]

[0222]

[0223]

[0224]

[0225]

[0226]

[0227]

[0228]

[0229]

[0230]

[0231]

[0232]

[0233]

[0234]

[0235]

[0236]

[0237]

[0238]

[0239]

[0240]

[0241]

[0242]

[0243]

[0244]

[0245]

[0246]

[0247]

[0248]

[0249] Table 12: Glossary of abbreviations

[0250]

[0251]

[0252]

[0253] Incorporated by Reference

[0254] Throughout this disclosure, references and citations are made to other documents, such as patents, patent applications, patent publications, journals, books, papers, web content, publicly accessible databases. All such documents are incorporated herein by reference in their entirety for all purposes.

[0255] Equivalent

[0256] Various modifications of the present invention and many other embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the entire contents of this document (including references to the scientific and patent literature cited herein). The subject matter herein contains important information, examples, and guidance that can be applicable to the practice of various embodiments of the present invention and their equivalents.

Claims

1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of flavonols by converting a carbon source by one or more enzymes through the engineered host cell.

2. The engineered host cell of claim 1, wherein the flavonol is selected from the group consisting of kaempferol, myricetin and quercetin.

3. The engineered host cell of claim 1, wherein the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL).

4. The engineered host cell of claim 1, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications for expressing one or more non-native genes in the engineered host cell; and (iv) combinations thereof.

5. The engineered host cell of claim 1, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homolog thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homolog thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3',5'-hydroxylase (F3'5'H) or a homolog thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homolog thereof, and (v) any combination thereof. The engineered host cell of claim 1 , wherein the engineered host cell is Escherichia coli.

7. The engineered host cell of claim 1, wherein the production of flavonols by enzymatic conversion of a carbon source comprises one or more chemical intermediates.

8. The engineered host cell of claim 1, wherein the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthases (FLS).

9. The engineered host cell of claim 1, wherein the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3',5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

10. The engineered host cell of claim 9, wherein 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.

11. The engineered host cell of claim 1, wherein the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthases (FLS).

12. The engineered host cell of claim 11, wherein the flavonol synthase has an amino acid sequence at least 80% identical to any one of the polypeptides of SEQ ID NOs. 99-122.

13. The engineered host cell of claim 1, wherein the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

14. An engineered host cell according to claim 13, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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.

15. The engineered host cell of claim 13, wherein the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate.

16. The engineered host cell of claim 1, wherein the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthases (FLS).

17. The engineered host cell of claim 1, wherein the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H).

18. The engineered host cell of claim 17, wherein the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptides shown in (i) SEQ ID NO:8, (ii) SEQ ID NO.49, (iii) SEQ ID NO.50, (iv) SEQ ID NO.51, and (v) SEQ ID NO.

52.

19. The engineered host cell of claim 1, wherein the flavonol is kaempferol; the carbon source is selected from the group consisting of: (i) dihydrokaempferol (DHK), (ii) naringenin, and (iii) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

20. The engineered host cell of claim 1, wherein the flavonol is quercetin; the carbon source is selected from the group consisting of: (i) dihydroquercetin (DHQ), (ii) dihydrokaempferol (DHK), (iii) eriodictyol (EDL), (iv) naringenin, (v) kaempferol, and (vi) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), and (iv) any combination thereof.

21. The engineered host cell of claim 1, wherein the flavonol is myricetin, the carbon source is selected from the group consisting of: (i) dihydromyricetin (DHM), (ii) dihydroquercetin (DHQ), (iii) dihydrokaempferol (DHK), (iv) eriodictyol (EDL), (v) naringenin, (vi) quercetin, (vii) kaempferol, and (viii) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H), and (v) any combination thereof.

22. A method for increasing the production of flavonols, comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase the production of flavonols by converting a carbon source by one or more enzymes through the engineered host cell.

23. The method of claim 22, wherein the flavonol is selected from the group consisting of kaempferol, myricetin, and quercetin.

24. The method of claim 22, wherein the carbon source is selected from the group consisting of naringenin, dihydrokaempferol (DHK), dihydromyricetin (DHM), dihydroquercetin (DHQ) and eriodictyol (EDL).

25. The method of claim 22, wherein the one or more genetic modifications are at least one genetic modification selected from the group consisting of: (i) one or more modifications for overexpressing one or more endogenous genes in the engineered host cell; (ii) one or more modifications for underexpressing one or more endogenous genes in the engineered host cell; (iii) one or more genetic modifications for expressing one or more non-native genes in the engineered host cell; and (iv) combinations thereof.

26. The method of claim 22, wherein the engineered host cell comprises at least one or more nucleic acid sequences selected from the group consisting of: (i) a nucleic acid sequence encoding a natural or modified flavanone-3-hydroxylase (F3H) or a homolog thereof, (ii) a nucleic acid sequence encoding a natural or modified flavanone-3'-hydroxylase (F3'H) or a homolog thereof, (iii) a nucleic acid sequence encoding a natural or modified flavonoid 3',5'-hydroxylase (F3'5'H) or a homolog thereof, (iv) a nucleic acid sequence encoding a natural or modified flavonol synthase (FLS) or a homolog thereof, and (v) any combination thereof.

27. The method of claim 22, wherein the engineered host cell is Escherichia coli.

28. The method of claim 22, wherein the production of flavonols by enzymatic conversion of a carbon source comprises one or more chemical intermediates.

29. The method of claim 22, wherein the flavonol is myricetin, the carbon source is dihydromyricetin (DHM), and the one or more enzymes are flavonol synthases (FLS).

30. The method of claim 22, wherein the flavonol is myricetin, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are selected from the group consisting of: (i) flavonoid 3',5'-hydroxylase (F3'5'H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

31. The method of claim 30, wherein 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.

32. The method of claim 22, wherein the flavonol is kaempferol, the carbon source is dihydrokaempferol (DHK), and the one or more enzymes are flavonol synthases (FLS).

33. The method of claim 32, wherein the flavonol synthase (FLS) has an amino acid sequence at least 80% identical to any one of the polypeptides of SEQ ID NOs. 99-122.

34. The method of claim 22, wherein the flavonol is quercetin, the carbon source is eriodictyol (EDL), and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

35. The method of claim 34, wherein the flavanone-3-hydroxylase (F3H) has an amino acid sequence 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.

36. The method of claim 34, wherein the production of quercetin results in the formation of dihydroquercetin (DHQ) as an intermediate.

37. The method of claim 22, wherein the flavonol is quercetin, the carbon source is dihydroquercetin (DHQ), and the one or more enzymes are flavonol synthases (FLS).

38. The method of claim 22, wherein the flavonol is quercetin, the carbon source is kaempferol, and the enzyme is flavanone-3'-hydroxylase (F3'H).

39. The method of claim 38, wherein the flavanone-3'-hydroxylase (F3'H) has an amino acid sequence at least 80% identical to the polypeptides shown in (i) SEQ ID NO: 8, (ii) SEQ ID NO. 49, (iii) SEQ ID NO. 50, (iv) SEQ ID NO. 51 and (v) SEQ ID NO.

52.

40. The method of claim 22, wherein the flavonol is kaempferol; the carbon source is selected from the group consisting of: (i) dihydrokaempferol (DHK), (ii) naringenin, and (iii) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavanone-3-hydroxylase (F3H), (ii) flavonol synthase (FLS), and (iii) any combination thereof.

41. The method of claim 22, wherein the flavonol is quercetin; the carbon source is selected from the group consisting of: (i) dihydroquercetin (DHQ), (ii) dihydrokaempferol (DHK), (iii) eriodictyol (EDL), (iv) naringenin, (v) kaempferol, and (vi) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), and (iv) any combination thereof.

42. The method of claim 22, wherein the flavonol is myricetin, the carbon source is selected from the group consisting of: (i) dihydromyricetin (DHM), (ii) dihydroquercetin (DHQ), (iii) dihydrokaempferol (DHK), (iv) eriodictyol (EDL), (v) naringenin, (vi) quercetin, (vii) kaempferol, and (viii) any combination thereof; and the one or more enzymes are selected from the group consisting of: (i) flavonol synthase (FLS), (ii) flavanone-3'-hydroxylase (F3'H), (iii) flavanone-3-hydroxylase (F3H), (iv) flavonoid-3'-5'-hydroxylase (F3'5'H), and (v) any combination thereof.

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