Flavonoid and anthocyanin bioproduction using microorganism hosts
12203104 ยท 2025-01-21
Assignee
Inventors
- Jingyi Li (San Diego, CA, US)
- Nicholas Brideau (San Diego, CA, US)
- Joshua Britton (San Diego, CA, US)
- Erik Holtzapple (San Diego, CA)
Cpc classification
C12N9/0071
CHEMISTRY; METALLURGY
C12Y114/11009
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
C12N9/0073
CHEMISTRY; METALLURGY
C12N9/1029
CHEMISTRY; METALLURGY
C12Y106/02004
CHEMISTRY; METALLURGY
C12N15/70
CHEMISTRY; METALLURGY
C12N15/8243
CHEMISTRY; METALLURGY
C12P19/44
CHEMISTRY; METALLURGY
International classification
C12N15/70
CHEMISTRY; METALLURGY
C12N15/82
CHEMISTRY; METALLURGY
C12N9/00
CHEMISTRY; METALLURGY
C12P17/06
CHEMISTRY; METALLURGY
C12P19/44
CHEMISTRY; METALLURGY
Abstract
The invention is directed to methods involved in the production of flavonoids, anthocyanins and other organic compounds. The invention provides cells engineered for the production of flavonoids, anthocyanins and other organic compounds, where the engineered cells include one or more genetic modifications that increase flavonoid production by increasing metabolic flux to flavonoid precursors and/or reducing carbon losses resulting from the production of byproducts.
Claims
1. An engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to an endogenous gene to increase the production and/or availability of malonyl-CoA in comparison to a wild-type host cell, wherein the engineered host cell expresses acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 85% identical to the polypeptide set forth in SEQ ID NO: 15.
2. The engineered host cell of claim 1, wherein the production and/or availability of malonyl-CoA is increased by transformation of acetyl-CoA to malonyl-CoA.
3. The engineered host cell of claim 1, wherein the engineered host cell comprises one or more genetic modifications to increase expression of acetyl-CoA carboxylase (ACC) in comparison to a wild-type host cell.
4. The engineered host cell of claim 1, wherein the engineered host cell is E. coli.
5. The engineered host cell of claim 4, wherein the E. coli further comprises genes from fungi.
6. The engineered host cell of claim 3, wherein the acetyl-CoA carboxylase is from a species selected from the group consisting of Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species.
7. The engineered host cell of claim 1, wherein the one or more genetic modification is deletion or attenuation of one or more fatty acid biosynthetic genes resulting in decrease in fatty acid biosynthesis in comparison to a wild-type host cell.
8. The engineered host cell of claim 1, wherein the engineered host cell further comprises-peptides selected from a group consisting of: (i) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (ii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iii) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (iv) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (v) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vi) any combinations thereof.
9. The engineered host cell of claim 1, wherein the engineered host cell expresses acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 90% identical to the polypeptide set forth in SEQ ID NO: 15.
10. The engineered host cell of claim 1, wherein the engineered host cell expresses acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 95% identical to the polypeptide set forth in SEQ ID NO: 15.
Description
VI. BRIEF DESCRIPTION OF THE SEVERAL VIEWS OF THE DRAWING(S)
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(2)
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VII. DETAILED DESCRIPTION OF THE INVENTION
(5) The present application provides engineered cells for producing one or more flavonoids, cultures that include the engineered cells, and methods of producing one or more flavonoids, or at least one anthocyanin. The terms flavonoid, flavonoid product, or flavonoid compound are used herein to refer to a member of a diverse group of phytonutrients found in almost all fruits and vegetables. As used herein, the terms flavonoid, flavonoid product, or flavonoid compound are used interchangeably to refer a molecule containing the general structure of a 15-carbon skeleton, which consists of two phenyl rings (A and B) and a heterocyclic ring. Flavonoids may include, but are not limited to, isoflavone type (e.g., genistein), flavone type (e.g., apigenin), flavonol type (e.g., kaempferol), flavanone type (e.g., naringenin), chalcone type (e.g., phloretin), anthocyanidin type (e.g., cyanidin), catechins, flavanones, and flavanonols. Flavonoid compounds of interest include, without limitation, naringenin, naringenin chalcone, eriodictyol, taxifolin, dihydrokaempferol, dihydroquercetin, dihydromyricetin, leucocyanidin, leucopelargonidin, leucodelphindin, pentahydroxyflavone, cyanidin, catechin, delphinidin, pelargonidin, and kaempferol. Anthocyanins are in the forms of anthocyanidin glycosides and acylated anthocyanins. Anthocyanin compounds of interest include, without limitation, cyanidin glycoside, delphinidin glycoside, pelargonidin glycoside, peonidin glycoside, and petunidin glycoside.
(6) The terms precursor or flavonoid precursor as used herein may refer to any intermediate present in the biosynthetic pathway that leads to the production of catechins or anthocyanins. flavonoid precursors may include, but are not limited to tyrosine, phenylalanine, coumaric acid, p-coumaroyl-CoA, malonyl-CoA, pyruvate, acetyl-CoA, and naringenin.
(7) Cells engineered for the production of a flavonoid or an anthocyanin can have one or multiple modifications, including, without limitation, the downregulation, disruption, or deletion of endogenous genes, the upregulation of an endogenous gene, and the introduction of exogenous genes.
(8) The term non-naturally occurring, when used in reference to an enzyme is intended to mean that nucleic acids or polypeptides include at least one genetic alteration not normally found in a naturally occurring polypeptide or nucleic acid sequence. Naturally occurring nucleic acids, and polypeptides can be referred to as wild-type or original. A host cell, organism, or microorganism that includes at least one genetic modification generated by human intervention can also be referred to as non-naturally occurring, engineered, genetically engineered, or recombinant.
(9) A host cell, organism, or microorganism engineered to express or overexpress a gene or nucleic acid sequence, or to overexpress an enzyme or polypeptide has been genetically engineered through recombinant DNA technology to include a gene or nucleic acid sequence that does not naturally encode the enzyme or polypeptide or to express an endogenous gene at a level that exceeds its level of expression in a non-altered cell. As nonlimiting examples, a host cell, organism, or microorganism engineered to express or overexpress a gene or a nucleic acid sequence, or to overexpress an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Overexpression of a gene can also be by increasing the copy number of a gene in the cell or organism. Similarly, a host cell, organism, or microorganism engineered to under-express or to have reduced expression of a gene, nucleic acid sequence, or to under-express an enzyme or polypeptide can have any modifications that affect a coding sequence of a gene, the position of a gene on a chromosome or regulatory elements associated with a gene. Specifically included are gene disruptions, which include any insertions, deletions, or sequence mutations into or of the gene or a portion of the gene that affect its expression or the activity of the encoded polypeptide. Gene disruptions include knockout mutations that eliminate expression of the gene. Modifications to under-express a gene also include modifications to regulatory regions of the gene that can reduce its expression.
(10) The term exogenous or heterologous is intended to mean that the referenced molecule or the referenced activity is introduced into the host microbial organism. The molecule can be introduced, for example, by introduction of an encoding nucleic acid into the host genetic material such as by integration into a host chromosome or as non-chromosomal genetic material that may be introduced on a vehicle such as a plasmid. Therefore, the term endogenous refers to a referenced molecule or activity that is naturally present in the host.
(11) Genes or nucleic acid sequences can be introduced stably or transiently into a host cell using techniques well known in the art including, but not limited to, conjugation, electroporation, chemical transformation, transduction, and transfection. Optionally, for exogenous expression in E. coli or other prokaryotic cells, some nucleic acid sequences in the genes or cDNAs of eukaryotic nucleic acids can encode targeting signals such as an N-terminal mitochondrial or other targeting signal, which can be removed before transformation into prokaryotic host cells, if desired. Furthermore, genes can be subjected to codon optimization with techniques well known in the art to achieve optimized expression of the proteins.
(12) The percent identity (% identity) between two sequences is determined when sequences are aligned for maximum homology. Algorithms well known to those skilled in the art, such as Align, BLAST, Clustal Omega, and others compare and determine a raw sequence similarity or identity, and also determine the presence or significance of gaps in the sequence which can be assigned a weight or score. Such algorithms also are known in the art and are similarly applicable for determining nucleotide or amino acid sequence similarity or identity and can be useful in identifying orthologs of genes of interest. Additional sequences added to a polypeptide sequence, such as but not limited to immunodetection tags, purification tags, localization sequences (presence or absence), etc., do not affect the % identity.
(13) A homolog is a gene or genes that have the same or identical functions in different organisms. Genes that are orthologous can encode proteins with sequence similarity of about 45% to 100% amino acid sequence identity, and more preferably about 60% to 100% amino acid sequence identity. Genes can also be considered orthologs if they share three-dimensional structure but not necessarily sequence similarity, of a sufficient amount to indicate that they have evolved from a common ancestor to the extent that the primary sequence similarity is not identifiable. Paralogs are genes related by duplication within a genome, and can evolve new functions, even if these are related to the original one.
(14) An engineered cell for producing flavonoids include an exogenous nucleic acid sequence encoding tyrosine ammonia lyase (TAL) activity (alternatively or in addition, an exogenous nucleic acid encoding phenylalanine ammonia-lyase (PAL) activity and an exogenous nucleic acid encoding cinnamate-4-hydroxylase (C4H) activity), an exogenous nucleic acid sequence encoding 4-coumarate-CoA ligase (4CL) activity, an exogenous nucleic acid sequence encoding chalcone synthase (CHS) activity, and an exogenous nucleic acid sequence encoding chalcone isomerase (CHI) activity. Optionally, the engineered cell can further include an exogenous nucleic acid sequence encoding an exogenous nucleic acid sequence encoding flavanone-3-hydroxylase (F3H) activity, an exogenous nucleic acid sequence encoding flavonoid 3-hydroxlase (F3H) activity or flavonoid 3,5-hydroxylase (F35H), an exogenous nucleic acid sequence encoding cytochrome P450 reductase (CPR) activity, an exogenous nucleic acid sequence encoding dihydroflavonol-4-reductase (DFR) activity, and/or an exogenous nucleic acid sequence encoding leucoanthocyanidin reductase (LAR) activity.
(15) Tyrosine ammonia-lyase (TAL) can be, for example, a member of the aromatic amino acid deaminase family that catalyzes the elimination of ammonia from L-tyrosine to yield p-coumaric acid. An exemplary tyrosine ammonia lyase is the Saccharothrix espanaensis tyrosine ammonia lyase (TAL; SEQ ID NO: 1). Also considered for use in the engineered cells provided herein are TALs with SEQ ID NOS. 23-26, TALs listed in Table 1, TAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID:1 that have the activity of a tyrosine ammonia lyase that produces p-coumaric acid from tyrosine.
(16) TABLE-US-00001 TABLE 1 Tyrosine ammonia-lyase Organism GenBank Accession Number Rhodotorula glutini AGZ04575.1 Flavobacterium johnsoniae WP_012023194.1 Herpetosiphon aurantiacus ABX02653.1 Rhodobacter capsulatus ADE83766.1 Saccharothrix espanaensis AKE50820.1 Trichosporon cutaneum AKE50834.1
(17) Similar to tyrosine ammonia-lyase, phenylalanine ammonia-lyase (PAL) can be a member of the aromatic amino acid deaminase family that catalyzes the non-oxidative deamination of L-phenylalanine to form trans-cinnamic acid. An exemplary phenylalanine ammonia-lyase is the Brevibacillus laterosporus phenylalanine ammonia-lyase (PAL; SEQ ID NO:2). Also considered for use in the engineered cells provided herein are PALs with SEQ ID NOS: 27-29, PAL homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to SEQ ID NO: 2 that have the activity of a phenylalanine ammonia lyase that produces trans-cinnamic acid from phenylalanine.
(18) Cinnamate-4-hydroxylase (C4H) belongs to the cytochrome P450-dependent monooxygenase family and catalyzes the formation of p-coumaric acid from trans-cinnamic acid. Considered for use in the engineered cells provided herein are C4H of Helianthus annuus L. (C4H; SEQ ID NO: 3), C4Hs with SEQ ID NOS: 30-32, and C4H homologs of other species, as well as variants of naturally occurring C4Hs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to the SEQ ID NO: 3 (C4H, Helianthus annuus L.) that have the activity of a C4H.
(19) 4-coumarate-CoA ligase (4CL) catalyzes the activation of 4-coumarate to its CoA ester. Considered for use in the engineered cells provided herein are 4CLs of Petroselinum crispum (SEQ ID NO: 4), 4CLs in Table 2, 4CLs with SEQ ID NOS: 33-36, and 4CL homologs of other species, as well as variants of naturally occurring 4CLs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID No: 4 (4CL, Petroselinum crispum) that have the activity of a 4CL.
(20) TABLE-US-00002 TABLE 2 4-coumarate-CoA ligases Organism GenBank Accession Number Petroselinum crispum CAA31697.1 Camellia sinensis ASU87409.1 Capsicum annuum KAF3620173.1 Castanea mollissima KAF3954751.1 Daucus carota AIT52344.1 Gynura bicolor BAJ17664.1 Ipomoea purpurea AHJ60263.1 Lonicera japonica AGE10594.1 Lycium chinense QDL52638.1 Nelumbo nucifera XP_010265453.1 Nyssa sinensis KAA8540582.1 Solanum lycopersicum NP_001333770.1 Striga asiatica GER48539.1
(21) The chalcone synthase (CHS) can be, for example, a type III polyketide synthase that sequentially condenses three molecules of malonyl-CoA with one molecule of p-coumaryol-CoA to produce the naringenin precursor naringenin chalcone or naringenin. An exemplary chalcone synthase is the chalcone synthase of Petunia x hybrida (CHS, SEQ TD NO: 5). Also considered for use in the engineered cells provided herein are the genes listed in Table 3, CHSs with SEQ ID: 37-40, and CHS homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75% at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97% at least 98%, or at least 99% amino acid identity to SEQ ID NO: 5 (CHS, Petunia x hybrida) that have the activity of a chalcone synthase.
(22) TABLE-US-00003 TABLE 3 Chalcone synthases Organism GenBank Accession Number Petunia hybrida AAF60297.1 Acer palmatum AWN08245.1 Callistephus chinensis CAA91930.1 Camellia japonica BAI66465.1 Capsicum annuum XP_016566084.1 Coffea arabica XP_027118978.1 Curcuma alismatifolia ADP08987.1 Dendrobium catenatum ALE71934.1 Garcinia mangostana ACM62742.1 Iochroma calycinum AIY22758.1 Iris germanica BAE53636.1 Lilium speciosum BAE79201.1 Lonicera caerulea ALU09326.1 Lycium ruthenicum ATB56297.1 Magnolia liliiflora AHJ60259.1 Matthiola incana BBM96372.1 Morus alba var. multicaulis AHL83549.1 Nelumbo nucifera NP_001305084.1 Nyssa sinensis KAA8548459.1 Paeonia lactiflora AEK70334.1 Panax notoginseng QKV26463.1 Ranunculus asiaticus AYV99476.1 Rosa chinensis AEC13058.1 Theobroma cacao XP_007032052.2
(23) Chalcone isomerase (CHI, also referred to as chalcone flavonone isomerase) catalyzes the stereospecific and intramolecular isomerization of naringenin chalcone into its corresponding (2S)-flavanones. Considered for use in the engineered cells provided herein are CHI of Medicago sativa (SEQ TD NO: 6), CHI of Table 4, CHIs with SEQ TD NOS: 41-44, and CHI homologs of other species, as well as variants of naturally occurring CHI having at least 50%, at least 55% at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95% at least 96%, at least 97% at least 98%, or at least 99% amino acid identity to SEQ ID NO: 6 (CHI, Medicago sativa) that have the activity of a chalcone isomerase.
(24) TABLE-US-00004 TABLE 4 Chalcone Isomerases Organism GenBank Accession Number Medicago sativa AGZ04578.1 Dendrobium hybrid cultivar AGY46120.1 Abrus precatorius XP_027366189.1 Antirrhinum majus BA032070.1 Arachis duranensis XP_015942246.1 Astragalus membranaceus ATY39974.1 Camellia sinensis XP_028119616.1 Castanea mollissima KAF3958409.1 Cephalotus follicularis GAV77263.1 Clarkia gracilis subsp. QPF47150.1 sonomensis Dianthus caryophyllus CAA91931.1 Glycyrrhiza uralensis AXO59749.1 Handroanthus impetiginosus PIN05040.1 Lotus japonicus CAD69022.1 Morus alba AFM29131.1 Phaseolus vulgaris XP_007142690.1 Punica granatum ANB66204.1 Rhodamnia argentea XP_030524476.1 Spatholobus suberectus TKY50621.1 Trifolium subterraneum GAU12132.1
(25) A nucleic acid sequence encoding a CHI can in some embodiments be fused to a nucleic acid sequence encoding a CHS in an engineered cell as provided herein, such that the CHI activity is fused to the chalcone synthase activity, i.e., a fusion protein is produced in the engineered cell that has both condensing and cyclization activities.
(26) Flavanone 3-hydroxylase (F3H) catalyzes the stereospecific hydroxylation of (2S)-naringenin to form (2R,3R)-dihydrokaempferol. Other substrates include (2S)-eriodictyol, (2S)-dihydrotricetin and (2S)-pinocembrin. Some F3H enzymes are bifunctional and also catalyzes as flavonol synthase (EC: 1.14.20.6). Considered for use in the engineered cells provided herein are F3H of Rubus occidentalis (SEQ ID NO: 7), F3Hs with SEQ ID NOS: 45-48, F3Hs listed in Table 5, and other F3H homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:7 (F3H, Rubus occidentalis) that have the activity of a F3H.
(27) TABLE-US-00005 TABLE 5 Flavanone 3-hydroxylases Organism GenBank Accession Number Rubus occidentalis ACM17897.1 Abrus precatorius XP_027347564.1 Nyssa sinensis KAA8547483.1 Camellia sinensis AAT68774.1 Morelia rubra KAB1219056.1 Rosa chinensis PRQ47414.1 Malus domestica AAD26206.1 Vitis amurensis ALB75302.1 Iochroma ellipticum AMQ48669.1 Hibiscus sabdariffa ALB35017 Cephalotus follicularis GAV71832
(28) Flavonoid 3-hydroxylases (F3H) belongs to the cytochrome P450 family with systematic name of flavonoid, NADPH:oxygen oxidoreductase (3-hydroxylating). In the flavonoid biosynthetic pathway, F3H converts dihydrokaempferol to dihydroquercetin (taxifolin) or naringenin to eriodictyol. Considered for use in the engineered cells provided herein are F3H of Brassica napus (F3H; SEQ ID NO: 8), F3H with SEQ ID NOS: 49-52, those listed in Table 6, and homologs and variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these F3H. F3H is a cytochrome P450 enzyme that requires a cytochrome P450 reductase (CPR) to function. Cytochrome P450 reductases are diflavin oxidoreductases that supply electrons to F3Hs. The P450 reductase can be from the same species as F3H or different species from F3H. Considered for use in the engineered cells provided herein are CPR of Catharanthus roseus (SEQ ID NO: 9), additional CPRs listed in Table 7, CPRs with SEQ ID NOS: 53-55, CPR homologs of other species, and variants of naturally occurring CPRs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these CPRs that have the activity of a CPR. In various embodiments, the N-terminal nucleic acid sequences in the genes of F3H and/or CPR originated from eukaryotic cells can encode targeting leader peptides, which can be removed before introduction into prokaryotic host cells, if desired. In some embodiments, the hydroxylase complex HpaBC from E. coli was used to hydroxylate naringenin to eriodictyol or dihydrokaempferol to dihydroquercetin (taxifolin).
(29) TABLE-US-00006 TABLE 6 Flavonoid 3-hydroxylases Organism GenBank Accession Number Brassica napus ABC58722.1 Gerbera hybrid cultivar D1 ABA64468.1 Cephalotus follicularis GAV84063.1 Theobroma cacao XP_007037548.1 Phoenix dactylifera XP_008791304.2
(30) TABLE-US-00007 TABLE 7 Cytochrome P450 reductases Organism GenBank Accession Number Catharanthus roseus CAA49446.1 Brassica napus XP_013706600.1 Cephalotus follicularis GAV59576.1 Camellia sinensis XP_028084858.1
(31) A nucleic acid sequence encoding a F3H can in some embodiments be fused to a nucleic acid sequence encoding a CPR in an engineered cell as provided herein, such that the F3H activity is fused to the CPR activity.
(32) In the cells engineered to produce dihydomyricetin, flavonoid 3, 5-hydroxylase (F35H) can be used to convert dihydrokaempferol to dihydromyricetin or naringenin to pentahydroxyflavone, which is further converted to dihydromyricetin by a F3H. F35H has the systematic name flavanone, NADPH: oxygen oxidoreductase and catalyzes the formation of 3,5-dihydroxyflavanone from flavanone. An exemplary F35H is the Delphinium grandiflorum F35H (SEQ ID NO: 10), Also considered for use in the engineered cells provided herein include F35H with SEQ ID NOS:56-57, F35H homologs of other species, and variants of naturally occurring F35H having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NOS:10 that have the activity of a F35H.
(33) Dihydroflavonol 4-reductase (DFR) acts on (+)-dihydrokaempferol (DHK), (+)-dihydroquercetin (Taxifolin, DHQ), or dihydromyricein (DHM) to reduce those compounds to the corresponding cis-flavan-3,4-diol (DHK to leucopelargonidin; Taxifolin to leucocyanidin; DHM to leucodelphinidin). An exemplary DFR is the Anthurium andraeanum DFR (SEQ ID NO: 11). Also considered for use in the engineered cells provided herein include DFRs in Table 8, DFRs with SEQ ID NOS: 58-61, and DFR homologs of other species, as well as variants of naturally occurring DFR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 11. Table 8. Dihydroflavonol 4-reductases
(34) TABLE-US-00008 TABLE 8 Dihydroflavonol 4-reductases Organism GenBank Accession Number Eustoma grandiflorum BAD34461.1 Anthurium andraeanum AAP20866.1 Camellia sinensis AAT66505.1 Morelia rubra KAB1203810.1 Dendrobium moniliforme AEB96144.1 Fragaria ananassa AHL46451.1 Rosa chinensis XP_024167119.1 Acer palmatum AWN08247.1 Nyssa sinensis KAA8531902.1 Vitis amurensis I82380.1 Abrus precatorius XP_027329642.1 Angelonia angustifolia AHM27144.1 Pyrus pyrifolia Q84KP0.1 Theobroma cacao XP_017985307 Theobroma cacao XP_007051597.2 Brassica oleracea var. capitata QKO29328.1 Rubus idaeus AXK92786.1 Citrus sinensis AAY87035.1 Gerbera hybrida P51105.1 Cephalotus follicularis GAV76940.1 Ginkgo biloba AGR34043.1 Dryopteris erythrosora QFQ61498.1 Dryopteris erythrosora QFQ61499.1 Cephalotus follicularis GAV76942.1
(35) Leucoanthocyanidin reductase (LAR) catalyzes the synthesis of catechin from 3,4-cis-leucocyanidin. LAR also synthesizes afzelechin and gallocatechin. Considered for use in the engineered cells provided herein are LAR of Desmodium uncinatum (SEQ ID NO: 12), LARs with SEQ ID NOS: 62-65, and LAR homologs of other species, as well as variants of naturally occurring LAR having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 12 (LAR, Desmodium uncinatum) that have the activity of a LAR.
(36) Optionally, the cells are further engineered to include an anthocyanin synthase (ANS) which catalyzes the conversion of leucoanthocyanidin or catechin to anthocyanidin, leucopelargonidin to pelargonidin, or leucodelphinidin to delphinidin. Considered for use in the engineered cells provided herein are ANS of Carica papaya (SEQ ID NO: 13), ANS with SEQ ID NOS: 66-69, and ANS homologs of other species, as well as variants of naturally occurring ANS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO:13 (ANS, Carica papaya) that have the activity of a ANS.
(37) Optionally, the cells are further engineered to include a flavonoid-3-glucosyl transferase (3GT) to generate anthocyanins by transfer of a sugar moiety such as, without limitation, UDP--D-glucose to anthocyanidins to form glycosylated anthocyanins. Considered for use in the engineered cells provided herein are 3GT of Vitis labrusca (SEQ ID NO:14), 3GT with SEQ ID NOS: 70-73, and 3GT homologs of other species, as well as variants of naturally occurring 3GT having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to SEQ ID NO: 14 (3GT, Vitis labrusca) that have the activity of a 3GT.
(38) In various aspects, host cells may be engineered for enhanced production of flavonoids or anthocyanins by introducing additional exogenous pathways and/or modifying endogenous metabolic pathways to remove or downregulate competitive pathways to reduce carbon loss, increase precursor supply, improve cofactor availability, reduce byproduct formation, or improve cell fitness. Enhancing or improving production of flavonoids or anthocyanins can be increasing yield, titer, or rate of production.
(39) Thus, a host cell engineered for the production of a flavonoid or anthocyanin can be engineered to include any or any combination of: overexpression of an acetyl-CoA carboxylase (ACC) or an ACC variant; expression or overexpression of at least one enzyme for increasing cell's malonyl-CoA supply that does not rely on the ACC step; expression or overexpression of at least one enzyme to increase tyrosine supply; expression or overexpression of at least one enzyme to increase CoA availability for synthesizing precursors malonyl-CoA or p-coumaryol-CoA; expression or overexpression at least one enzyme to increase heme biosynthesis; deletion or downregulation of at least one fatty acid synthesis enzyme; at least one alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, phosphate acetyl transferase, or acetate kinase; at least one enzyme of a fatty acid degradation pathway, at least one thioesterase, or at least one TCA gene. The foregoing list of modifications is nonlimiting.
(40) Malonyl-CoA is the direct precursor for chalcone synthase to perform sequential condensations with p-coumaryol-CoA. Malonyl-CoA supply can be increased by one or more modifications. Malonyl-CoA is synthesized by acetyl-CoA carboxylase (ACC) via the ATP-dependent carboxylation of acetyl-CoA in a multistep reaction. First, the biotin carboxylase domain catalyzes the ATP-dependent carboxylation of biotin using bicarbonate as a CO.sub.2 donor. In the second reaction, the carboxyl-group is transferred from biotin to acetyl-CoA to form malonyl-CoA. In most eukaryotes, including fungi, both reactions are catalyzed by a large single chain protein, but in E. coli and other bacteria, the activity is catalyzed by a multi-subunit enzyme. Host cells can be engineered for example to express an exogenous acetyl-CoA carboxylase or a variant ACC to increase malonyl-CoA synthesis from acetyl-CoA. For example, Mucor circinelloides (SEQ ID NO: 15) acetyl-CoA carboxylase can be introduced into the host cells. Additional examples of ACC genes that may be used in the engineered cells provided herein include, without limitation, the genes listed in Table 9, genes with SEQ ID NOS: 74-76, naturally occurring orthologs of these ACCs, or variants having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to referenced genes. Further, naturally occurring acetyl-CoA carboxylase genes can be further engineered to introduce single or multiple amino acid mutations to increase catalytic activity and/or remove feedback inhibition.
(41) TABLE-US-00009 TABLE 9 Acetyl-CoA carboxylases Organism GenBank Accession Number Lipomyces starkeyi AJT60321.1 Rhodotorula toruloides GEM08739.1 Ustilago maydis XP_011390921.1 Mucor circinelloides EPB82652.1 Kalaharituber pfeilii KAF8466702.1 Aspergillus fumigatus KEY77072.1 Rhodotorula diobovata TNY18634.1 Leucosporidium creatinivorum ORY74050.1 Microbotryum intermedium SCV70467.1 Mixia osmundae GAA98306.1 Puccinia graminis KAA1079218.1 Suillus occidentalis KAG1764021.1 Gymnopilus junonius KAF8909366.1
(42) Additional strategies for increasing malonyl-CoA include increasing acetyl-CoA, which is converted to malonyl-CoA by acetyl-CoA carboxylase (ACC). Acetyl-CoA can be synthesized from acetate by an acyl-CoA ligase in an ATP-dependent reaction. Acetyl-CoA synthetase (ACS) or acetate-CoA ligase (EC 6.2.1.1.) catalyzes the formation of a new chemical bond between acetate and CoA coenzyme A (CoA). ACSs with native activity on acetate will provide the function of increasing acetyl-CoA supply when cells are either supplied with acetate as a co-feed, or where acetate is produced as a by-product. Other acyl-CoA ligases, having their main activity on other acid substrates, may also have substantial activity on acetate, and are viable candidates for providing acetate-CoA ligase activity in the engineered cells provided herein. The ACSs expressed in the host cells can be prokaryotic or eukaryotic. Cultures of engineered host cells that overexpress a nucleic acid sequence encoding ACS can optionally include acetate in the culture medium. Examples of acetyl-CoA synthase that can be expressed in a host cell engineered to produce a flavonoid or anthocyanin include, without limitation, the ACS gene of E. coli, the ACS of Salmonella typhimurium (SEQ ID NO:16), and orthologs of these ACSs in other species having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% amino acid identity to these ACSs.
(43) Alternatively, or in addition, an engineered host cell can overexpress a gene encoding pyruvate dehydrogenase (PDH), which converts pyruvate to acetyl-CoA, to increase acetyl-CoA supply. PDH catalyzes an irreversible metabolic step, and the control of its activity is complex and involves control by its substrates and products. Nicotinamide adenine dinucleotide hydrogen (NADH), a product of the PDH reaction, is a competitive inhibitor of the PDH complex. The NADH sensitivity of the PDH complex has been demonstrated to reside in LPD, the enzyme that interacts with NAD+ as a substrate. Thus, a variant of the Lpd subunit of PDH can be expressed that includes one or more mutations that reduces inhibition of PDH by NADH. Such an example is a LPD variant in E. coli that contains E354K mutation, and the mutated enzyme was less sensitive to NADH inhibition than the native LPD.
(44) Alternatively, or in addition to strategies for increasing ACC activity and strategies for increasing acetyl-CoA, strategies for increasing malonyl-CoA by mechanisms that do not rely on the activity of an ACC can be employed. For example, a cell engineered to produce a flavonoid or an anthocyanin as provided herein can include an exogenous nucleic acid sequence encoding a malonyl-CoA synthetase (EC 6.2.1.14) that generates malonyl-CoA from malonate. Acyl-CoA synthetase catalyzes the conversion of a carboxylic acid to its acyl-CoA thioester through an ATP-dependent two-step reaction. In the first step, the free fatty acid is converted to an acyl-AMP intermediate with the release of pyrophosphate. In the second step, the activated acyl group is coupled to the thiol group of CoA, releasing AMP and the acyl-CoA product. Nonlimiting examples of malonyl-CoA synthetases include the malonyl-CoA synthetases of Streptomyces coelicolor (SEQ ID NO:17), matB of Rhodopseudomonas palustris (SEQ ID NO: 77), matB of Rhizobium sp, BUS003 (SEQ ID NO: 78), matB of Ochrobacrum sp. (SEQ ID NO: 79), or other homologs having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced sequences. Malonate can optionally be added to the culture medium of a culture that includes a cell engineered to express a malonyl-CoA synthetase. In Rhizobium trifolii, the matB gene is part of the matABC operon, with matA encoding a malonyl-CoA decarboxylase and matC encoding a putative dicarboxylate carrier protein or malonate transporter. An engineered cell that includes an exogenous gene encoding a malonyl-CoA synthetase can also include an exogenous nucleic acid sequence encoding a malonate transporter, such as a malonate transporter encoded by a matC gene, for example of Streptomyces coelicolor (SEQ ID NO:18), of Rhizobiales bacterium (SEQ ID NO:80), of Rhizobium leguminosarum (SEQ ID NO:81), of Agrobacterium vitis (SEQ ID NO: 82), of Neorhizobium sp. (SEQ ID NO: 83), or a malonate transporter encoded by DctPQM of Sinorhizobium medicae, or encoding a malonyl-CoA transporter having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to a naturally-occurring malonate transporter. Cell cultures of a host cell engineered to express a malonyl-CoA synthetase and a malonate transporter can include a culture medium that includes malonate.
(45) In additional embodiments, a cell engineered to produce a flavonoid or an anthocyanin is further engineered to include an exogenous nucleic acid sequence encoding malonate CoA-transferase (EC:2.8.3.3; also referred to as the alpha subunit of malonate decarboxylase) that makes malonyl-CoA by direct transfer of the CoA from acetyl-CoA. For example, the alpha subunit of malonate decarboxylase from the mdcACDE gene cluster in Acinetobacter calcoaceticus has the malonate CoA-transferase activity. The mdcA gene product, the a subunit, is malonate CoA-transferase, and mdcD gene product, the subunit, is a malonyl-CoA decarboxylase. The mdcE gene product, the subunit, may play a role in subunit interaction to form a stable complex or as a codecarboxylase. The mdcC gene product, the subunit, was an acyl-carrier protein, which has a unique CoA-like prosthetic group. When the subunit is removed from the complex and incubated with malonate and acetyl-CoA, the acetyl-CoA moiety of the prosthetic group binds on an subunit to exchange the acetyl group for a malonyl group. As the thioester transfer should be thermodynamically favorable, the engineered cells can include a nucleic acid encoding a malonate CoA-transferase to increase malonyl-CoA supply. Examples of mdcAs that can be expressed in an engineered cell as provided herein include, without limitation, mdcA of Acinetobacter calcoaceticus (SEQ ID NO: 19), mdcAs of Table 10, mdcAs with SEQ ID NOS: 84-87, or a transferase having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally occurring malonate CoA-transferases.
(46) TABLE-US-00010 TABLE 10 Malonate CoA-transferases (malonate decarboxylase subunit alpha) Organism GenBank Accession Number Acinetobacter calcoaceticus AAB97627.1 Geobacillus sp. QNU36929.1 Acinetobacter johnsonii WP_087014029.1 Acinetobacter marinus WP_092618543.1 Acinetobacter rudis WP_016655668.1 Psychrobacter sp. G WP_020444454.1 Moraxella catarrhalis WP_064617969.1 Zoogloea sp. MBL0283742.1 Dechloromonas sp. KAB2923906.1 Stenotrophomonas rhizophila WP_123729366.1 Xanthomonas cucurbitae WP_159407614.1
(47) In some embodiments, a cell engineered to produce flavonoids or anthocyanins is further engineered to increase the supply of coenzyme A (CoA) to increase its availability for producing acetyl-CoA, malonyl-CoA, and/or p-coumaroyl-CoA. Strategies for increasing CoA supply include expressing or overexpressing at least one enzyme of a CoA biosynthesis pathway. Pantothenate kinase (EC 2.7.1.33, PanK; CoaA) is the first enzyme in the coenzyme CoA biosynthetic pathway. It phosphorylates pantothenate (vitamin B5) to form 4-phosphopantothenate at the expense of a molecule of adenosine triphosphate (ATP). It is the rate-limiting step in the biosynthesis of CoA. Three distinct types of PanK have been identifiedPanK-I (found in bacteria), PanK-II (mainly found in eukaryotes, but also in the Staphylococci) and PanK-III, also known as CoaX (found in bacteria). In E. coli, pantothenate kinase is competitively inhibited by CoA itself, as well as by some CoA esters. The type III enzymes CoaX are not subject to feedback inhibition by CoA. In some embodiments, a host cell can be engineered to include a nucleic acid sequence encoding type III pantothenate kinase that is not feedback inhibited by coenzyme A, such as, without limitation, CoaX gene of Pseudomonas aeruginosa (EC:2.7.1.33, SEQ ID NO: 20), CoaX of Streptomyces sp. CLI2509 (SEQ ID NO: 88), CoaX of Streptomyces cinereus (SEQ ID:89), or CoaX of Kitasatospora kifunensis (SEQ ID NO: 90) Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes pantothenate, a precursor of CoA biosynthesis, and can optionally also include cysteine, used in the CoA biosynthesis.
(48) Additional strategies to increase malonyl-CoA flux to the flavonoid pathway include mutation or downregulation of one or more genes that function in fatty acid biosynthesis. Fatty acid biosynthesis directly competes with flavonoid biosynthesis for the precursor malonyl-CoA and thus limits flavonoid formation. Without limiting the embodiments to any particular mechanism, limiting fatty acid biosynthesis can increase the malonyl-CoA supply available for flavonoid biosynthesis. In some embodiments, the gene beta-ketoacyl-ACP synthase II (E. coli fabF) can be disrupted, attenuated or deleted to reduce fatty acid biosynthesis. Another example of a fatty acid biosynthesis gene of a host cell that may be mutated or downregulated is a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD). Other fatty acid biosynthesis genes of the engineered host cell that can be downregulated include a beta-ketoacyl-ACP synthase I enzyme (E. coli fabB) and/or acyl carrier protein (E. coli acpP).
(49) Additional genetic modifications that may be present in a host cell engineered to produce flavonoids or anthocyanins include downregulation, disruption, or deletion of the gene targets that divert carbon flux to form byproducts such as ethanol, acetate, and lactate. They include genes encoding alcohol dehydrogenase, lactate dehydrogenase, pyruvate oxidase, acetyl phosphate transferase and acetate kinase. In an E. coli host cell, genes that are downregulated, disrupted, or deleted can include adhE, ldhA, poxB, and ackA-pta.
(50) Further, a cell engineered for the production of flavonoids or anthocyanins can have one or more genes encoding thioesterases downregulated, disrupted, or deleted to prevent hydrolysis of precursors malonyl-CoA, acetyl-CoA, and/or p-coumaryol-CoA. Acyl-CoA thioesterase enzymes (ACOTs) catalyze the hydrolysis of acyl-CoAs (short-, medium-, long- and very long-chain), bile acid-CoAs, and methyl branched-CoAs, to the free fatty acid and coenzyme A. For example, in an E. coli host one or more of the thioesterase genes tesA, tesB, yciA, and/or ybgC can be downregulated, disrupted, or deleted.
(51) In further embodiments, a cell engineered for the production of flavonoids or anthocyanins can have one or more of fatty acid degradation genes downregulated, disrupted, or deleted to improve precursor supply to the flavonoid pathway. In E. coli, for example, the acyl-coenzyme A dehydrogenase (fade) gene encoding acyl-CoA dehydrogenase, adhesion A (fadA) gene encoding 3-ketoacyl-CoA thiolase, and/or gene encoding fatty acid oxidation complex subunit alpha (fadB) can be downregulated, disrupted, or deleted.
(52) Alternatively, or in addition, genes encoding enzymes of the tricarboxylic acid cycle (TCA), such as succinate dehydrogenase, can be disrupted or downregulated to increase alpha-ketoglutarate supply which serves as a cofactor for the flavonoid and anthocyanin pathway enzymes. Other TCA enzymes that can be downregulated include citrate synthase that converts acetyl-CoA to citrate.
(53) Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins to upregulate the endogenous biosynthesis of amino acid tyrosine. Tyrosine is one of the precursors for the flavonoid biosynthesis and its conversion to 4-coumaric acid is the first committed step of the pathway. Efficient biosynthesis of L-tyrosine from feedstock such as glucose or glycerol is necessary to make biological production economically viable. L-tyrosine is one of the three aromatic amino acids derived from the shikimate pathway. The shikimate pathway is the central metabolic route leading to formation of tryptophan (TRP), tyrosine (TYR), and phenylalanine (PHE), this pathway exclusively exists in plants and microorganisms. It starts with the condensation of intermediates of glycolysis and pentosephosphate-pathway, phosphoenolpyruvate (PEP), and erythrose-4-phosphate (E4P), respectively, which enter the pathway through a series of condensation and redox reactions via 3-deoxy-d-arabino-heptulosonate-7-phosphate (DAHP), 3-dehydroquinate (DHQ), 3-dehydroshikimate (DHS) to shikimate. From there the central branch point metabolite chorismate is obtained via shikimate-3-phosphate under ATP hydrolysis and introduction of a second PEP. The initial step of the shikimate pathway is catalyzed by DAHP synthase isozymes and regulated through feedback-inhibition. In E. coli three DAHP synthase isozymes exist (aroF, aroG, aroH), which are each feedback inhibited by one of the three aromatic amino acids (TYR, PHE, TRP), in contrast the two DAHP synthases of plants are not subject to feedback-inhibition. In plants and bacteria, the subsequent five steps are catalyzed by single enzymes. From the central intermediate chorismate the pathway branches off to anthranilate and prephenate leading to aromatic amino acid, para-hydroxybenzoic acid (pHBA) and para-aminobenzoic acid (pABA) synthesis, the latter being a precursor for folate metabolism. Strategies to increase L-tyrosine production can include, without limitation, transcriptional deregulation, removing feedback inhibition, overexpression of rate-limiting enzymes, and/or deletion of the L-phenylalanine branch of the aromatic acid biosynthetic pathway. For example, in an E. coli host the tyrR gene can be disrupted, feedback-inhibition-resistant versions of the DAHP synthase (aroG) and chorismate mutase (tyrA) can be introduced, and/or rate-limiting enzymes, shikimate kinase (aroK or aroL) and quinate (QUIN)/shikimate dehydrogenase (ydiB) can be overexpressed. Further, the ppsA, aroG, and/or transketolase (tktA) can be overexpressed or exogenously introduced to enhance tyrosine production.
(54) Also considered, in further embodiments, is an engineered host cell for the production of flavonoids or anthocyanins further engineered to upregulate the endogenous biosynthesis of cofactor heme. Cytochrome P450 (CYPs), one of the exogenous genes in the engineered cells provided herein, contain heme as a cofactor. Improving heme supply can be an effective strategy to increase flavonoid biosynthesis. 5-aminolevulinic acid (ALA) is the first committed precursor to the heme pathway. There exist two known alternate routes by which this committed intermediate is generated. One route is the C4 pathway (Shemin pathway), which involves the condensation of succinyl-CoA and glycine to D-aminolevulinic acid by ALA synthase (ALAS). The C4 pathway is restricted to mammals, fungi and purple nonsulfur bacteria. The second route is the C5 pathway, which involves three enzymatic reactions resulting in the biosynthesis of ALA from the five-carbon skeleton of glutamate. The C5 pathway is active in most bacteria, all archaea and plants. Seven additional reactions, including assembly of eight ALA molecules into a cyclic tetrapyrrole, modification of the side chains, and incorporation of reduced iron into the molecule, are required to convert ALA to heme. In an E. coli host, the three enzymes involved in ALA biosynthesis are glutamyl-tRNA synthetase (GltX), glutamyl-tRNA reductase (hemA), and glutamate-1-semialdehyde aminotransferase (hemL). In an E. coli host, the engineered cells provided herein can be further engineered to express or overexpress hemA or its variants, and/or hemL to increase the heme precursor ALA production. The nonlimiting examples of hemA gene that can be overexpressed include, without limitation, a mutated hemA gene from Salmonella typhimurium (EC:1.1.1.70, SEQ ID NO: 21) and hemA with SEQ ID NOS: 91-93. Alternatively, or in addition, a heterologous ALAS gene can be introduced to produce ALA via the C4 pathway. Nonlimiting examples of heterologous ALAS that can be expressed in E. coli include ALAS of Rhodobacter capsulatus (SEQ ID:22), ALAS with SEQ ID NOS: 94-97, or an ALAS having at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to any of these or other naturally-occurring ALAS. Further, one or more of the downstream genes (E. coli hemB, hemC, hemD, hemE, hemF, hemG, hemI, or hemH) that catalyze the synthesis of heme from ALA can be overexpressed to drive the flux from ALA to heme production. Cultures of cells engineered for the production of flavonoids or anthocyanins can in some embodiments include a medium that includes succinate and/or glycine, precursors of heme biosynthesis via the C4 pathway.
(55) Engineered cells that produce a flavonoid can be engineered to include multiple pathways to enhance flavonoid production. Those skilled in the art will recognize that the embodiments described herein can be combined in multiple ways. Examples of engineered cells having multiple genetic modifications are exemplary only and do not limit the scope of the invention.
(56) Enzymes to be expressed or overexpressed in engineered cells according to the invention are set forth in Table 11.
(57) Host Cells
(58) A host cell as provided herein can be a prokaryotic cell or a eukaryotic cell. Eukaryotic cells may be microbial eukaryotic cells, such as, for example, fungal cells or yeast cells. Prokaryotic cells that can be engineered as provided herein include bacterial cells and cyanobacterial cells.
(59) Host can be selected based on their ability to take up and utilize particular carbon sources, nitrogen sources, or precursor molecules or may be engineered to take up and utilize molecules that may be added to the culture medium.
(60) Nonlimiting examples of suitable microbial hosts for the bio-production of a flavonoid include, but are not limited to, any gram-negative organisms, more particularly a member of the family Enterobacteriaceae, such as E. coli, any gram-positive microorganism, for example Bacillus subtilis, Lactobacillus sp. or Lactococcus sp.; a yeast, for example Saccharomyces cerevisiae, Pichia pastoris or Pichia stipitis; and other groups or microbial species. More particularly, suitable microbial hosts for the bio-production of a flavonoid generally include, but are not limited to, members of the genera Clostridium, Zymomonas, Escherichia, Salmonella, Rhodococcus, Pseudomonas, Bacillus, Lactobacillus, Enterococcus, Alcaligenes, Klebsiella, Paenibacillus, Arthrobacter, Corynebacterium, Brevibacterium, Pichia, Candida, Hansenula, and Saccharomyces.
(61) Culture Medium
(62) In yet another aspect, methods for producing a flavonoid or an anthocyanin that include incubating a culture of an engineered host cell as provided herein to produce a flavonoid or an anthocyanin. The methods can further include recovering the flavonoid or anthocyanin from the culture medium, whole culture, or cells.
(63) The culture comprises cells engineered for the production of flavonoids or anthocyanins in a culture medium. In various embodiments the engineered cells can be prokaryotic or eukaryotic cells. The culture medium includes at least one carbon source that is also an energy source. Exemplary carbon sources include glucose, glycerol, sucrose, fructose, and xylose. Such carbon sources may be purified or crude, including a biomass comprising glycerol, for example, crude glycerol produced as a byproduct of biodiesel production from corn waste. In addition, the culture medium can include one or more other carbon sources or compounds to increase precursor generation or cofactor supply such as, without limitation, tyrosine, phenylalanine, coumaric acid, acetate, malonate, succinate, glycine, bicarbonate, biotin, naringenin, 5-aminolevulinic acid, thiamine, pantothenate, alpha-ketoglutarate, and ascorbate. In some embodiments, tyrosine and coumaric acid are provided in the culture medium. In some embodiments, tyrosine, alpha-ketoglutarate, 5-aminolevulinic acid, and ascorbate are provided in the culture medium.
(64) Culture conditions can include aerobic, microaerobic or any combination alternating aerobic/microaerobic growth conditions. Further, culture conditions can include shake flasks, fermentation, and other large scale culture procedures. An exemplary growth condition for achieving a flavonoid product include aerobic or microaerobic fermentation conditions. The culture conditions can be scaled up and grown continuously for manufacturing flavonoid product. Exemplary growth procedures include, for example, fed-batch fermentation and batch separation. In an exemplary batch fermentation protocol, the cells are grown in a bioreactor that is well controlled for growth temperature, oxygen, pH, carbon sources, and other compounds. The desired temperature can be from, for example, 20-37 C., depending on the growth characteristics of the production cells and desired conditions for the fermented products. The pH of the bioreactor can be controlled to range from 5-8 or left uncontrolled in some cases. The batch fermentation period can last in the range of several hours to several days, for examples, 8 to 96 hours. Upon completion of the cultivation period, the fermenter contents can be passed through a cell separation unit to remove cells and cell debris. The cells can be lysed or disrupted enzymatically or chemically prior to or after separation of cells from the fermentation broth, as desired, in order to release additional product. To purify the flavonoids and/or anthocyanins to homogeneity the solution containing the flavonoids and/or anthocyanins was concentrated and the product purified via ion exchange or silica-based chromatography. The resulting solution was either lyophilized to yield the products in a solid form or was concentrated into a liquid solution.
(65) In some embodiments, a method of producing a flavonoid or an anthocyanin comprises culturing an engineered cell disclosed herein in a culture medium to produce a flavonoid or an anthocyanin. In some embodiments, glycerol is used as a carbon feedstock. In some embodiments, the glycerol is crude glycerol. In some embodiments, the method comprises isolating naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside. In some embodiments the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of greater than 50%, greater than 55%, greater than 60%, greater than 65%, greater than 70%, greater than 75%, greater than 80%, greater than 85%, greater than 90%, or greater than 95%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to about 99%, e.g., from about 50% to about 95% (for example from: about 50%, 55%, 60%, 65%, 70%, 75%, 80% to about: 85%, 90%, 95%, 97.5%, 99% or 99.9%). In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 50% to: about 55%, about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 55% to: about 60%, about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 60% to: about 65%, about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 65% to: about 70%, about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 70% to: about 75%, about 80%, about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 75% to: about 80%, about 85%, about 90%, about 95%, or about 99%, from about 80% to about 85%, about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 85% to: about 90%, about 95%, or about 99%. In some embodiments, the naringenin, dihydrokaempferol, taxifolin, eriodictyol, leucocyanidin, leucodelphinidin, leucopelargonidin, (+)-catechin, cyanidin, delphinidin, pelargonidin, cyanidin glucoside, delphinidin glucoside or pelargonidin glucoside may be isolated at a purity of from about 90% to about 95%, or about 99%, or from about 95% to about 99% or greater.
VIII. EXAMPLES
(66) Using the Modified Cell to Create Products
Example 1Production of Naringenin in E. coli
(67) An E. coli cell derived from MG1655 was engineered to overexpress ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) to produce naringenin when substrates tyrosine and coumaric acid were supplied in culture medium. ACC was expressed on a medium-copy plasmid (15-20 copies) while TAL, 4CL, CHS, and CHI were expressed on the chromosome. Cells of an OD 2.5 were cultured in a 48-well plate at 30 degree for 24 hours with a shaking speed of 600 RPM in minimal medium supplied with trace element, vitamins, 1 mM tyrosine, 1 mM coumaric acid, and 2% glycerol. Cell cultures were extracted with DMSO at 1:1 ratio and centrifuged for 15 mins. The supernatant was analyzed for naringenin with HPLC. The cells produced 232 M naringenin.
(68) Variants of the foregoing host cell may be prepared using one or more of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), and CHI (SEQ ID NO: 6) with one or more homologs of ACC (SEQ ID NO: 15), TAL (SEQ ID NO: 1), 4CL (SEQ ID NO: 4), CHS (SEQ ID NO: 5), or CHI (SEQ ID NO: 6), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
Example 2Production of Dihydrokaempferol in E. coli
(69) An E. coli cell derived from MG1655 was engineered to overexpress F3H (SEQ ID NO: 7) on the chromosome to produce dihydrokaempferol when substrate naringenin was supplied in culture medium. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glycerol, trace elements, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with DMSO and centrifuged for 15 minutes. The supernatant was analyzed for dihydrokaempferol with HPLC. The cells produced 315 M dihydrokaempferol.
(70) Variants of the foregoing host cell may be prepared using a homolog of F3H (SEQ ID NO: 7), wherein the homologous enzyme has at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzyme.
Example 3Production of Taxifolin in E. coli
(71) An E. coli strain derived from MG1655 was engineered to overexpress F3H (SEQ ID NO: 7), F3H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) to produce taxifolin when the substrate naringenin was supplied in culture medium. F3H was overexpressed on the chromosome while F3H and CPR were overexpressed on a medium-copy plasmid. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 2% glucose, 0.8 mM naringenin, 65 mg/L 5-aminoleuvinic acid, 0.1 mM ferrous sulfate, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were extracted with 50% DMSO and centrifuged for 15 minutes. The supernatant was analyzed for taxifolin with HPLC. The cells produced 500 M taxifolin.
(72) Variants of the foregoing host cell may be prepared using one or more of F3H (SEQ ID NO: 7), F3H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9) along with one or more homologs of F3H (SEQ ID NO: 7), F3H (SEQ ID NO: 8), and CPR (SEQ ID NO: 9), or combinations of two or more thereof, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
Example 4Production of Anthocyanidins and Anthocyanins
(73) An E. coli strain derived from MG1655 was engineered to overexpress ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) to produce cyanidin-3-O-glucoside when the substrate (+)-catechin was supplied in culture medium. ANS and 3GT were overexpressed on the chromosome. Cells of an OD 0.5-0.7 were cultured in a 24-well plate at 30 degree for 18 hours with a shaking speed of 200 RPM in minimal medium supplied with 1.0% glucose, 2.0 mM (+)-catechin, 0.1 mM 2-oxoglutarate, and 2.5 mM ascorbic acid. Cell cultures were acidified with 2M HCL and extracted with 100% Ethanol. The supernatant was analyzed for cyanidin-3-O-glucoside by HPLC. The cells produced 50 mg/L cyanidin-3-O-glucoside.
(74) Variants of the foregoing host cell may be prepared using one or both of ANS (SEQ ID NO: 13) and 3GT (SEQ ID NO: 14) along with a homolog of ANS (SEQ ID NO: 13), 3GT (SEQ ID NO: 14), or both, wherein the homologous enzymes have at least 50%, at least 55%, at least 60%, at least 65%, at least 70%, at least 75%, at least 80%, at least 85%, at least 90%, at least 95%, at least 96%, at least 97%, at least 98%, or at least 99% identity to the referenced enzymes.
(75) Analytical Methods
Example 5Flavonoid Precursors and Flavonoids
(76) For sampling naringenin, eriodictyol, dihydrokaempferol and taxifolin, extraction of total flavonoids from E. coli were performed on whole cell broth. 500 L of whole cell broth was vortexed for 30 seconds with 500 L of DMSO (dimethyl sulfoxide) and centrifuged for 15 minutes. For HPLC analysis, 50 L of supernatant was transferred to an HPLC vial.
(77) The HPLC method was as follows: An Agilent 1200 HPLC was fitted with an Ascentis C18 Column 150 mm4.6 mm, 3 m, equipped with a R-18 (3 m) guard column. The column was heated to 30 C. with the sample block being maintained at 25 C. For each sample, 5 L was injected and the product was eluted at a flow rate of 1.5 mL/min using 0.1% phosphoric acid in water (solvent A), acetonitrile (solvent B), and methanol (solvent C) with the following gradient:
(78) TABLE-US-00011 Time A (%) B (%) C (%) 0 85 10 5 2.5 85 10 5 7.5 70 25 5 12.5 50 45 5 15 85 10 5
(79) The run time was a total of 15 minutes with naringenin, eriodictyol, dihydrokaempferol and taxifolin eluting at 12.50, 11.56, 10.20, and 8.85 minutes respectively. A diode array detector (DAD) was used for the detection of the molecule of interest at 288 nm.
Example 6Anthocyanidins and Anthocyanins
(80) For sampling (+)-catechin, cyanidin, and cyanidin-3-glucoside the reaction fluid was acidified with 13 M HCl (1:40 v/v), and extracted with 100% ethanol followed by mixing, centrifugation and filtration through a 0.45 m filter. The HPLC method was as follows: An Agilent 1200 HPLC was fitted with a LiChrospher RP-8 Column 250 mm4.6 mm, 5 m, equipped with a LiChrospher 100 RP-8 (5 m) LiChroCART 4-4 guard column. The column was heated to 25 C. with the sample block being maintained at 25 C. For each sample, 10 L was injected and the product was eluted at a flow rate of 1.0 ml/min using 0.1% phosphoric acid in water (solvent A) and acetonitrile (solvent B) with the following gradient: 90% A to 10% A for 12 min, 90% A for 0.5 min, and 90% A for 3.5 min for column equilibration. The run time was a total of 16 minutes with cyanidin-3-glycoside eluting at 6.95 mins and cyanidin eluting at 8.9 minutes. A diode array detector (DAD) was used for the detection of the molecule of interest at either 280 nm or 530 nm.
Example 7Flavonoid Production
(81) The example provides a combination of modifications to the E. coli host genome including deletions and overexpression of enzymes from other organisms to recapitulate the bioproduction pathway described in
(82) As shown in
(83) As shown in
(84) As shown in
(85) As shown in
(86) As shown in
(87) The compositions as described above, can be used in methods described herein for increasing the production of flavonoids or anthocyanins. Such methods involve providing any of the compositions described above to result in enzymatic transformation by the engineered host cell of glycerol through multiple chemical intermediates into a flavonoid or anthocyanin (such as shown in part or in whole in
(88) In yet another aspect, it is envisioned that the pathway illustrated in
(89) Aspects of the invention are now described with reference herein to
(90) In another aspect, the invention provides a method of increasing the production of flavonoids comprising an engineered host cell, wherein the one or more engineered host cells comprise one or more genetic modifications to increase production and/or availability of malonyl-CoA. In certain embodiments, the engineered host cell comprises one or more genetic modifications selected from: (i) expression of acetyl-CoA carboxylase (ACC); and (ii) overexpression of acetyl-CoA carboxylase. In another embodiment, the engineered host cell is an E. coli. In certain embodiments, the acetyl-CoA carboxylase is from: Mucor circinelloides, Rhodotorula toruloides, Lipomyces starkeyi, and Ustilago maydis, and orthologs of acetyl-CoA carboxylase having at least 50% amino acid identity to the acetyl-CoA carboxylase of these aforementioned species. In certain embodiments, one or more genetic modification is deletion or attenuation of one or more fatty biosynthetic genes resulting in decrease in fatty acid biosynthesis. In certain embodiments, one or more genetic modification is overexpression of acetyl-CoA synthase (ACS). In certain embodiments, the acetyl-CoA synthase is selected from: acetyl-CoA synthase gene of E. coli, acetyl-CoA synthase gene of Salmonella typhimurium, and orthologs of acetyl-CoA synthase gene in any other species having at least 50% amino acid identity to the acetyl-CoA synthase gene of E. coli and Salmonella typhimurium. In certain embodiments, one or more genetic modification is selected from a group consisting of: (i) overexpression a gene encoding pyruvate dehydrogenase (PDH), wherein the PDH may include E354K mutation; (ii) exogenous nucleic acid sequence encoding a malonyl-CoA synthetase; (iii) upregulation of endogenous pantothenate kinase (PanK), wherein PanK is not feedback inhibited by coenzyme A; (iv) exogenous nucleic acid sequence encoding a malonate transporter; and (v) any combinations thereof. In certain embodiments, the malonyl-CoA synthetase is selected from of malonyl-CoA synthetases of Streptomyces coelicolor, Rhodopseudomonas palustris, or a malonyl-CoA synthetase having at least 50% identity to any of these or other naturally occurring malonyl-CoA synthetases. In certain embodiments, one or more genetic modifications to decrease fatty acid biosynthesis is selected from: (i) mutation or downregulation of a gene encoding malonyl-CoA-ACP transacylase (E. coli fabD); (ii) modifications to the gene beta-ketoacyl-ACP synthase II (E. coli fabF); (iii) downregulation of beta-ketoacyl-ACP synthase I enzyme (E. coli fabB); (iv) downregulation of acyl carrier protein (E. coli acpP); and (v) any combinations thereof. In certain embodiments, the engineered host cell comprises peptides selected from: (i) acetyl-CoA carboxylase (ACC) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 15 or SEQ ID NO: 16; (ii) malonate CoA-transferase having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 19; (iii) acetyl-CoA synthase (ACS) having an amino acid sequence at least 80% identical to the polypeptide set forth in SEQ ID NO: 16; (iv) malonyl-CoA synthase having an amino acid sequence at least 80% identical SEQ ID NO: 77, SEQ ID NO: 78, or SEQ ID NO: 79; (v) malonate transporter having an amino acid sequence at least 80% identical to SEQ ID NO: 80, SEQ ID NO: 81, SEQ ID NO: 82, SEQ ID NO: 83, SEQ ID NO: 84, SEQ ID NO: 85, SEQ ID NO: 86, or SEQ ID NO: 87; (vi) pantothenate kinase having an amino acid sequence at least 80% identical to SEQ ID NO: 88, SEQ ID NO: 89, or SEQ ID NO: 90; and (vii) any combinations thereof. Step 7: conversion of mal-CoA to malonyl-ACP (acyl carrier protein). malonyl-coA-ACP transacylase (fabD) is downregulated to increase carbon flux. Step 8: conversion of malonyl-ACP to 3-ketyoacyl-ACP. beta-ketoacyl-ACP synthase II (fabF) is downregulated to increase carbon flux. Step 9: conversion to mal-CoA to naringenin chalcone; conversion of coumaryl-CoA to naringenin chalcone. A heterologous CHS is overexpressed. Step 10: conversion to naringenin chalcone to naringenin. A heterologous CHI is overexpressed.
(91) Steps 11, 12, and 13: conversion of naringenin to dihydrokaempferol (DHK); conversion of naringenin to eriodictyol (EDL); conversion of eriodictyol (EDL) to dihydroquercetin (DHQ); conversion of (DHK) to dihydroquercetin (DHQ); conversion of dihydrokaempferol (DHK) to dihydromyricetin (DHM); conversion of pentahydroxyflayaone (PHF) to dihydromyricein (DHM). Heterologous F35H, F3H, F3H, and/or CPR are overexpressed. Accordingly, as shown in
(92) As shown in
(93) In another aspect, the invention provides a method for increasing the production of flavonoids comprising an engineered host cell, wherein the engineered host cell comprises one or more genetic modifications to increase transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase. In certain embodiments, the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof. In certain embodiments, one or more engineered host cells comprises flavonoid-3-glucosyl transferase (3GT). In certain embodiments, flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications comprises overexpression of anthocyanin synthase and flavonoid-3-glucosyl transferase (3GT). In certain embodiments, the one or more genetic modifications are selected from a group consisting of: (i) anthocyanin synthase, (ii) flavonoid-3-glucosyl transferase (3GT), and (iii) a combination thereof.
(94) In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising anthocyanin synthase, wherein the anthocyanin synthase is selected from: (i) anthocyanin synthase of Carica papaya (SEQ. ID NO:13); (ii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 66, SEQ. ID NO: 67, SEQ. ID NO: 68, or SEQ. ID NO: 69; (iii) the anthocyanin synthase has an amino acid sequence at least 80% identical to SEQ. ID NO: 13; and (iv) any combinations thereof.
(95) In another aspect, the invention provides a method of increasing the transformation of leucocyanidin or catechin to cyanidin-3-glucoside (Cy3G) comprising flavonoid-3-glucosyl transferase (3GT), wherein the flavonoid-3-glucosyl transferase is selected from: (i) flavonoid-3-glucosyl transferase in Vitis labrusca (SEQ. ID NO:14); (ii) the flavonoid-3-glucosyl transferase has an amino acid sequence at least 80% identical to SEQ. ID NO: 70, SEQ. ID NO: 71, SEQ. ID NO: 72, or SEQ. ID NO: 73; and (iii) any combinations thereof. Step 17: conversion of pelargonidin to callistephin; conversion of delphinidin to myrtillin (De3G); conversion of cyanidin to Cy3G. Heterologous 3GT was overexpressed in E. coli. Step 17 could be carried in vivo or as a cell-free reaction. Step 18: conversion of pyruvate to phosphoenolpyruvate (PEP). ppsA is overexpressed to upregulate tyrosine. Step 19: conversion of fructose-6-phosphate (F6P) to erythrose-4-phosphate (E4P). tktA is overexpressed to upregulate tyrosine. Step 20: conversion of phosphoenolpyruvate (PEP) to deoxy-d-arabino-heptulosonate-7-phosphate (DAHP). aroG variant is overexpressed to upregulate tyrosine. Step 21: conversion of deoxy-d-arabino-heptulosonate-7-phosphate (DAHP) to dehydroquinate (DHQ); conversion of erythrose-4-phosphate (E4P) to dehydroquinate (DHQ). Step 22: conversion of dehydroquinate (DHQ) to 3-dehydroshikimate (DHS). Step 23: conversion of 3-dehydroshikimate (DHS) to shikimic acid (SHK). aroE is overexpressed to upregulate tyrosine. Step 24: conversion of shikimic acid (SHK) to shikimate-3-phosphate (S3P). Step 25: conversion of shikimate-3-phosphate (S3P) to 5-enolpyruvylshikimate-3-phosphate (EPSP). Step 26: conversion of 5-enolpyruvylshikimate-3-phosphate (EPSP) to chorismic acid (CHA). Step 27: conversion of chorismic acid (CHA) to prephenate (PPA); conversion of prephenate (PPA) to 4-hydroxy-phenylpyruvate (HPP). tryA variant is overexpressed. Step 28: conversion of 4-hydroxy-phenylpyruvate (HPP) to tyrosine; conversion of phenylpyruvate (POPP) to phenylalanine (Phe). Accordingly, as shown in
(96) As shown in
(97) TABLE-US-00012 TABLE11 EnzymeSequences: Enzyme: Sequence: SEQID: Tyrosineammonia- MTQVVERQADRLSSREYLARVVRSAGWDAGLTSCTD 1 lyase(TAL) EEIVRMGASARTIEEYLKSDKPIYGLTQGFGPLVLFDA Saccharothrix DSELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNM espanaensis RKGYSAVSPVFWQKLADLWNKGFTPAIPRHGTVSAS Accession: GDLQPLAHAALAFTGVGEAWTRDADGRWSTVPAVD ABC88669.1 ALAALGAEPFDWPVREALAFVNGTGASLAVAVLNHR SALRLVRACAVLSARLATLLGANPEHYDVGHGVARG QVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSLRCA PQVLGAVLDQLDGAGDVLAREVDGCQDNPITYEGEL LHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGL LVSPVTNGDLPPMLTPRAGRGAGLAGVQISATSFVSRI RQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEAL ELGWLTVGSLAVGVAQLAAMTGHAAEGVWAELAGI CPPLDADRPLGAEVRAARDLLSAHADQLLVDEADGK DFG Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 2 ammonia-lyase GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV (PAL) TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD Brevibacillus EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG laterosporusLMG ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE 15441 REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC Accession: LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ WP_003337219.1 KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD RALDGDIEKVVQLIRSGNLKKEIHDQNVND Cinnamate-4- MDLLLIEKTLLALFAAIIGAIVISKLRGKRFKLPPGPLP 3 hydroxylase(C4H) VPIFGNWLQVGDDLNHRNLTDLAKKFGEIFLLRMGQ Helianthusannuus RNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFTG L. KGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYR Accession: YGWEAEAAAVVEDVKKNPAAATEGVVIRRRLQLMM QJC72299.1 YNNMFRIMFDRRFESEDDPLFVKLKALNGERSRLAQS FEYNYGDFIPILRP FLKGYLKLCKEVKEKRFQLFKDYFVDERKKLESTKSV DNNQLKCAIDHILDAKEKGEINEDNVLYIVENINVAAI ETTLWSIEWGIAELVNHPEIQAKLRNELDTKLGPGVQ VTEPDLHKLPYLQAVIKETLRLRMAIPLLVPHMNLHD AKLGGYDIPAESKILVNAWWLANNPEQWKKPEEFRP ERFFEEESKVEANGNDFRYLPFGVGRRSCPGIILALPIL GITIGRLVQNFELLPPPGQSKVDTTEKGGQFSLHILKHS TIVAKPRAL 4-coumarate-CoA MGDCVAPKEDLIFRSKLPDIYIPKHLPLHTYCFENISKV 4 ligase(4CL) GDKSCLINGATGETFTYSQVELLSRKVASGLNKLGIQ Petroselinum QGDTIMLLLPNSPEYFFAFLGASYRGAISTMANPFFTS crispum AEVIKQLKASQAKLIITQACYVDKVKDYAAEKNIQIIC Accession: IDDAPQDCLHFSKLMEADESEMPEVVINSDDVVALPY P14912.1 SSGTTGLPKGVMLTHKGLVTSVAQQVDGDNPNLYM HSEDVMICILPLFHIYSLNAVLCCGLRAGVTILIMQKF DIVPFLELIQKYKVTIGPFVPPIVLAIAKSPVVDKYDLS SVRTVMSGAAPLGKELEDAVRAKFPNAKLGQGYGM TEAGPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIV DPETNASLPRNQRGEICIRGDQIMKGYLNDPESTRTTI DEEGWLHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVA PAELEALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRT NGFTTTEEEIKQFVSKQVVFYKRIFRVFFVDAIPKSPSG KILRKDLRARIASGDLPK Chalconesynthase MVTVEEYRKAQRAEGPATVMAIGTATPTNCVDQSTY 5 (CHS) PDYYFRITNSEHKTDLKEKFKRMCEKSMIKKRYMHLT Petuniaxhybrida EEILKENPSMCEYMAPSLDARQDIVVVEVPKLGKEAA Accession: QKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLTKL AAF60297.1 LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK GARVLVVCSEITAVTFRGPNDTHLDSLVGQALFGDGA GAIIIGSDPIPGVERPLFELVSAAQTLLPDSHGAIDGHL REVGLTFHLLKDVPGLISKNIEKSLEEAFRPLSISDWNS LFWIAHPGGPAILDQVEIKLGLKPEKLKATRNVLSNY GNMSSACVLFILDEMRKASAKEGLGTTGEGLEWGVL FGFGPGLTVETVVLHSVAT Chalconeisomerase MAASITAITVENLEYPAVVTSPVTGKSYFLGGAGERG 6 (CHI) LTIEGNFIKFTAIGVYLEDIAVASLAAKWKGKSSEELL Medicagosativa ETLDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVMENC Accession: VAHLKSVGTYGDAEAEAMQKFAEAFKPVNFPPGASV P28012.1 FYRQSPDGILGLSFSPDTSIPEKEAALIENKAVSSAVLE TMIGEHAVSPDLKRCLAARLPALLNEGAFKIGN Flavanone3- MAPTPTTLTAIAGEKTLQQSFVRDEDERPKVAYNQFS 7 hydroxylase(F3H) NEIPIISLSGIDEVEGRRAEICNKIVEACEDWGVFQIVD Rubusoccidentalis HGVDAKLISEMTRLARDFFALPPEEKLRFDMSGGKKG Accession: GFIVSSHLQGEAVQDWREIVTYFSYPVRHRDYSRWPD ACM17897.1 KPEGWRAVTQQYSDELMGLACKLLEVLSEAMGLEKE ALTKACVDMDQKVVVNFYPKCPQPDLTLGLKRHTDP GTITLLLQDQVGGLQATRDGGKTWITVQPVEGAFVV NLGDHGHFLSNGRFKNADHQAVVNSNHSRLSIATFQ NPAQEAIVYPLKVREGEKPILEEPITYTEMYKKKMSK DLELARLKKLAKEQQPEDSEKAKLEVKQVDDIFA Flavonoid3 MTNLYLTILLPTFIFLIVLVLSRRRNNRLPPGPNPWPIIG 8 hydroxylase(F3H) NLPHMGPKPHQTLAAMVTTYGPILHLRLGFADVVVA Brassicanapus ASKSVAEQFLKVHDANFASRPPNSGAKHMAYNYQDL Accession: VFAPYGQRWRMLRKISSVHLFSAKALEDFKHVRQEE ABC58723.1 VGTLMRELARANTKPVNLGQLVNMCVLNALGREMI GRRLFGADADHKAEEFRSMVTEMMALAGVFNIGDFV PALDCLDLQGVAGKMKRLHKRFDAFLSSILEEHEAM KNGQDQKHTDMLSTLISLKGTDFDGEGGTLTDTEIKA LLLNMFTAGTDTSASTVDWAIAELIRHPEIMRKAQEE LDSVVGRGRPINESDLSQLPYLQAVIKENFRLHPPTPLS LPHIASESCEINGYHIPKGSTLLTNIWAIARDPDQWSDP LTFRPERFLPGGEKAGVDVKGNDFELIPFGAGRRICAG LSLGLRTIQLLTATLVHGFEWELAGGVTPEKLNMEET YGITLQRAVPLVVHPKLRLDMSAYGLGSA CytochromeP450 MDSSSEKLSPFELMSAILKGAKLDGSNSSDSGVAVSPA 9 reductase(CPR) VMAMLLENKELVMILTTSVAVLIGCVVVLIWRRSSGS Catharanthus GKKVVEPPKLIVPKSVVEPEEIDEGKKKFTIFFGTQTGT roseus AEGFAKALAEEAKARYEKAVIKVIDIDDYAADDEEYE Accession: EKFRKETLAFFILATYGDGEPTDNAARFYKWFVEGND Q05001 RGDWLKNLQYGVFGLGNRQYEHFNKIAKVVDEKVA EQGGKRIVPLVLGDDDQCIEDDFAAWRENVWPELDN LLRDEDDTTVSTTYTAAIPEYRVVFPDKSDSLISEANG HANGYANGNTVYDAQHPCRSNVAVRKELHTPASDRS CTHLDFDIAGTGLSYGTGDHVGVYCDNLSETVEEAER LLNLPPETYFSLHADKEDGTPLAGSSLPPPFPPCTLRTA LTRYADLLNTPKKSALLALAAYASDPNEADRLKYLAS PAGKDEYAQSLVANQRSLLEVMAEFPSAKPPLGVFFA AIAPRLQPRFYSISSSPRMAPSRIHVTCALVYEKTPGGR IHKGVCSTWMKNAIPLEESRDCSWAPIFVRQSNFKLP ADPKVPVIMIGPGTGLAPFRGFLQERLALKEEGAELGT AVFFFGCRNRKMDYIYEDELNHFLEIGALSELLVAFSR EGPTKQYVQHKMAEKASDIWRMISDGAYVYVCGDA KGMARDVHRTLHTIAQEQGSMDSTQAEGFVKNLQM TGRYLRDVW Flavonoid3,5- MSTSLLLAAAAILFFITHLFLRFLLSPRRTRKLPPGPKG 10 hydroxylase WPVVGALPMLGNMPHAALADLSRRYGPIVYLKLGSR (F35H) GMVVASTPDSARAFLKTQDLNFSNRPTDAGATHIAYN Delphinium SQDMVFADYGPRWKLLRKLSSLHMLGGKAVEDWAV grandiflorum VRRDEVGYMVKAIYESSCAGEAVHVPDMLVFAMAN Accession: MLGQVILSRRVFVTKGVESNEFKEMVIELMTSAGLFN BAO66642 VGDFIPSIAWMDLQGIVRGMKRLHKKFDALLDKILRE HTATRRERKEKPDLVDVLMDNRDNKSEQERLTDTNI KALLLNLFSAGTDTSSSTIEWALTEMIKNPSIFGRAHA EMDQVIGRNRRLEESDIPKLPYLQAICKETFRKHPSTP LNLPRVAIEPCEVEGYHIPKGTRLSVNIWAIGRDPNVW ENPLEFNPDRFLTGKMAKIDPRGNNFELIPFGAGRRIC AGTRMGIVLVEYILGSLVHAFEWKLRDGETLNMEETF GIALQKAVPLAAVVTPRLPPSAYVV Dihydroflavonol4- MMHKGTVCVTGAAGFVGSWLIMRLLEQGYSVKATV 11 reductase(DFR) RDPSNMKKVKHLLDLPGAANRLTLWKADLVDEGSFD Anthurium EPIQGCTGVFHVATPMDFESKDPESEMIKPTIEGMLNV andraeanum LRSCARASSTVRRVVFTSSAGTVSIHEGRRHLYDETS Accession: WSDVDFCRAKKMTGWMYFVSKTLAEKAAWDFAEK AAP20866.1 NNIDFISIIPTLVNGPFVMPTMPPSMLSALALITRNEPH YSILNPVQFVHLDDLCNAHIFLFECPDAKGRYICSSHD VTIAGLAQILRQRYPEFDVPTEFGEMEVFDIISYSSKKL TDLGFEFKYSLEDMFDGAIQSCREKGLLPPATKEPSYA TEQLIATGQDNGH Leucoanthocyanidin MTVSGAIPSMTKNRTLVVGGTGFIGQFITKASLGFGYP 12 reductase(LAR) TFLLVRPGPVSPSKAVIIKTFQDKGAKVIYGVINDKEC Desmodium MEKILKEYEIDVVISLVGGARLLDQLTLLEAIKSVKTIK uncinatum RFLPSEFGHDVDRTDPVEPGLTMYKEKRLVRRAVEEY Accession: GIPFTNICCNSIASWPYYDNCHPSQVPPPMDQFQIYGD Q84V83.1 GNTKAYFIDGNDIGKFTMKTIDDIRTLNKNVHFRPSSN CYSINELASLWEKKIGRTLPRFTVTADKLLAHAAENII PESIVSSFTHDIFINGCQVNFSIDEHSDVEIDTLYPDEKF RSLDDCYEDFVPMVHDKIHAGKSGEIKIKDGKPLVQT GTIEEINKDIKTLVETQPNEEIKKDMKALVEAVPISAM G Anthocyanin MFSSVAVPRVEILASSGIESIPKEYVRPQEELTTIGNIFD 13 dioxygenase(ANS) EEKKDEGPQVPTIDLRDIDSDDQQVRQRCRDELKKAA Caricapapaya VDWGVMHLVNHGIPDHLIDRVKKAGQAFFELPVEVK Accession: EKYANDQASGNIQGYGSKLANNASGQLEWEDYYFHL XP_021901846.1 IFPEEKRDLAIWPNNPADYIEVTSEYARQLRRLVSKIL GVLSLGLGLEEGRLEKEVGGLDELLLQMKINYYPTCP QPELALGVEAHTDISALTFILHNMVPGLQLFYEGKWV TAKCVPNSIVMHVGDTIEILSNGKYKSILHRGLVNKEK VRISWAVFCEPPKEKIILKPLPETVSENEPPLFPPRTFAQ HIQHKLFRKNQENLEAK Anthocyanidin-3- MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAVAAPH 14 O-glycotransferase AVFSFFSTSESNASIFHDSMHTMQCNIKSYDVSDGVPE (3GT) GYVFTGRPQEGIDLFMRAAPESFRQGMVMAVAETGR Vitislabrusca PVSCLVADAFIWFAADMAAEMGVAWLPFWTAGPNS Accession: LSTHVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFR ABR24135 DLQEGIVFGNLNSLFSRLLHRMGQVLPKATAVFINSFE ELDDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCL QWLKERKPTSVVYISFGTVTTPPPAELVALAEALEASR VPFIWSLRDKARMHLPEGFLEKTRGHGMVVPWAPQA EVLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFF GDQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQI LSQEKGKKLRENLRALRETADRAVGPKGSSTENFKTL VDLVSKPKDV Acetyl-CoA MVEHRSLPGHFLGGNSLESAPQGPVKDFVQAHEGHT 15 carboxylase(ACC) VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF Mucor TVMATPEDLKANAEYIRMADNFVEVPGGSNNNNYAN circinelloides VELIVDVAERTAVHAVWAGWGHASENPRLPEMLAKS 1006PhL KHKCLFIGPPASAMRSLGDKISSTIVAQSAQVPTMGW Accession: SGDGITETEFDAAGHVIVPDNAYNEACVKTAEQGLKA EPB82652.1 AEKIGFPVMIKASEGGGGKGIRMVKDGSNFAQLFAQV QGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLFG RDCSVQRRHQKIIEEAPVTIAKPDVFEQMEKAAVRLG KLVGYVSAGTVEYLYSHHDDQFYFLELNPRLQVEHPT TEMVSGVNLPAAQLQIAMGIPLHRIRDIRVLYGVQPNS ASEIDFGFEHPTSLTSHRRPTPKGHVIACRITAENPDAG FKPSSGIMQELNFRSSTNVWGYFSVVSAGGLHEYADS QFGHIFAYGENRQQARKNMVIALKELSIRADFRSTVE YIIRLLETPDFEENTINTGWLDMLISKKLTAERPDTML AVFCGAVTKAHMASLDCFQQYKQSLEKGQVPSKGSL KTVFTVDFIYEEVRYNFTVTQSAPGIYTLYLNGTKTQV GIRDLSDGGLLISIDGKSHTTYSRDEVQATRMMVDGK TCLLEKESDPTQLRSPSPGKLVNLLVENGDHLNAGDA YAEIEVMKMYMPLIATEDGHVQFIKQAGATLEAGDII GILSLDDPSRVKHALPFNGTVPAFGAPHITGDKPVQRF NATKLTLQHILQGYDNQALVQTVVKDFADILNNPDLP YSELNSVLSALSGRIPQRLEASIHKLADESKAANQEFP AAQFEKLVEDFAREHITLQSEATAYKNSVAPLSSIFAR YRNGLTEHAYSNYVELMEAYYDVEILFNQQREEEVIL SLRDQHKDDLDKVLAVTLSHAKVNIKNNVILMLLDLI NPVSTGSALDKYFTPILKRLSEIESRATQKVTLKAREL LILCQLPSYEERQAQMYQILKNSVTESVYGGGSEYRTP SYDAFKDLIDTKFNVFDVLPHFFYHADPYIALAAIEVY CRRSYHAYKILDVAYNLEHKPYVVAWKFLLQTAANG IDSNKRIASYSDLTFLLNKTEEEPIRTGAMTACNSLAD LQAELPRILTAFEEEPLPPMLQRNAAPKEERMENILNI AVRADEDMDDTAFRTKICEMITANADVFRQAHLRRL SVVVCRDNQWPDYYTFRERENYQEDETIRHIEPAMA YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE IVSHEYKNSDCNHLFINFIPTFAIEADDVEHALKDFVD RHGKRLWKLRVTGAEIRFNVQSKKPDAPIIPMRFTVD NVSGFILKVEVYQEVKTEKSGWILKSVNKIPGAMHM QPLSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQ SVQNQWTQAIKRNPLLKQPSHLVEAKELVLDEDDVL QEIDRAPGTNTVGMVAWIMTIRTPEYPSGRRIIAIANDI TFKIGSFGVAEDQVFYKASELARALGIPRIYLSANSGA RIGLADELISQFRAAWKDASNPTAGFKYLYLTPAEYD VLAQQGDAKSVLVEEIQDEGETRLRITDVIGHTDGLG VENLKGSGLIAGATSRAYDDIFTITLVTCRSVGIGAYL VRLGQRTIQNEGQPIILTGAPALNKVLGREVYTSNLQL GGTQIMYKNGVSHLTAENDLEGIAKIVQWLSFVPDVR NAPVSMRLGADPIDRDIEYTPPKGPSDPRFFLAGKSEN GKWLSGFFDQDSFVETLSGWARTVVVGRARLGGIPM GVVSVETRTVENIVPADPANSDSTEQVFMEAGGVWFP NSAYKTAQAINDFNKGEQLPLMIFANWRGFSGGQRD MYNEVLKYGAQIVDALSNYKQPVFVYIIPNGELRGGA WVVVDPTINKDMMEMYADNNARGGVLEPEGIVEIKY RKPALLATMERLDATYASLKKQLAEEGKTDEEKAAL KVQVEAREQELLPVYQQISIQFADLHDRAGRMKAKG VIRKALDWRRARHYFYWRVRRRLCEEYTFRKIVTATS AAPMPREQMLDLVKQWFTNDNETVNFEDADELVSE WFEKRASVIDQRISKLKSDATKEQIVSLGNADQEAVIE GFSQLIENLSEDARAEILRKLNSRF Acetyl-CoA MSQTHKHAIPANIADRCLINPEQYETKYKQSINDPDTF 16 synthase(ACS) WGEQGKILDWITPYQKVKNTSFAPGNVSIKWYEDGT Salmonella LNLAANCLDRHLQENGDRTAIIWEGDDTSQSKHISYR typhimurium ELHRDVCRFANTLLDLGIKKGDVVAIYMPMVPEAAV Accession: AMLACARIGAVHSVIFGGFSPEAVAGRIIDSSSRLVITA NP_463140.1 DEGVRAGRSIPLKKNVDDALKNPNVTSVEHVIVLKRT GSDIDWQEGRDLWWRDLIEKASPEHQPEAMNAEDPL FILYTSGSTGKPKGVLHTTGGYLVYAATTFKYVFDYH PGDIYWCTADVGWVTGHSYLLYGPLACGATTLMFEG VPNWPTPARMCQVVDKHQVNILYTAPTAIRALMAEG DKAIEGTDRSSLRILGSVGEPINPEAWEWYWKKIGKE KCPVVDTWWQTETGGFMITPLPGAIELKAGSATRPFF GVQPALVDNEGHPQEGATEGNLVITDSWPGQARTLF GDHERFEQTYFSTFKNMYFSGDGARRDEDGYYWITG RVDDVLNVSGHRLGTAEIESALVAHPKIAEAAVVGIP HAIKGQAIYAYVTLNHGEEPSPELYAEVRNWVRKEIG PLATPDVLHWTDSLPKTRSGKIMRRILRKIAAGDTSNL GDTSTLADPGVVEKLLEEKQAIAMPS Malonyl-CoA MSSLFPALSPAPTGAPADRPALRFGERSLTYAELAAA 17 synthase(matB) AGATAGRIGGAGRVAVWATPAMETGVAVVAALLAG Streptomyces VAAVPLNPKSGDKELAHILSDSAPSLVLAPPDAELPPA coelicolor LGALERVDVDVRARGAVPEDGADDGDPALVVYTSGT Accession: TGPPKGAVIPRRALATTLDALADAWQWTGEDVLVQG WP_011028356 LPLFHVHGLVLGILGPLRRGGSVRHLGRFSTEGAAREL NDGATMLFGVPTMYHRIAETLPADPELAKALAGARL LVSGSAALPVHDHERIAAATGRRVIERYGMTETLMNT SVRADGEPRAGTVGVPLPGVELRLVEEDGTPIAALDG ESVGEIQVRGPNLFTEYLNRPDATAAAFTEDGFFRTG DMAVRDPDGYVRIVGRKATDLIKSGGYKIGAGEIENA LLEHPEVREAAVTGEPDPDLGERIVAWIVPADPAAPP ALGTLADHVAARLAPHKRPRVVRYLDAVPRNDMGKI MKRALNRD Malonate MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT 18 transporter(matC) LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT Streptomyces TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV coelicolor SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG Accession: GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA NP_626686.1 AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY GGIVVAAVPALAWLVLVVPGFG MalonateCoA- MVKKRLWDKQRTRRQEKLNLAQQKGFAKQVEHARA 19 transferase(MdcA) IELLETVIASGDRVCLEGNNQKQADFLSKCLSQCNPD Acinetobacter AVNDLHIVQSVLALPSHIDVFEKGIASKVDFSFAGPQS calcoaceticus LRLAQLVQQQKISIGSIHTYLELYGRYFIDLTPNICLITA Accession: HAADREGNLYTGPNTEDTPAIVEATAFKSGIVIAQVNE AAB97627.1 IVDKLPRVDVPADWVDFYIESPKHNYIEPLFTRDPAQI TEVQILMAMMVIKGIYAPYQVQRLNHGIGFDTAAIEL LLPTYAASLGLKGQICTNWALNPHPTLIPAIESGFVDS VHSFGSEVGMEDYIKERPDVFFTGSDGSMRSNRAFSQ TAGLYACDSFIGSTLQIELQGNSSTATVDRISGFGGAP NMGSDPHGRRHASYAYTKAGREATDGKLIKGRKLVV QTVETYREHMHPVFVEELDAWQLQDKMDSELPPIMI YGEDVTHIVTEEGIANLLLCRTDEEREQAIRGVAGYTP VGLKRDAAKVEELRQRGIIQRPEDLGIDPTQVSRDLLA AKSVKDLVKWSGGLYSPPSRFRNW Pantothenatekinase MILELDCGNSLIKWRVIEGAARSVAGGLAESDDALVE 20 (CoaX) QLTSQQALPVRACRLVSVRSEQETSQLVARLEQLFPV Pseudomonas SALVASSGKQLAGVRNGYLDYQRLGLDRWLALVAA aeruginosa HHLAKKACLVIDLGTAVTSDLVAADGVHLGGYICPG Accession: MTLMRSQLRTHTRRIRYDDAEARRALASLQPGQATA Q9HWCL1 EAVERGCLLMLRGFVREQYAMACELLGPDCEIFLTGG DAELVRDELAGARIMPDLVFVGLALACPIE glutamyl-tRNA MTKKLLALGINHKTAPVSLRERVTFSPDTLDQALDSL 21 reductase(hemA.sup.m) LAQPMVQGGVVLSTCNRTELYLSVEEQDNLQEALIR Salmonella WLCDYHNLNEDDLRNSLYWHQDNDAVSHLMRVASG typhimurium LDSLVLGEPQILGQVKKAFADSQKGHLNASALRRMF Accession: QKSFSVAKRVRTETDIGASAVSVAFAACTLARQIFESL AAA88610.1 STVTVLLVGAGETIELVARHLREHKVQKMIIANRTRE RAQALADEVGAEVISLSDIDARLQDADIIISSTASPLPII GKGMVERALKSRRNQPMLLVDIAVPRDVEPEVGKLA NAYLYSVDDLQSIISHNLAQRQAAAVEAETIVEQEASE FMAWLRAQGASETIREYRSQSEQIRDELTTKALSALQ QGGDAQAILQDLAWKLTNRLIHAPTKSLQQAARDGD DERLNILRDSLGLE 5-aminolevulinic MDYNLALDKAIQKLHDEGRYRTFIDIEREKGAFPKAQ 22 acidsynthase WNRPDGGKQDITVWCGNDYLGMGQHPVVLAAMHE (ALAS) ALEAVGAGSGGTRNISGTTAYHRRLEAEIADLHGKEA Rhodobacter ALVFSSAYIANDATLSTLRLLFPGLIIYSDSLNHASMIE capsulatus GIKRNAGPKRIFRHNDVAHLRELIAADDPAAPKLIAFE Accession: SVYSMDGDFGPIKEICDIADEFGALTYIDEVHAVGMY CAA37857 GPRGAGVAERDGLMHRIDIFNGTLAKAYGVFGGYIA ASAKMVDAVRSYAPGFIFSTSLPPAIAAGAQASIAFLK TAEGQKLRDAQQMHAKVLKMRLKALGMPIIDHGSHI VPVVIGDPVHTKAVSDMLLSDYGVYVQPINFPTVPRG TERLRFTPSPVHDLKQIDGLVHAMDLLWARCA Tyrosineammonia- MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTP 23 lyase(TAL) ALRERCARAHARLEHAIAEQRHIYGITTGFGPLANRLI Rhodobacter GADQGAELQQNLIYHLATGVGPKLSWAEARALMLAR capsulatusSB1003 LNSILQGASGASPETIDRIVAVLNAGFAPEVPAQGTVG Accession: ASGDLTPLAHMVLALQGRGRMIDPSGRVQEAGAVM ADE84832.1 DRLCGGPLTLAARDGLALVNGTSAMTAIAALTGVEA ARAIDAALRHSAVLMEVLSGHAEAWHPAFAELRPHP GQLRATERLAQALDGAGRVCRTLTAARRLTAADLRP EDHPAQDAYSLRVVPQLVGAVWDTLDWHDRVVTCE LNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDAL NAALVTLAGLVERQIARLTDEKLNKGLPAFLHGGQA GLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNG ANQDVVSMGTIAARRARAQLLPLSQIQAILALALAQA MDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRP LAGHIEAVAQGLRHPSAAADPPA Tyrosineammonia- MITETNVAKPASTKVMNGDAAKAAPVEPFATYAHSQ 24 lyase(TAL) ATKTVVIDGHNMKVGDVVAVARHGAKVELAASVAG Trichosporon PVQASVDFKESKKHTSIYGVTTGFGGSADTRTSDTEA cutaneum LQISLLEHQLCGYLPTDPTYEGMLLAAMPIPIVRGAM Accession: AVRVNSCVRGHSGVRLEVLQSFADFINIGLVPCVPLR XP_018276715 GTISASGDLSPLSYIAGAICGHPDVKVFDTAASPPTVLT APEAIAKYKLKTVRLASKEGLGLVNGTAVSAAAGAL ALYDAECLAMMSQTNTALTVEALDGHVGSFAPFIQEI RPHVGQIEAAKNIRHMLSNSKLAVHEEPELLADQDAG ILRQDRYALRTSAQWIGPQLEMLGLARQQIETELNSTT DNPLIDVEGGMFHHGGNFQAMAVTSAMDSTRIVLQN LGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYHCK GLDIHCGAYCAELGFLANPMSNHVQSTEMHNQSVNS MAFASARKTMEANEVLSLLLGSQMYCATQALDLRV MEVKFKMAIVKLLNDTLTKHFSTFLTPEQLAKLNTTA AITLYKRLNQTPSWDSAPRFEDAAKHLVGCIMDALM VNDDITDLTNLPKWKKEFAKDAGDLYRSILTATTADG RNDLEPAEYLGQTRAVYEAIRSDLGVKVRRGDVAEG KSGKSIGSNVARIVEAMRDGRLMGAVSKMFF Tyrosineammonia- MNTINEYLSLEEFEAIIFGNQKVTISDVVVNRVNESFNF 25 lyase(TAL) LKEFSGNKVIYGVNTGFGPMAQYRIKESDQIQLQYNLI Flavobacterium RSHSSGTGKPLSPVCAKAAILARLNTLSLGNSGVHPSV johnsoniae INLMSELINKDITPLIFEHGGVGASGDLVQLSHLALVLI Accession: GEGEVFYKGERRPTPEVFEIEGLKPIQVEIREGLALING WP_012023194 TSVMTGIGVVNVYHAKKLLDWSLKSSCAINELVQAY DDHFSAELNQTKRHKGQQEIALKMRQNLSDSTLIRKR EDHLYSGENTEEIFKEKVQEYYSLRCVPQILGPVLETI NNVASILEDEFNSANDNPIIDVKNQHVYHGGNFHGDY ISLEMDKLKIVITKLTMLAERQLNYLLNSKINELLPPFV NLGTLGFNFGMQGVQFTATSTTAESQMLSNPMYVHSI PNNNDNQDIVSMGTNSAVITSKVIENAFEVLAIEMITIV QAIDYLGQKDKISSVSKKWYDEIRNIIPTFKEDQVMYP FVQKVKDHLINN Tyrosineammonia- MSTTLILTGEGLGIDDVVRVARHQDRVELTTDPAILA 26 lyase(TAL) QIEASCAYINQAVKEHQPVYGVTTGFGGMANVIISPEE Herpetosiphon AAELQNNAIWYHKTGAGKLLPFTDVRAAMLLRANSH aurantiacusDSM MRGASGIRLEIIQRMVTFLNANVTPHVREFGSIGASGD 785 LVPLISITGALLGTDQAFMVDFNGETLDCISALERLGL Accession: PRLRLQPKEGLAMMNGTSVMTGIAANCVHDARILLA ABX04526.1 LALEAHALMIQGLQGTNQSFHPFIHRHKPHTGQVWA ADHMLELLQGSQLSRNELDGSHDYRDGDLIQDRYSL RCLPQFLGPIIDGMAFISHHLRVEINSANDNPLIDTASA ASYHGGNFLGQYIGVGMDQLRYYMGLMAKHLDVQI ALLVSPQFNNGLPASLVGNIQRKVNMGLKGLQLTANS IMPILTFLGNSLADRFPTHAEQFNQNINSQGFGSANLA RQTIQTLQQYIAITLMFGVQAVDLRTHKLAGHYNAAE LLSPLTAKIYHAVRSIVKHPPSPERPYIWNDDEQVLEA HISALAHDIANDGSLVSAVEQTLSGLRSIILFR Phenylalanine MHDDNTSPYCIGQLGNGAVHGADPLNWAKTAKAME 27 ammonia-lyase CSHLEEIKRMVDTYQNATQVMIEGATLTVPQVAAIAR (PAL) RPEVHVVLDAANARSRVDESSNWVLDRIMGGGDIYG Physcomitrella VTTGFGATSHRRTQQGVELQRELIRFLNAGVLSKGNS patens LPSETARAAMLVRTNTLMQGYSGIRWEILHAMEKLL Accession: NAHVTPKLPLRGTITASGDLVPLSYIAGLLTGRPNSKA XP_001758374.1 VTEDGREVSALEALRIAGVEKPFELAPKEGLALVNGT AVGSALASTVCYDANIMVLLAEVLSALFCEVMQGKP EFADPLTHKLKHHPGQMEAAAVMEWVLDGSSFMKA AAKFNETDPLRKPKQDRYALRTSPQWLGPQVEVIRNA THAIEREINSVNDNPIIDAARGIALHGGNFQGTPIGVSM DNMRLSLAAIAKLMFAQFSELVNDYYNNGLPSNLSG GPNPSLDYGMKGAEIAMASYLSEINYLANPVTTHVQS AEQHNQDVNSLGLVSARKTEEAMEILKLMSATFLVG LCQAIDLRHVEETMQSAVKQVVTQVAKKTLFMGSDG SLLPSRFCEKELLMVVDRQPVFSYIDDSTSDSYPLMEK LRGVLVSRALKSADKETSNAVFRQIPVFEAELKLQLSR VVPAVREAYDTKGLSLVPNRIQDCRTYPLYKLVRGDL KTQLLSGQRTVSPGQEIEKVFNAISAGQLVAPLLECVQ GWTGTPGPFSARASC Phenylalanine MIETNHKDNFLIDGENKNLEINDIISISKGEKNIIFTNEL 28 ammonia-lyase LEFLQKGRDQLENKLKENVAIYGINTGFGGNGDLIIPF (PAL) DKLDYHQSNLLDFLTCGTGDFFNDQYVRGIQFIIIIALS Dictyostelium RGWSGVRPMVIQTLAKHLNKGIIPQVPMHGSVGASG discoideumAX4 DLVPLSYIANVLCGKGMVKYNEKLMNASDALKITSIE Accession: PLVLKSKEGLALVNGTRVMSSVSCISINKFETIFKAAIG XP_644510.1 SIALAVEGLLASKDHYDMRIHNLKNHPGQILIAQILNK YFNTSDNNTKSSNITFNQSENVQKLDKSVQEVYSLRC APQILGIISENISNAKIVIKREILSVNDNPLIDPYYGDVL SGGNFMGNHIARIMDGIKLDISLVANHLHSLVALMMH SEFSKGLPNSLSPNPGIYQGYKGMQISQTSLVVWLRQE AAPACIHSLTTEQFNQDIVSLGLHSANGAASMLIKLCD IVSMTLIIAFQAISLRMKSIENFKLPNKVQKLYSSIIKIIPI LENDRRTDIDVREITNAILQDKLDFFNLNL Phenylalanine MSQVALFEQELMLHGKHTLLLNGNDLTITDVAQMAK 29 ammonia-lyase GTFEAFTFHISEEANKRIEECNELKHEIMNQHNPIYGV (PAL) TTGFGDSVHRQISGEKAWDLQRNLIRFLSCGVGPVAD Brevibacillus EAVARATMLIRTNCLVKGNSAVRLEVIHQLIAYMERG laterosporusLMG ITPIIPERGSVGASGDLVPLSYLASILVGEGKVLYKGEE 15441 REVAEALGAEGLEPLTLEAKEGLALVNGTSFMSAFAC Accession: LAYADAEEIAFIADICTAMASEALLGNRGHFYSFIHEQ WP_003337219.1 KPHLGQMASAKNIYTLLEGSQLSKEYSQIVGNNEKLD SKAYLELTQSIQDRYSIRCAPHVTGVLYDTLDWVKK WLEVEINSTNDNPIFDVETRDVYNGGNFYGGHVVQA MDSLKVAVANIADLLDRQLQLVVDEKFNKDLTPNLIP RFNNDNYEIGLHHGFKGMQIASSALTAEALKMSGPVS VFSRSTEAHNQDKVSMGTISSRDARTIVELTQHVAAIH LIALCQALDLRDSKKMSPQTTKIYNMIRKQVPFVERD RALDGDIEKVVQLIRSGNLKKEIHDQNVND Cinnamate-4- MDLLLMEKTLLGLFVAVVVAITVSKLRGKKFKLPPGP 30 hydroxylase(C4H) IPVPVFGNWLQVGDDLNHRNLTEMAKKFGEVFMLR Rubussp.SSL-2007 MGQRNLVWSSPDLAKEVLHTQGVEFGSRTRNVVFDI Accession: FTGKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQ ABX74781.1 QYRYGWESEAAAVVEDVKKHPEAATNGMVLRRRLQ LMMYNNMYRIMFDRRFESEDDPLFVKLKGLNGERSR LAQSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLF KDYFVDERKKLSSTQATTNEGLKCAIDHILDAQQKGE INEDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQ KKLRDELDTVLGRGVQITEPEIQKLPYLQAVVKETLR LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF GVGRRSCPGIILALPILGITLGRLVQNFELLPPPGQTQL DTTEKGGQFSLHILKHSPIVMKPRT Cinnamate-4- MDLLLLEKTLIGLFIAIVVAIIVSKLRGKKFKLPPGPIPV 31 hydroxylase(C4H) PVFGNWLQVGDDLNHRNLTDMAKKFGDVFMLRMG Fragariavesca QRNLVVVSSPDLAKEVLHTQGVEFGSRTRNVVFDIFT Accession: GKGQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQY XP_004294725.1 RHGWEAEAAAVVEDVKKHPEAATSGMVLRRRLQLM MYNNMYRIMFDRRFESEEDPLFVKLKGLNGERSRLA QSFEYNYGDFIPVLRPFLRGYLKICKEVKEKRIQLFKD YFVDERKKLASTQVTTNEGLKCAIDHILDAQQKGEIN EDNVLYIVENINVAAIETTLWSIEWGIAELVNHPEIQK KLRDELDTVLGHGVQVTEPELHKLPYLQAVVKETLR LRMAIPLLVPHMNLHDAKLGGFDIPAESKILVNAWWL ANNPAHWKKPEEFRPERFLEEESKVEANGNDFRYLPF GVGRRSCPGIILALPILGVTLGRLVQNFEMLPPPGQTQ LDTTEKGGQFSLHILKHSTIVMKPRA Cinnamate-4- MDLLLLEKTLIGLFFAILIAIIVSKLRSKRFKLPPGPIPVP 32 hydroxylase(C4H) VFGNWLQVGDDLNHRNLTEYAKKFGDVFLLRMGQR Solanumtuberosum NLVVVSSPELAKEVLHTQGVEFGSRTRNVVFDIFTGK Accession: GQDMVFTVYGEHWRKMRRIMTVPFFTNKVVQQYRG ABC69046.1 GWESEAASVVEDVKKNPESATNGIVLRKRLQLMMYN NMFRIMFDRRFESEDDPLFVKLRALNGERSRLAQSFE YNYGDFIPILRPFLRGYLKICKEVKEKRLKLFKDYFVD ERKKLANTKSMDSNALKCAIDHILEAQQKGEINEDNV LYIVENFNVAAIETTLWSIEWGIAELVNHPHIQKKLRD EIDTVLGPGMQVTEPDMPKLPYLQAVIKETLRLRMAI PLLVPHMNLHDAKLAGYDIPAESKILVNAWWLANNP AHWKKPEEFRPERFFEEEKHVEANGNDFRFLPFGVGR RSCPGIILALPILGITLGRLVQNFEMLPPPGQSKLDTSE KGGQFSLHILKHSTIVMKPRSF 4-coumarate-CoA MGDCAAPKQEIIFRSKLPDIYIPKHLPLHSYCFENISKV 33 ligase(4CL) SDRACLINGATGETFSYAQVELISRRVASGLNKLGIHQ Daucuscarota GDTMMILLPNTPEYFFAFLGASYRGAVSTMANPFFTS Accession: PEVIKQLKASQAKLIITQACYVEKVKEYAAENNITVVC AIT52344.1 IDEAPRDCLHFTTLMEADEAEMPEVAIDSDDVVALPY SSGTTGLPKGVMLTHKGLVTSVAQRVDGENPNLYIHS EDVMICILPLFHIYSLNAVLCCGLRAGATILIMQKFDIV PFLELIQKYKVTIGPFVPPIVLAIAKSPVVDNYDLSSVR TVMSGAAPLGKELEDAVRAKFPNAKLGQGYGMTEA GPVLAMCLAFAKEPYEIKSGACGTVVRNAEMKIVDPE THASLPRNQSGEICIRGDQIMKGYLNDPESTKTTIDEE GWLHTGDIGFIDEDDELFIVDRLKEIIKYKGFQVAPAEI EALLLTHPTISDAAVVPMIDEKAGEVPVAFVVRLNGS TTTEEEIKQFVSKQVVFYKRVFRVFFVDAIPKSPSGKIL RKELRARIASGDLPK 4-coumarate-CoA MEPTTKSKDIIFRSKLPDIYIPKHLPLHTYCFENISRFGS 34 ligase(4CL) RPCLINGSTGEILTYDQVELASRRVGSGLHRLGIRQGD Strigaasiatica TIMLLLPNSPEFVLAFLGASHIGAVSTMANPFFTPAEV Accession: VKQAAASRAKLIVTQACHVDKVRDYAAEHGVKVVC GER48539.1 VDGAPPEECLPFSEVASGDEAELPAVKISPDDVVALPY SSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYIHS DDVIMCVLPLFHIYSLNSIMLCGLRVGAAILIMQKFEIV PFLELIQRYRVTIGPFVPPIVLAIEKSPVVEKYDLSSVRT VMSGAAPLGRELEDAVRLKFPNAKLGQGYGMTEAGP VLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVDTETG ASLGRNQPGEICIRGDQIMKGYLNDPESTERTIDKEGW LHTGDIGFIDDDDELFIVDRLKEIIKYKGFQVAPAELEA LLLNHPNISDAAVVSMKDEQAGEVPVAYVVKSNGSTI TEDEIKQFVSKQVIFYKRINRVFFIDAIPKSPSGKILRKD LRARLAAGVPN 4-coumarate-CoA MPMENEAKQGDIIFRSKLPDIYIPNHLSLHSYCFENISE 35 ligase(4CL) FSSRPCLINGANNQIYTYADVELNSRKVAAGLHKQFGI Capsicumannuum QQKDTIMILLPNSPEFVFAFLGASYLGAISTMANPLFTP Accession: AEVVKQVKASNAEIIVTQACHVNKVKDYALENDVKI KAF3620179.1 VCIDSAPEGCVHFSELIQADEHDIPEVQIKPDDVVALP YSSGTTGLPKGVMLTHKGLVTSVAQQVDGENPNLYI HSEDVMLCVLPLFHIYSLNSVLLCGLRVGAAILIMQKF DIVPFLELIQNYKVTIGPFVPPIVLAIAKSPMVDNYDLS SVRTVMSGAAPLGKELEDTVRAKFPNAKLGQGYGMT EAGPVLAMCLAFAKEPFEIKSGACGTVVRNAEMKIVD PDTGNSLHRNQSGEICIRGDQIMKGYLNDPEATAGTID KEGWLHTGDIGYIDNDDELFIVDRLKELIKYKGFQVA PAELEALLLNHPNISDAAVVPMKDEQAGEVPVAFVVR SNGSTITEDEVKEFISKQVIFYKRIKRVFFVDAVPKSPS GKILRKDLRAKLAAGFPN 4-coumarate-CoA MDTKTTQQEIIFRSKLPDIYIPKQLPLHSYCFENISQFSS 36 ligase(4CL) KPCLINGSTGKVYTYSDVELTSRKVAAGFHNLGIQQR Camelliasinensis DTIMLLLPNCPEFVFAFLGASYLGAIITMANPFFTPAET Accession: IKQAKASNSKLIITQSSYTSKVLDYSSENNVKIICIDSPP ASU87409.1 DGCLHFSELIQSNETQLPEVEIDSNEVVALPYSSGTTGL PKGVMLTHKGLVTSVAQQVDGENPNLYIHSEDMMM CVLPLFHIYSLNSVLLCGLRVGAAILIMQKFEIGSFLKL IQRYKVTIGPFVPPIVLAIAKSEVVDDYDLSTIRTMMS GAAPLGKELEDAVRAKFPHAKLGQGYGMTEAGPVLA MCLAFAKKPFEIKSGACGTVVRNAEMKIVDPDAGFSL PRNQPGEICIRGDQIMKGYLNDPEATERTIDKQGWLH TGDIGYIDDDDELFIVDRLKELIKYKGFQVAPAELEAL LLNHPTISDAAVVPMKDESAGEVPVAFVVRTNGFEVT ENEIKKYISEQVVFYKINRVYFVDAIPKAPSGKILRK DLRARLAAGIPS Chaiconesynthase MVTVEEYRKAQRAEGPATVMAIGTATPSNCVDQSTY 37 (CHS) PDYYFRITNSEHKTELKEKFKRMCEKSMIKTRYMHLT Capsicumannuum EEILKENPNMCAYMAPSLDARQDIVVVEVPKLGKEA Accession: AQKAIKEWGQPKSKITHLVFCTTSGVDMPGCDYQLA XP_016566084.1 KLLGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAEN NKGARVLVVCSEITAVTFRGPSESHLDSLVGQALFGD GAAAIIMGSDPIPGVERPLFQLVSAAQTLLPDSEGAID GHLREVGLTFHLLKDVPGLISKNIEKSLVEAFQPLGISD WNSLFWIAHPGGPAILDQVELKLGLKPEKLKATREVL SNYGNMSSACVLFILDEMRKASTKEGLGTSGEGLEW GVLFGFGPGLTVETVVLHSVAI Chalconesynthase MVTVEEVRKAQRAEGPATVLAIGTATPPNCIDQSTYP 38 (CHS) DYYFRITKSEHKAELKEKFQRMCDKSMIKKRYMYLT Rosachinensis EEILKENPSMCEYMAPSLDARQDMVVVEIPKLGKEAA Accession: TKAIKEWGQPKSKITHLVFCTTSGVDMPGADYQLTKL AEC13058.1 LGLRPSVKRLMMYQQGCFAGGTVLRLAKDLAENNK GARVLVVCSEITAVTFRGPSDTHLDSLVGQALFGDGA AAIIVGSDPLPEVEKPLFELVSAAQTILPDSDGAIDGHL REVGLTFHLLKDVPGLISKNIEKSLNEAFKPLNITDWN SLFWIAHPGGPAILDQVEAKLGLKPEKLEATRHILSEY GNMSSACVLFILDEVRRKSAANGHKTTGEGLEWGVL FGFGPGLTVETVVLHSVAA Cha:conesynthase MSMTPSVHEIRKAQRSEGPATVLSIGTATPTNFVPQAD 39 (CHS) YPDYYFRITNSDHMTDLKDKFKRMCEKSMITKRHMY Morusalbavar. LTEEILKENPKMCEYMAPSLDARQDIVVVEVPKLGKE multicaulis AAAKAIKEWGQPKSKITHLIFCTTSGVDMPGADYQLT Accession: KLLGLRPSVKRFMMYQQGCFAGGTVLRLAKDLAENN AHL83549.1 KGARVLVVCSEITAVTFRGPSHTHLDSLVGQALFGDG AAAVILGADPDTSVERPIFELVSAAQTILPDSEGAIDGH LREVGLTFHLLKDVPGLISKNIEKSLVEAFTPIGISDWN SIFWIAHPGGPAILDQVEAKLGLKQEKLSATRHVLSEY GNMSSACVLFILDEVRKKSVEEGKATTGEGLEWGVLF GFGPGLTVETIVLHSLPAV Chalconesynthase MAPPAMEEIRRAQRAEGPATVLAIGASTPPNALYQAD 40 (CHS) YPDYYFRITKSEHLTELKEKFKQMCDKSMIRKRYMYL Dendrobium TEEILKENPNICAFMAPSLDARQDIVVTEVPKLAREAS catenatum ARAIKEWGQPKSRITHLIFCTTSGVDMPGADYQLTRL Accession: LGLRPSVNRIMLYQQGCFAGGTVLRLAKDLAENNAG ALE71934.1 ARVLVVCSEITAVTFRGPSESHLDSLVGQALFGDGAA AIIVGSDPDLTTERPLFQLVSASQTILPESEGAIDGHLRE MGLTFHLLKDVPGLISKNIQKSLVETFKPLGIHDWNSI FWIAHPGGPAILDQVEIKLGLKEEKLASSRNVLAEYG NMSSACVLFILDEMRRRSAEAGQATTGEGLEWGVLF GFGPGLTVETVVLRSVPIAGAV Chalconeisomerase MSAITAIHVENIEFPAVITSPVTGKSYFLGGAGERGLTI 41 (CHI) EGNFIKFTAIGVYLEDVAVASLATKWKGKSSEELLET Trifoliumpratense LDFYRDIISGPFEKLIRGSKIRELSGPEYSRKVTENCVA Accession: HLKSVGTYGDAEVEAMEKFVEAFKPINFPPGASVFYR PNX83855.1 QSPDGILGVSISIHFFP Chalconeisomerase MAAASLTAVQVENLEFPAVVTSPATGKTYFLGGAGV 42 (CHI) RGLTIEGNFIKFTGIGVYLEDQAVASLATKWKGKSSEE Abrusprecatorius LVESLDFFRDIISGPFEKLIRGSKIRQLSGPEYSKKVME Accession: NCVAHMKSVGTYGDAEAAGIEEFAQAFKPVNFPPGA XP_027366189.1 SVFYRQSPDGVLGLSFSQDATIPEEEAAVIKNKPVSAA VLETMIGEHAVSPDLKRSLAARLPAVLSHGVFKIGN Chalconeisomerase MAAEPSITAIQFENLVFPAVVTPPGSSKSYFLAGAGER 43 (CHI) GLTIDGKFIKFTGIGVYLEDKAVPSLAGKWKDKSSQQ Arachisduranensis LLQTLHFYRDIISGPFEKLIRGSKILALSGVEYSRKVME Accession: NCVAHMKSVGTYGDAEAEAIQQFAEAFKNVNFKPGA XP_015942246.1 SVFYRQSPLGHLGLSFSQDGNIPEKEAAVIENKPLSSA VLETMIGEHAVSPDLKCSLAARLPAVLQQGIIVTPPQH N Chalconeisomerase MGPSPSVTELQVENVTFPPSVKPPGSTKTLFLGGAGER 44 (CHI) GLEIQGKFIKFTAIGVYLEGDAVASLAVKWKGKSKEE Cephalotus LTDSVEFFRDIVTGPFEKFTQVTTILPLTGQQYSEKVSE follicularis NCVAFWKSVGIYTDAEAKAIEKFIEVFKEETFPPGSSIL Accession: FTQSPNGALTIAFSKDGVIPEVGKAVIENKLLAEGLLE GAV77263.1 SIIGKHGVSPVAKQCLATRLSELL Flavanone3- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 45 hydroxylase(F3H) RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD Abrusprecatorius EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI Accession: MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC XP_027329642.1 WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK Flavanone3- MKDSVASATASAPGTVCVTGAAGFIGSWLVMRLLER 46 hydroxylase(F3H) GYIVRATVRDPANLKKVKHLLDLPKADTNLTLWKAD Camelliasinensis LNEEGSFDEAIEGCSGVFHVATPMDFESKDPENEVIKP Accession: TINGVLSIIRSCTKAKTVKRLVFTSSAGTVNVQEHQQP AAT66505.1 VFDENNWSDLHFINKKKMTGWMYFVSKTLAEKAAW EAAKENNIDFISIIPTLVGGPFIMPTFPPSLITALSPITRN EGHYSIIKQGQFVHLDDLCESHIFLYERPQAEGRYICSS HDATIHDLAKLMREKWPEYNVPTEFKGIDKDLPVVSF SSKKLIGMGFEFKYSLEDMFRGAIDTCREKGLLPHSFA ENPVNGNKV Flavanone3- MVDMKDDDSPATVCVTGAAGFIGSWLIMRLLQQGYI 47 hydroxylase(F3H) VRATVRDPANMKKVKHLQELEKADKNLTLWKADLT Nyssasinensis EEGSFDEAIKGCSGVFHVATPMDFESKDPENEVIKPTI Accession: NGVLSIVRSCVKAKTVKRLVFTSSAGTVNLQEHQQLV KAA8531902.1 YDENNWSDLDLIYAKKMTGWMYFVSKILAEKAAWE ATKENNIDFISIIPTLVVGPFITPTFPPSLITALSLITGNEA HYSIIKQGQFVHLDDLCEAHIFLYEQPKAEGRYICSSH DATIYDLAKMIREKWPEYNVPTELKGIEKDLQTVSFSS KKLIGMGFEFKYSLEDMYKGAIDTCREKGLLPYSTHE TPANANANANANVKKNQNENTEI Flavanone3- MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV 48 hydroxylase(F3H) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD Rosachinensis EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI Accession: MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN XP_024167119.1 WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE VDESSVVGVKVTG Flavonoid3 MSPLILYSIALAIFLYCLRTLLKRHPHRLPPGPRPWPIIG 49 hydroxylase(F3H) NLPHMGQMPHHSLAAMARTYGPLMHLRLGFVDVIV Cephalotus AASASVASQLLKTHDANFSSRPHNSGAKYIAYNYQDL follicularis VFAPYGPRWRMLRKISSVHLFSGKALDDYRHVRQEE Accession: VAVLIRALARAESKQAVNLGQLLNVCTANALGRVML GAV84063.1 GRRVFGDGSGVSDPMAEEFKSMVVEVMALAGVFNIG DFIPALDWLDLQGVAAKMKNLHKRFDTFLTGLLEEH KKMLVGDGGSEKHKDLLSTLISLKDSADDEGLKLTDT EIKALLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQV LKELDTVVGRDRLVTDLDLPQLTYLQAVIKETFRLHP STPLSLPRVAAESCEIMGYHIPKGSTLLVNVWAIARDP KEWAEPLEFRPERFLPGGEKPNVDIKGNDFEVIPFGAG RRICAGMSLGLRMVQLLTATLVHAFDWDLTSGLMPE DLSMEEAYGLTLQRAEPLMVHPRPRLSPNVY Flavonoid3 MASFLLYSILSAVFLYFIFATLRKRHRLPLPPGPKPWPII 50 hydroxylase(F3H) GNLPHMGPVPHHSLAALAKVYGPLMHLRLGFVDVV Theobromacacao VAASASVAAQFLKVHDANFSSRPPNSGAKYVAYNYQ Accession: DLVFAPYGPRWRMLRKISSVHLFSGKALDDFRHVRQ EOY22049.1 DEVGVLVRALADAKTKVNLGQLLNVCTVNALGRVM LGKRVFGDGSGKADPEADEFKSMVVELMVLAGVVNI GDFIPALEWLDLQGVQAKMKKLHKRFDRFLSAILEEH KIKARDGSGQHKDLLSTFISLEDADGEGGKLTDTEIKA LLLNMFTAGTDTSSSTVEWAIAELIRHPKILAQVRKEL DSVVGRDRLVSDLDLPNLTYFQAVIKETFRLHPSTPLS LPRMASESCEINGYHIPKGATLLVNVWAIARDPDEWK DPLEFRPERFLPGGERPNADVRGNDFEVIPFGAGRRIC AGMSLGLRMVQLLAATLVHAFDWELADGLMPEKLN MEEAFGLTLQRAAPLMVHPRPRLSPRAY Flavonoid3 MTPLTLLIGTCVTGLFLYVLLNRCTRNPNRLPPGPTPW 51 hydroxylase(F3H) PVVGNLPHLGTIPHHSLAAMAKKYGPLMHLRLGFVD Gerberahybrida VVVAASASVAAQFLKTHDANFADRPPNSGAKHIAYN Accession: YQDLVFAPYGPRWRMLRKICSVHLFSTKALDDFRHV ABA64468.1 RQEEVAILARALVGAGKSPVKLGQLLNVCTTNALAR VMLGRRVFDSGDAQADEFKDMVVELMVLAGEFNIG DFIPVLDWLDLQGVTKKMKKLHAKFDSFLNTILEEHK TGAGDGVASGKVDLLSTLISLKDDADGEGGKLSDIEI KALLLNLFTAGTDTSSSTIEWAIAELIRNPQLLNQARK EMDTIVGQDRLVTESDLGQLTFLQAIIKETFRLHPSTPL SLPRMALESCEVGGYYIPKGSTLLVNVWAISRDPKIW ADPLEFQPTRFLPGGEKPNTDIKGNDFEVIPFGAGRRIC VGMSLGLRMVQLLTATLIHAFDWELADGLNPKKLNM EEAYGLTLQRAAPLVVHPRPRLAPHVYETTKV Flavonoid3 MAPLLLLFFTLLLSYLLYYYFFSKERTKGSRAPLPPGP 52 hydroxylase(F3H) RGWPVLGNLPQLGPKPHHTLHALSRAHGPLFRLRLGS Phoenixdactylifera VDVVVAASAAVAAQFLRAHDANFSNRPPNSGAEHIA Accession: YNYQDLVFAPYGPGWRARRKLLNVHLFSGKALEDLR XP_008791304.2 PVREGELALLVRALRDRAGANELVDLGRAANKCATN ALARAMVGRRVFQEEEDEKAAEFENMVVELMRLAG VFNVGDFVPGIGWLDLQGVVRRMKELHRRYDGFLDG LIAAHRRAAEGGGGGGKDLLSVLLGLKDEDLDFDGE GAKLTDTDIKALLLNLFTAGTDTTSSTVEWALSELVK HPDILRKAQLELDSVVGGDRLVSESDLPNLPFMQAIIK ETFRLHPSTPLSLPRMAAEECEVAGYCIPKGATLLVNV WAIARDPAVWRDPLEFRPARFLPDGGCEGMDVKGND FGIIPFGAGRRICAGMSLGIRMVQFMTATLAHAFHWD LPEGQMPEKLDMEEAYGLTLQRATPLMVHPVPRLAP TAYQS CytochromeP450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI 53 reductase(CPR) IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE Camelliasinensis AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK Accession: AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG XP_028084858 DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR DVW CytochromeP450 MASNSNLIRAIESALGVSFGSELVSDTAIVVVTTSVAVI 54 reductase(CPR) IGLLFFLLKRSSDRSKESKPVVISKPLLVEEEEEEDEVE Cephalotus AGSGKTKVTMFYGTQTGTAEGFAKSLAKEIKARYEK follicularis AIVKVVDLDDYAADDDQYEQKLKKETLVFFMLATYG Accession: DGEPTDDAARFYKWFTEENERGAWLQQLTYGVFSLG GAV59576.1 NRQYEHFNKIGKVVDEQLSKQGAKRLIPVGLGDDDQ CIEDDFAAWRETLWPELDQLLRDEDDANTVSTPYAA AIPEYRVVIHDPLSGRGEAPSFSIDSHLTICEIWSTSREG SNQQISEYFWTSNSLKTMASNSNLIRSIESALGVSFGSE SVSDTAIVVVTTSVAVIIGLLFFLLKRSSDRSKESKPVV ISKPLLVEEEEDEVEAGSGKTKVTLFYGTQTGTAEGFA KSLAEEIKARYEKAIVKVVDLDDYAADDDQYEQKLK KETLVFFMLATYGDGEPTDNAARFYKWFTEENERGA WLQQLTYGVFSLGNRQYEHFNKIGKVVDEQLSKQGA KRLIPVGLGDDDQCIEDDFAAWRETLWPELDQLLRDE DDANTVSTPYTAAIPEYRVVIHDPTTTSYEDKNLNMA NGNASYDIHHPCRVNVAVQRELHKPESDRSCIHLEFDI SGTGIIYETGDHVGVYADNFDEVVEEAANLLGQPLEL LFSVHADKDDGTSLGGSLPPPFPGPCTLRDALAHYAD LLNPPRKAALSALAAHAVEPSEAERLKFLSSPQGKED YSQWVVASQRSLLEIMAEFPSAKPPLGVFFAAVAPRL QPRYYSISSSPRFVPNRVHVTCALVYGPSPTGRIHKGV CSTWMKNAVPLEKSHDCSSAPIFTRTSNFKLPTDPSIPI IMVGPGTGLAPFRGFLQERLALKEDGVQLGHAMLFFG CRNRRMDFIYEDELNNFVDQGAVSELVVAFSREGPEK EYVQHKLNAKAAQVWGLISQGGYLYVCGDAKGMAR DVHRMLHTIVEQQENVDSRKAEVIVKKLQMEGRYLR DVW CytochromeP450 MSSSSSSPFDLMSAIIKGEPVVVSDPANASAYESVAAE 55 reductase(CPR) LSSMLIENRQFAMIISTSIAVLIGCIVMLLWRRSGGSGS Brassicanapus SKRAETLKPLVLKPPREDEVDDGRKKVTIFFGTQTGT Accession: AEGFAKALGEEARARYEKTRFKIVDLDDYAADDDEY XP_013706600.1 EEKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEG DDRGEWLKNLKYGVFGLGNRQYEHFNKVAKVVDDI LVEQGAQRLVHVGLGDDDQCIEDDFTAWREALWPEL DTILREEGDTAVTPYTAAVLEYRVSIHNSADALNEKN LANGNGHAVFDAQHPYRANVAVRRELHTPESDRSCT HLEFDIAGSGLTYETGDHVGVLSDNLNETVEEALRLL DMSPDTYFSLHSDKEDGTPISSSLPPTFPPCSLRTALTR YACLLSSPKKSALLALAAHASDPTEAERLKHLASPAG KDEYSKWVVESQRSLLEVMAEFPSAKPPLGVFFAAV APRLQPRFYSISSSPKIAETRIHVTCALVYEKMPTGRIH KGVCSTWMKSAVPYEKSENCCSAPIFVRQSNFKLPSD SKVPIIMIGPGTGLAPFRGFLQERLALVESGVELGPSVL FFGCRNRRMDFIYEEELQRFLESGALSELSVAFSREGP TKEYVQHKMMDKASDIWNMISQGAYVYVCGDAKG MARDVHRSLHTIAQEQGSMDSTKAESFVKNLQMSGR YLRDVW Flavonoid3,5- MALDTFLLRELAAAAVLFLISHYLIHSLLKKSTPPLPPG 56 hydroxylase PKGWPFVGALPLLGTMPHVALAQMAKKYGPVMYLK (F35H) MGTCGMVVASTPDAARAFLKTLDLNFSNRPPNAGAT Cephalotus HLAYNAQDMVFADYGPRWKLLRKLSNLHMLGGKAL follicularis EDWTQVRTVELGHMIQAMCEASRAKEPVVVPEMLTY Accession: AMANMIGKVILGHRVFVTQGSESNEFKDMVVELMTS GAV62131 AGYFNIGDFIPSIAWMDLQGIERGMKKLHKRFDALLT KMFEEHMATAHERKGNPDLLDIVMANRDNSEGERLT TTNIKALLLNLFSAGTDTSSSIIEWSLAEMLKNPSILKR AHEEMDQVIGRNRRLEESDIKKLPYLQAICKESFRKHP STPLNLPRVSSQACQVNGYYIPKDTRLSVNIWAIGRDP EVWENPLDFTPERFLSGKNAKIDPRGNDFELIPFGAGR RICAGTRMGIVLVEYILGTLVHSFDWSLPHGVKLNMD EAFGLALQKAVPLAAIVSPRLAPTAYVV Flavonoid3,5- MSIFLITSLLLCLSLHLLLRRRHISRLPLPPGPPNLPIIGA 57 hydroxylase LPFIGPMPHSGLALLARRYGPIMFLKMGIRRVVVASSS (F35H) TAARTFLKTFDSHFSDRPSGVISKEISYNGQNMVFADY Dendrobium GPKWKLLRKVSSLHLLGSKAMSRWAGVRRDEALSMI moniliforme QFLKKHSDSEKPVLLPNLLVCAMANVIGRIAMSKRVF Accession: HEDGEEAKEFKEMIKELLVGQGASNMEDLVPAIGWL AEB96145 DPMGVRKKMLGLNRRFDRMVSKLLVEHAETAGERQ GNPDLLDLVVASEVKGEDGEGLCEDNIKGFISDLFVA GTDTSAIVIEWAMAEMLKNPSILRRAQEETDRVIGRH RLLDESDIPNLPYLQAICKEALRKHPPTPLSIPHYASEP CEVEGYHIPGETWLLVNIWAIGRDPDVWENPLVFDPE RFLQGEMARIDPMGNDFELIPFGAGRRICAGKLAGMV MVQYYLGTLVHAFDWSLPEGVGELDMEEGPGLVLPK AVPLAVMATPRLPAAAYGLL Dihydroflavonol4- MGSEAETVCVTGASGFIGSWLIMRLLERGYTVRATVR 58 reductase(DFR) DPDNEKKVKHLVELPKAKTHLTLWKADLSDEGSFDE Acerpalmatum AIHGCTGVFHVATPMDFESKDPENEVIKPTINGVLGIM Accession: KACKKAKTVKRLVFTSSAGTVDVEEHKKPVYDENSW AWN08247.1 SDLDFVQSVKMTGWMYFVSKTLAEKAAWKFAEENSI DFISVIPPLVVGPFLMPSMPPSLITALSPITRNEGHYAII KQGNYVHLDDLCMGHIFLYEHAESKGRYFCSSHSATI LELSKFLRERYPEYDLPTEYKGVDDSLENVVFCSKKIL DLGFQFKYSLEDMFTGAVETCREKGLIPLTNIDKKHV AAKGLIPNNSDEIHVAAAEKTTATA Dihydroflavonol4- MGSASETVCVTGAAGFIGSWLVMRLIQNGYKVRATV 59 reductase(DFR) RDPANMKKVKHLLELPNAKTNLSLWKADLAEEGSFD Abrusprecatorius EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGLIDI Accession: MKACMKAKTVRRLVFTSSAGTVDVTEHPKPLFDESC XP_027329642.1 WSDVQFCRRVRMTGWMYFVSKTLAEQEAWKFAKEN NIDFISVIPPLVVGPFLVPTMPPSLITALSLITGNESHYAI IKQGQFVHLDDLCLAHIFLFQHPKAQGRYICCSHEATI HDIASLLNQKYPEFNVPTKFKNIPDQLEIIRFSSKKITDL GFKFKYSLEDMFTGAVETCKEKRLLSETAEISGTTQK Dihydroflavonol4- MENEKKGPVVVTGASGYVGSWLVMKLLQKGYEVRA 60 reductase(DFR) TVRDPTNLKKVKPLLDLPRSNELLSIWKADLDGIEGSF Dendrobium DEVIRGSIGVFHVATPMNFQSKDPENEVIQPAINGLLGI moniliforme LRSCKNAGSVQRVIFTSSAGTVNVEEHQAAAYDETC Accession: WSDLDFVNRVKMTGWMYFLSKTLAEKAAWEFVKD AEB96144.1 NHIHLITIIPTLVVGSFITSEMPPSMITALSLITGNDAHY SILKQIQFVHLDDLCDAHIFLFEHPKANGRYICSSYDST IYGLAEMLKNRYPTYAIPHKFKEIDPDIKCVSFSSKKL MELGFKYKYTMEEMFDDAIKTCREKKLIPLNTEEIVL AAEKFEEVKEQIAVK Dihydroflavonol4- MASESESVCVTGASGFVGSWLVMRLLDRGYTVRATV 61 reductase(DFR) RDPANKKKVKHLLDLPKAATHLTLWKADLAEEGSFD Rosachinensis EAIKGCTGVFHVATPMDFESKDPENEVIKPTINGVLDI Accession: MKACLKAKTVRRLVFTASAGSVNVEETQKPVYDESN XP_024167119.1 WSDVEFCRRVKMTGWMYFASKTLAEQEAWKFAKEN NIDFITIIPTLVIGPFLMPAMPPSLITGLSPLTGNESHYSII KQGQFIHLDDLCQSHIYLYEHPKAEGRYICSSHDATIH EIAKLLREKYPEYNVPTTFKGIEENLPKVHFSSKKLLE TGFEFKYSLEDMFVGAVDACKAKGLLPPPTERVEKQE VDESSVVGVKVTG Leucoanthocyanidin MTVSSPCVGEGQGRVLIIGASGFIGEFIAQASLDSGRTT 62 reductase(LAR) FLLVRSLDKGAIPSKSKTINSLHDKGAILIHGVIEDQEF Camelliasinensis VEGILKDHKIDIVISAVGGANILNQLTIVKAIKAVGTIK Accession: RFLPSEFGHDVDRANPVEPGLAMYKEKRMVRRLIEES XP_028127206.1 GVPYTYICCNSIASWPYYDNTHPSEVIPPLDRFQIYGD GTVKAYFVDGSDIGKFTMKVVDDIRTLNKSVHFRPSC NFLNMNELSSLWEKKIGYMLPRLTVTEDDLLAAAAE NIIPQSIVASFTHDIFIKGCQVNFSIDGPNEVEVSNLYPD ETFRTMDECFDDFVMKMDRWN Leucoanthocyanidin MTRSPSPNGQAEKGSRILIIGATGFIGHFIAQASLASGK 63 reductase(LAR) STYILSRAAARCPSKARAIKALEDQGAISIHGSVNDQE Coffeaarabica FMEKTLKEHEIDIVISAVGGGNLLEQVILIRAMKAVGT Accession: IKRFLPSEFGHDVDRAEPVEPGLTMYNEKRRVRRLIEE XP_027097479.1 SGVPYTYICCNSIASWPYYDNTHPSEVSPPLDQFQIYG DGSVKAYFVAGADIGKFTVKATEDVRTLNKIVHFRPS CNFLNINELATLWEKKIGRTLPRVVVSEDDLLAAAEE NIIPQSVVASFTHDIFIKGCQVNFPVDGPNEIEVSSLYP DEPFQTMDECFNEFAGKIEEDKKHVVGTKGKNIAHRL VDVLTAPKLCA Leucoanthocyanidin MKSTNMNGSSPNVSEETGRTLVVGSGGFMGRFVTEA 64 reductase(LAR) SLDSGRPTYILARSSSNSPSKASTIKFLQDRGATVIYGSI Theobromacacao TDKEFMEKVLKEHKIEVVISAVGGGSILDQFNLIEAIR Accession: NVDTVKRFLPSEFGHDTDRADPVEPGLTMYEQKRQIR ADD51357.1 RQIEKSGIPYTYICCNSIAAWPYHDNTHPADVLPPLDR FKIYGDGTVKAYFVAGTDIGKFTIMSIEDDRTLNKTVH FQPPSNLLNINEMASLWEEKIGRTLPRVTITEEDLLQM AKEMRIPQSVVAALTHDIFINGCQINFSLDKPTDVEVC SLYPDTPFRTINECFEDFAKKIIDNAKAVSKPAASNNAI FVPTAKPGALPITAICT Leucoanthocyanidin MTVSPSIASAAKSGRVLIIGATGFIGKFVAEASLDSGLP 65 reductase(LAR) TYVLVRPGPSRPSKSDTIKSLKDRGAIILHGVMSDKPL Fragariax MEKLLKEHEIEIVISAVGGATILDQITLVEAITSVGTVK ananassa RFLPSEFGHDVDRADPVEPGLTMYLEKRKVRRAIEKS Accession: GVPYTYICCNSIASWPYYDNKHPSEVVPPLDQFQIYGD ABH07785.2 GTVKAYFVDGPDIGKFTMKTVDDIRTMNKNVHFRPSS NLYDINGLASLWEKKIGRTLPKVTITENDLLTMAAEN RIPESIVASFTHDIFIKGCQTNFPIEGPNDVDIGTLYPEE SFRTLDECFNDFLVKVGGKLETDKLAAKNKAAVGVE PMAITATCA Anthocyanin MTQNKEPVNQGKSEHDEQRVESLASSGIESIPKEYVRL 66 dioxygenase(ANS) NEELTSMGNVFEEEKKEEGSQVPTIDIKDIASEDPEVR Chenopodium GKAIQELKRAAMEWGVMHLVNHGISDELIDRVKVAG quinoa QTFFELPVEEKEKYANDQASGNVQGYGSKLANSASG Accession: RLEWEDYYFHLSYPEDKRDLSIWPETPADYIPAVSEYS XP_021735950.1 KELRYLATKILSALSLALGLEEGRLEKEVGGLEELLLQ FKINYYPKCPQPELALGVEAHTDVSALTFILHNMVPG LQLFYEGKWVTAKCVPNSIIMHIGDTIEILSNGKYKSIL HRGLVNKEKVRISWAVFCEPPKEKIILKPLPDLVSDEE PARYPPRTFAQHVQYKLFRKTQGPQTTITKN Anthocyanin MASSKVMPAPARVESLASSGLASIPTEYVRPEWERDD 67 dioxygenase(ANS) SLGDALEEIKKTEEGPQIPIVDLRGFDSGDEKERLHCM Irissanguinea EEVKEAAVEWGVMHIVNHGIAPELIERVRAAGKGFFD Accession: LPVEAKERYANNQSEGKIQGYGSKLANNASGQLEWE QCI56004.1 DYFFHLIFPSDKVDLSIWPKEPADYTEVMMEFAKQLR VVVTKMLSILSLGLGFEEEKLEKKLGGMEELLMQMKI NYYPKCPQPELALGVEAHTDVSSLSFILHNGVPGLQV FHGGRWVNARLVPGSLVVHVGDTLEILSNGRYKSVL HRGLVNKEKVRISWAVFCEPPKEKIVLEPLAELVDKR SPAKYPPRTFAQHIQHKLFKKAQEQLAGGVHIPEAIQN Anthocyanin MATQVASIPRVEMLASAGIQAIPTEYVRPEAERNSIGD 68 dioxygenase(ANS) VFEEEKKLEGPQIPVVDLMGLEWENEEVFKKVEEDM Magnoliasprengeri KKAASEWGVMHIFNHGISMELMDRVRIAGKAFFDLPI Accession: EEKEMYANDQASGKIAGYGSKLANNASGQLEWEDYF AHU88620.1 FHLIFPEDKRDMSIWPKQPSDYVEATEEFAKQLRGLV TKVLVLLSRGLGVEEDRLEKEFGGMEELLLQMKINYY PKCPQPDLALGVEAHTDVSALTFILHNMVPGLQVFFD DKWVTAKCIPGALVVHIGDSLEILSNGKYRSILHRGLV NKEKVRISWAIFCEPPKEKVVLQPLPELVSEAEPARFT PRTFSQHVRQKLFKKQQDALENLKSE Anthocyanin MVSSAAVVATRVERLATSGIKSIPKEYVRPQEELTNIG 69 dioxygenase(ANS) NVFEEEKKEGPEVPTIDLTEIESEDEVVRARCHETLKK Prosopisalba AAQEWGVMNLVNHGIPEELLNQLRKAGETFFSLPIEE Accession: KEKYANDQASGKIQGYGSKLANNASGQLEWEDYFFH XP_028787846.1 LVFPEDKCDLSIWPRTPSDYIEVTSEYARQLRGLATKI LGALSLGLGLEKGRLEEEVGGMEELLLQMKINYYPIC PQPELALGVEAHTDVSSLTFLLHNMVPGLQLFYNGQ WITAKCVPNSIFMHIGDTVEILSNGRYKSILHRGLVNK EKVRISWAVFCEPPKEKIILKPLPELVTDDEPARFPPRT FAQHIQHKLFRKCQEGLSK Anthocyanidin-3- MPQFTTNEPHVAVLAFPFGTHAAPLITIIHRLAVASPN 70 O-glycotransferase THFSFLNTSQSNNSIFSSDVYNRQPNLKAHNVWDGVP (3GT) EGYVFVGKPQESIELFVKAAPETFRKGVEAAVAETGR Cephalotus KVSCLVTDAFFWFAAEIAGELGVPWVPFWTAGPCSLS follicularis THVYTDLIRKTIGVGGIEGREDESLEFIPGMSQVVIRDL Accession: QEGIVFGNLESVFSDMVHRMGIVLPQAAAIFINSFEEL GAV66155.1 DLTITNDLKSKFKQFLSIGPLNLASPPPRVPDTNGCLP WLDQQKVASVAYISFGTVMAPSPPELVALAEALEASK IPFIWSLGEKLKVHLPKGFLDKTRTHGIVVPWAPQSDV LENGAVGVFITHCGWNSLLESIAGGVPMICRPFFGDQ RLNGRMVQDVWEIGVTATGGPFTTEGVMGDLDLILS QARGKKMKDNISVLKTLAQTAVGPEGSSAKNYEALL NLVRLSI Anthocyanidin-3- MAPQPIDDDHVVYEHHVAALAFPFSTHASPTLALVRR 71 O-glycotransferase LAAASPNTLFSFFSTSQSNNSLFSNTITNLPRNIKVFDV (3GT) ADGVPDGYVFAGKPQEDIELFMKAAPHNFTTSLDTCV Prunuscerasifera AHTGKRLTCLITDAFLWFGAHLAHDLGVPWLPLWLS Accession: GLNSLSLHVHTDLLRHTIGTQSIAGRENELITKNVNIPG AKV89253.1 MSKVRIKDLPEGVIFGNLDSVFSRMLHQMGQLLPRAN AVLVNSFEELDITVTNDLKSKFNKLLNVGPFNLAAAA SPPLPEAPTAADDVTGCLSWLDKQKAASSVVYVSFGS VARPPEKELLAMAQALEASGVPFLWSLKDSFKTPLLN ELLIKASNGMVVPWAPQPRVLAHASVGAFVTHCGWN SLLETIAGGVPMICRPFFGDQRVNARLVEDVLEIGVTV EDGVFTKHGLIKYFDQVLSQQRGKKMRDNINTVKLL AQQPVEPKGSSAQNFKLLLDVISGSTKV Anthocyanidin-3- MVFQSHIGVLAFPFGTHAAPLLTVVQRLATSSPHTLFS 72 O-glycotransferase FFNSAVSNSTLFNNGVLDSYDNIRVYHVWDGTPQGQ (3GT) AFTGSHFEAVGLFLKASPGNFDKVIDEAEVETGLKISC Scutellaria LITDAFLWFGYDLAEKRGVPWLAFWTSAQCALSAHM baicalensis YTHEILKAVGSNGVGETAEEELIQSLIPGLEMAHLSDL Accession: PPEIFFDKNPNPLAITINKMVLKLPKSTAVILNSFEEIDP A0A482AQV3 IITTDLKSKFHHFLNIGPSILSSPTPPPPDDKTGCLAWLD SQTRPKSVVYISFGTVITPPENELAALSEALETCNYPFL WSLNDRAKKSLPTGFLDRTKELGMIVPWAPQPRVLA HRSVGVFVTHCGWNSILESICSGVPLICRPFFGDQKLN SRMVEDSWKIGVRLEGGVLSKTATVEALGRVMMSEE GEIIRENVNEMNEKAK1AVEPKGSSFKNFNKLLEIINAP QSS Anthocyanidin-3- MSQTTTNPHVAVLAFPFSTHAAPLLAVVRRLAAAAPH 73 O-glycotransferase AVFSFFSTSQSNASIFHDSMHTMQCNIKSYDISDGVPE (3GT) GYVFAGRPQEDIELFTRAAPESFRQGMVMAVAETGRP Vitisvinifera VSCLVADAFIWFAADMAAEMGLAWLPFWTAGPNSLS Accession: THVYIDEIREKIGVSGIQGREDELLNFIPGMSKVRFRDL P51094 QEGIVFGNLNSLFSRMLHRMGQVLPKATAVFINSFEEL DDSLTNDLKSKLKTYLNIGPFNLITPPPVVPNTTGCLQ WLKERKPTSVVYISFGTVTTPPPAEVVALSEALEASRV PFIWSLRDKARVHLPEGFLEKTRGYGMVVPWAPQAE VLAHEAVGAFVTHCGWNSLWESVAGGVPLICRPFFG DQRLNGRMVEDVLEIGVRIEGGVFTKSGLMSCFDQIL SQEKGKKLRENLRALRETADRAVGPKGSSTENFITLV DLVSKPKDV Acetyl-CoA MPPPDHKAVSQFIGGNPLETAPASPVADFIRKQGGHS 74 carboxylase(ACC) VITKVLICNNGIAAVKEIRSIRKWAYETFGDERAIEFTV Ustilagomaydis MATPEDLKVNADYIRMADQYVEVPGGSNNNNYANV 521 DLIVDVAERAGVHAVWAGWGHASENPRLPESLAASK Accession: HKIIFIGPPGSAMRSLGDKISSTIVAQHADVPCMPWSG XP_011390921.1 TGIKETMMSDQGFLTVSDDVYQQACIHTAEEGLEKAE KIGYPVMIKASEGGGGKGIRKCTNGEEFKQLYNAVLG EVPGSPVFVMKLAGQARHLEVQLLADQYGNAISIFGR DCSVQRRHQKIIEEAPVTIAPEDARESMEKAAVRLAK LVGYVSAGTVEWLYSPESGEFAFLELNPRLQVEHPTT EMVSGVNIPAAQLQVAMGIPLYSIRDIRTLYGMDPRG NEVIDFDFSSPESFKTQRKPQPQGHVVACRITAENPDT GFKPGMGALTELNFRSSTSTWGYFSVGTSGALHEYAD SQFGHIFAYGADRSEARKQMVISLKELSIRGDFRTTVE YLIKLLETDAFESNKITTGWLDGLIQDRLTAERPPADL AVICGAAVKAHLLARECEDEYKRILNRGQVPPRDTIK TVFSIDFIYENVKYNFTATRSSVSGWVLYLNGGRTLV QLRPLTDGGLLIGLSGKSHPVYWREEVGMTRLMIDSK TCLIEQENDPTQIRSPSPGKLVRFLVDSGDHVKANQAI AEIEVMKMYLPLVAAEDGVVSFVKTAGVALSPGDIIG ILSLDDPSRVQHAKPFAGQLPDFGMPVIVGNKPHQRY TALVEVLNDILDGYDQSFRMQAVIKELIETLRNPELPY GQASQILSSLGGRIPARLEDVVRNTIEMGHSKNIEFPA ARLRKLTENFLRDSVDPAIRGQVQITIAPLYQLFETYA GGLKAHEGNVLASFLQKYYEVESQFTGEADVVLELR LQADGDLDKVVALQTSRNGINRKNALLLTLLDKHIKG TSLVSRTSGATMIEALRKLASLQGKSTAPIALKAREVS LDADMPSLADRSAQMQAILRGSVTSSKYGGDDEYHA PSLEVLRELSDSQYSVYDVLHSFFGHREHHVAFAALC TYVVRAYRAYEIVNFDYAVEDFDVEERAVLTWQFQL PRSASSLKERERQVSISDLSMMDNNRRARPIRELRTGA MTSCADVADIPELLPKVLKFFKSSAGASGAPINVLNV AVVDQTDFVDAEVRSQLALYTNACSKEFSAARVRRV TYLLCQPGLYPFFATFRPNEQGIWSEEKAIRNIEPALA YQLELDRVSKNFELTPVPVSSSTIHLYFARGIQNSADT RFFVRSLVRPGRVQGDMAAYLISESDRIVNDILNVIEV ALGQPEYRTADASHIFMSFIYQLDVSLVDVQKAIAGFL ERHGTRFFRLRITGAEIRMILNGPNGEPRPIRAFVTNET GLVVRYETYEETVADDGSVILRGIEPQGKDATLNAQS AHFPYTTKVALQSRRSRAHALQTTFVYDFIDVLGQAV RASWRKVAASKIPGDVIKSAVELVFDEQENLREVKRA PGMNNIGMVAWLVEVLTPEYPAGRKLVVIGNDVTIQ AGSFGPVEDRFFAAASKLARELGVPRLYISANSGARIG LATEALDLFKVKFVGDDPAKGFEYIYLDDESLQAVQA KAPNSVMTKPVQAADGSVHNIITDIIGKPQGGLGVEC LSGSGLIAGETSRAKDQIFTATIITGRSVGIGAYLARLG ERVIQVEGSPLILTGYQALNKLLGREVYTSNLQLGGPQ IMYKNGVSHLTAQDDLDAVRSFVNWISYVPAQRGGP LPIMPTTDSWDRAVTYQPPRGPYDPRWLINGTKAEDG TKLTGLFDEGSFVETLGGWATSVVTGRARLGGIPVGV IAVETRTLERVVPADPANPNSTEQRIMEAGQVWYPNS AYKTAQAIWDFDKEGLPLVILANWRGFSGGQQDMYD EILKQGSKIVDGLSSYKQPVFVHIPPMGELRGGSWVV VDSAINDNGMIEMSADVNSARGGVLEASGLVEIKYRA DKQRATMERLDSVYAKLSKEAAEATDFTAQTTARKA LAEREKQLAPIFTAIATEYADAHDRAGRMLATGVLRS ALPWENARRYFYWRLRRRLTEVAAERTVGEANPTLK HVERLAVLRQFVGAAASDDDKAVAEHLEASADQLLA ASKQLKAQYILAQISTLDPELRAQLAASLK Acetyl-CoA MVDHKSLPGHFLGGNSVDTAPQDPVCEFVKSHQGHT 75 carboxylase(ACC) VISKVLIANNGMAAMKEIRSVRKWAYETFGNERAIEF Hesseltinella TVMATPEDLKANAEYIRMADNYIEVPGGTNNNNYAN vesiculosa VELIVDVAERTGVHAVWAGWGHASENPRLPEMLAKS Accession: KNKCVFIGPPASAMRSLGDKISSTIVAQSADVPTMGW ORX57605.1 SGDGVSETTTDHNGHVLVNDDVYNSACVKTAEAGLA SAEKIGFPVMIKASEGGGGKGIRKVEDPSTFKQAFAQ VQGEIPGSPIFIMKLAGNARHLEVQLLADQYGNAISLF GRDCSVQRRHQKIIEEAPVTIAKPDIFEQMEKAAVRLG KLVGYVSAGTVEYLYSHHDEKFYFLELNPRLQVEHPT TEMVSGVNLPAAQLQIAMGIPMHRIRDIRVLYGVQPN SASEIDFDLEHPTALQSQRRPMPKGHVIAVRITAENPD AGFKPSGGVMQELNFRSSTNVWGYFSVVSSGAMHEY ADSQFGHIFAYGENRQQARKNMVIALKELSIRGDFRT TVEYIIRLLETPDFTDNTINTGWLDMLISKKLTAERPDT MLAVFCGAVTKAHLASVECWQQYKNSLERGQIPSKE SLKTVFTVDFIYENIRYNFTVTRSAPGIYTLYLNGTKT QVGVRDLSDGGLLISLNGRSHTTYNREEVQATRLMID GKTCLLEKESDPTQLRSPSPGKLVSLLLENGDHIRTGQ AYAEIEVMKMYMPLVASEDGHVQFIKQVGATLEAGD IIGILSLDDPSRVKHALPFTGQVPKYGLPHLTGDKPHQ RFTHLKQTLEYVLQGYDNQGLIQTIVKELSEVLNNPEL PYSELSASMSVLSGRIPGRLEQQLHDLINQAHAQNKG FPAVDIQQAIDTFARDHLTTQAEVNAYKTAVAPIMTIA ASYSNGLKQHEHSVYVDLMEQYYNVEVLFNSNQSRD EEVILALRDQHKDDLEKVINIILSHAKVNIKNNLILMLL DIIYPATSSEALDRCFLPILKHLSEIDSRGTQKVTLKAR EYLILCQLPSLEERQSQMYNILKSSVTESVYGGGTEYR TPSYDAFKDLIDTKFNVFDVLPNFFYHPDSYVSLAALE VYCRRSYHAYKILDVAYNLEHQPYIVAWKFLLQSSA GGGFNNQRIASYSDLTFLLNKTEEEPIRTGAMVALKTL EELEAELPRIMTAFEEEPLPPMLMKQPPPDKTEERMEN ILNISIQGQDMEDDTLRKNMTTLIQAHSDAFRKAALR RITLVVCRDNQTPDYYTFRERNGYEEDETIRHIEPALA YQLELARLSNFDIKPCFIENRQMHVYYAVAKENPSDC RFFIRALVRPGRVKSSMRTADYLISESDRLLTDILDTLE IVSHDYKNSDCNHLFINFIPTFAIEADEVETALKDFVDR HGKRLWKLRVTGAEIRFNIQSKRPDAPVIPLRFTVDNV SGYILKVDVYQEVKTDKNGWILKSVGKIPGAMHMQP LSTPYPTKEWLQPRRYKAHLMGTTYVYDFPELFRQAI HNLWAQACKADAAVKIPSQVIEAKELVLDDDNQLQA IDRAPGTNTVGMVAWLLTLRTPDYPRGRRVIAIANDI TFKIGSFGVQEDLVFYKASEYARELGVPRVYLSANSG ARIGLADELISRFHVAWKDEDQPGSGFEYLYLLPEEY DALIQQGDAQSVLVQEVQDKGERRFRITDIIGHTDGL GVENLRGSGLIAGATSRAYDDIFTITLVTCRSVGIGAY LVRLGQRTVQNEGQPIILTGAPALNKVLGREVYTSNL QLGGTQIMYKNGVSHLTAENDLEGINKIMQWLSFVPE CRGAPLPMRAGADPIDREIEYLPPKGPSDPRFFLAGKQ ENGKWLSGFFDHGSFVETLSGWARTVVVGRARLGGI PMGVVAVETRTVENIVPADPANADSQEQVVMEAGGV WFPNSAYKTAQAINDFNKGEQLPLMIFANWRGFSGG QRDMYNEVLKYGAQIVDALSNYKQPVFVYVVPNGEL RGGAWVVVDSTINEDMMEMYADTQARGGVLEPEGI VEIKYRRPQLLATMERLDPVYSDLKRRLAALDDSQKE QADELIAQVEAREQALLPVYQQVAIQFADLHDRSGR MEAKGVIRKTLEWRTARHYFYWRVRRRLLEEYAIRK MDESRDQAKTLLQQWFQADTNLDDFDKNDQAVVA WFDAKNLLLDQRIAKLKSEKLKDHVVQLASVDQDAV VEGFSKLMESLSVDQRKEVLHKLATRF Acetyl-CoA MASTTPHDSRVVSVSSGKKLYIEVDDGAGKDAPAIVF 76 carboxylase(ACC) MHGLGSSTSFWEAPFSRSNLSSRFRLIRYDFDGHGLSP Rhodotorula VSLLDAADDGAMIPLVDLVEDLAAVMEWTGVDKVA toruloides GIVGHSMSGLVASTFAAKYPQKVEKLVLLGAMRSLN NBRC10032 PTVQTNMLKRADTVLESGLSAIVAQVVSAALSDKSKQ Accession: DSPLAPAMVRTLVLGTDPLGYAAACRALAGAKDPDY GEM08739.1 STIKAKTLVVSGESDYLSNKETTEALVNDIPGAKEVQ MDGVGHWHAVEDPAGLAKILDGFFLQGKFSGEAKA VNGSHAVDETPKKPKYDHGRVVKYLGGNSLESAPPS NVADWVRERGGHTVITKILIANNGIAAVKEIRSVRKW AYETFGSERAIEFTVMATPEDLKVNADYIRMADQYVE VPGGTNNNNYANVDVIVDVAERAGVHAVWAGWGH ASENPRLPESLAASKHKIVFIGPPGSAMRSLGDKISSTI VAQHAEVPCMDWSGQGVDQVTQSLEGYVTVADDVY QQACVHDADEGLARASRIGYPVMIKASEGGGGKGIR KVEREQDFKQAFQAVLTEVPGSPVFIMKLAGAARHLE VQVLADQYGNAISLFGRDCSVQRRHQKIIEEAPVTIAK PDTFEQMEKSAVRLAKLVGYVSAGTVEFLYSAADDK FAFLELNPRLQVEHPTTEMVSGVNLPAAQLQVAMGV PLHRIRDIRTLYGKAPNGSSEIDFEFENPESAKTQRKPS PKGHVVAVRITAENPDAGFKPSMGTLQELNFRSSTNV WGYFSVGSAGGLHEFADSQFGHIFAYGSDRSESRKN MVVALKELSIRGDFRTTVEYLIKLLETDAFEQNTITTA WLDSLISARLTAERPDTTLAIICGAVTKAHLASEANIA EYKRILEKGQSPPKELLATVVPLEFVLEDVKYRATASR SSPSSWSIYVNGSNVSVGIRPLADGGLLILLDGRSYTC YAKEEVGALRLSIDSRTVLVAQENDPTQLRSPSPGKL VRYFIESGEHISKGEAYAEIEVMKMIMPLIAAEDGIAQ FIKQPGATLEAGDILGILSLDDPSRVHHAKPFDGQLPA LGLPSIIGTKPHQRFAYLKDVLSNILMGYDNQAIMQSS IKELISVLRNPELPYGEANAVLSTLSGRIPAKLEQTLRQ YIDSAHESGAEFPSAKCRKAIDTTLEQLRPAEAQTVRN FLVAFDDIVYRYRSGLKHHEWSTLAGIFAAYAETEKP FSGKDSDVVLELRDAHRDSLDSVVKIVLSHYKAASKN SLVLALLDVVKDSDSVPLIEQVVSPALKDLADLDSKA TTKVALKAREVLIHIQLPSLDERLGQLEQILKASVTPT VYGEPGHDRTPRGEVLKDVIDSRFTVFDVLPSFFQHQ DQWVSLAALDTYVRRAYRSYNLLNIEHIEADAAEDEP ATVAWSFRMRKAASESEPPTPTTGLTSQRTASYSDLT FLLNNAQSEPIRYGAMFSVRSLDGFRQELGTVLRHFP DSNKGKLQQQPAASSSQEQWNVINVALTVPASAQVD EDALRADFAAHVNAMSAEIDARGMRRLTLLICREGQ YPSYYTVRKQDGTWKELETIRDIEPALAFQLELGRLSN FHLEPCPVENRQVHIYYATAKGNSSDCRFFVRALVRP GRLRGNMKTADYLVSEADRLVTDVLDSLEVASSQRR AADGNHISLNFLYSLRLDFDEVQAALAGFIDRHGKRF WRLRVTGAEIRIVLEDAQGNIQPIRAIIENVSGFVVKYE AYREVTTDKGQVILKSIGPQGALHLQPVNFPYPTKEW LQPKRYKAHVVGTTYVYDFPDLFRQAIRKQWKAVGK TAPAELLVAKELVLDEFGKPQEVARPPGTNNIGMVG WIYTIFTPEYPSGRRVVVIANDITFKIGSFGPEEDRYFY AVTQLARQLGLPRVYLSANSGARLGIAEELVDLFSVA WADSSRPEKGFKYLYLTAEKLGELKNKGEKSVITKRI EDEGETRYQITDIIGLQEGLGVESLKGSGLIAGETSRAY DDIFTITLVTARSVGIGAYLVRLGQRAVQVEGQPIILTG AGALNKVLGREVYSSNLQLGGTQIMYKNGVSHLTAA NDLEGVLSIVQWLAFVPEHRGAPLPVLPSPVDPWDRSI DYTPIKGAYDPRWFLAGKTDEADGRWLSGFFDKGSF QETLSGWAQTVVVGRARLGGIPMGAIAVETRTIERIIP ADPANPLSNEQKIMEAGQVWYPNSSFKTGQAIFDFNR EGLPLIIFANWRGFSGGQQDMFDEVLKRGSLIVDGLS AYKQPVFVYIVPNGELRGGAWVVLDPSINAEGMMEM YVDETARAGVLEPEGIVEIKLRKDKLLALMDRLDPTY HALRVKSTDASLSPTDAAQAKTELAAREKQLMPIYQQ VALQFADSHDKAGRILSKGCAREALEWSNARRYFYA RLRRRLAEEAAVKRLGEADPTLSRDERLAIVHDAVGQ GVDLNNDLAAAAAFEQGAAAITERVKLARATTVAST LAQLAQDDKEAFAASLQQVLGDKLTAADLARILA Malonyl-CoA MNANLFSRLFDGLVEADKLAIETLEGERISYGDLVAR 77 synthase(matB) SGRMANVLVARGVKPGDRVAAQAEKSVAALVLYLA Rhodopseudomonas TVRAGAVYLPLNTAYTLHELDYFIGDAEPKLVVCDPA palustris KREGIAALAQKVGAGVETLDAKGQGSLSEAAAQASV Accession: DFATVPREGDDLAAILYTSGTTGRSKGAMLSHDNLAS WP_011661926.1 NSLTLVEFWRFTPDDVLIHALPIYHTHGLFVASNVTLF ARASMIFLPKFDPDAIIQLMSRASVLMGVPTFYTRLLQ SDGLTKEAARHMRLFISGSAPLLADTHREWASRTGHA VLERYGMTETNMNTSNPYDGARVPGAVGPALPGVSL RVVDPETGAELSPGEIGMIEVKGPNVFQGYWRMPEKT KAEFRDDGFFITGDLGKIDADGYVFIVGRGKDLVITGG FNVYPKEVESEIDAISGVVESAVIGVPHADLGEGVTAV VVRDKGASVDEAAVLGALQGQLAKFKMPKRVLFVD DLPRNTMGKVQKNVLREAYAKLYAK Malonyl-CoA MVNHLFDAIRLSITSPESTFIELEDGKVWTYGAMFNCS 78 synthase(matB) ARITHVLVKLGVSPGDRVAVQVEKSAQALMLYLGCL Rhizobium RAGAVYLPLNTAYTPAELEYFLGDATPKLVVVSPCAA sp.BUS003 EQLEPLARRVGTRLLTLGVNGDGSLMDMASLEPVEF Accession: ADIERKADDLAAILYTSGTTGRSKGAMLTHDNLLSNA NKF42351.1 QTLREHWRFTSADRLIHALPIFHTHGLFVATNVTLLAG GAIYLLSKFDPDQIFALMTRATVMMGVPTFYTRLLQD ERLNKANTRHMRLFISGSAPLLAETHRLFEEYTGHAIL ERYGMTETNMITSNPCDGARVPGTVGYALPGVSVRIT DPVSGEPLAAGEPGMIEVKGPNVFQGYWNMPDKTKE EFRSDGYFTTGDIGVMETDGRISIVGRGKDLIISGGYNI YPKEIENEIDAIEGVVESAVIGVPHPDLGEGVTAIVVG QPKAHLDLTTITNNLQGRLARFKQPKNVIFVDELPRNT MGKVQKNVLRDRYRDLYLK Malonyl-CoA MANHLFDLVRANATDLTKTFIETETGLKLTYDDLMT 79 synthase(matB) GTARYANVLVGLGVKPGDRVAVQVEKSAGAIFLYLA Ochrobactrumsp. CVRAGAVFLPLNTAYTLTEIEYFLGDAEPALVVCDPA 3-3 RRDGITEVAKKTGVPAVETLGKGQDGSLFDKAAAAP Accession: ETFADVARGPGDLAAILYTSGTTGRSKGAMLSHDNLA WP_114216069.1 SNALTLKDYWRFGADDVLLHALPIFHTHGLFVATNTI LVAGASMLFLPKFDADKVFELMPRATTMMGVPTFYV RLVQDARLTREATKHMRLFISGSAPLLAETHKLFREK TGVSILERYGMTETNMNTSNPYDGDRVAGTVGFPLPG VALRVADPETGAAIPQGEIGVIEVKGPNVFSGYWRMP EKTAAEFRQDGFFITGDLGKIDDQGYVHIVGRGKDLV ISGGYNVYPKEVETEIDGMAGVVESAVIGVPHPDFGE GVTAVVVAEKGASLDEATIIKTLEQRLARYKLPKRVI VVDDLPRNTMGKVQKNLLRDAYKGLYGG Malonate MSPELISILVLVVVFVIATTRSVNMGALAFAAAFGVGT 80 transporter(matC) LVADLDADGIFAGFPGDLFVVLVGVTYLFAIARANGT Rhizobiales TDWLVHAAVRLVRGRVALIPWVMFALTGALTAIGAV bacterium SPAAVAIVAPVALSFATRYSISPLLMGTMVVHGAQAG Accession: GFSPISIYGSIVNGIVEREKLPGSEIGLFLASLVANLLIA MBN8942514.1 AVLFAVLGGRKLWARGAVTPEGDGAPGKAGTGTTGS GSDTGTGTGTGTGTSAGTGGTAPTAVAVRSDRETGG AEGTGVRLTPARVATLVALVALVVAVLGFDLDAGLT AVTLAVVLSTAWPDDSRRAVGEIAWSTVLLICGVLTY VGVLEEMGTITWAGEGVGGIGVPLLAAVLLCYIGAIV SAFASSVGIMGALIPLAVPFLAQGEIGAVGMVAALAV SATVVDVSPFSTNGALVLAAAPDVDRDRFFRQLMVY GGIVVAAVPALAWLVLVVPGFG Malonate MGIELLSIGLLIAMFIIATIQPINMGALAFAGAFVLGSMI 81 transporter(matC) IGMKTNEIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL Rhizobium VECAVRLVRGRIGLIPWVMFLVAAIITGFGALGPAAV leguminosarum AILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSPI Accession: SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVFF AAC83457.1 VFGGARVMKHDPASLGPLPELHPEGVSASIRGHGGTP AKPIREHAYGTAADTATTLRLNNERITTLIGLTALGIG ALVFKFNVGLVAMTVAVVLALLSPKTQKAAIDKVSW STVLLIAGIITYVGVMEKAGTVDYVANGISSLGMPLLV ALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAI GVVAAIAISTTIVDTSPFSTNGALVVANAPDDSREQVL RQLLIYSALIAIIGPIVAWLVFVVPGLV Malonate MNIEILSIGLLVAIFIIATIQPINMGVLAFGCTFVLGSLII 82 transporter(matC) GMKPADIFAGFPADLFLTLVAVTYLFAIAQINGTIDWL Agrobacteriumvitis VERSVRMVRGRVGWIPWVMFLVAAIITGFGALGPAA Accession: VAILAPVALSFAVQYRIHPVLMGLMVIHGAQAGGFSPI WP_180575084.1 SIYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLIFFI FGGLSILKQRSSVKGPLPELHPEGISASIKGHGGTPAKP FREHAYGTAADTQSKVRLTTEKVTTLIGLTALGVGAL VFKFNVGLVAITVAVLLALLSPTTQKAAIDKVSWSTV LLISGIITYVGVMEKAGTIDYVAHGISSLGMPLLVALL LCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHISAVGV VAAIAISTTIVDTSPFSTNGALVVANAPDDQRDKVMR QMLIYSALIALIGPVIAWLVFVVPGII Malonate MSIEILSILLLVAMFVIATIQPINMGALAFACTFVLGSLI 83 transporter(matC) IGMKTSDIFAGFPSDLFLTLVAVTYLFAIAQINGTIDWL Neorhizobiumsp. VECAVRMVRGHVAWIPWVMFVVAAITGFGALGPAA Accession: VAILAPVALSFAVQYRIHPVMMGLMVIHGAQAGGFSP WP_105370917.1 ISVYGGITNQIVAKAGLPFAPTSLFLSSFFFNLAIAVLVF FVFGGARIMKQAAGPTGPLPELHPEGVSAAIRGHGGT PAKPIREHAYGTAADTLQTLRLTPEKVFTLIGLTALGI GALVFKFNVGLVAITVAVALALISPKTQKAAVDKVS WSTVLLIAGIITYVGVLEKAGTVNYVANGISSLGMPLL VALLLCFTGAIVSAFASSTALLGAIIPLAVPFLLQGHIS AVGVVAAIAISTTIVDTSPFSTNGALVVANAPDETREQ VLRQLLIYSALIAIIGPVVAWLVFVVPGLV MalonateCoA- MTTWNQKQQRKAQKLAKACDSGFDKYVPHERIIALL 84 transferase(MdcA) ETVIDRGDRVCLEGNNQKQADFLSKSLSSCNPDIVNG Moraxella LHIVQSVLALPSHIDVFERGIASKVDFSFAGPQSLRLAQ catarrhalis LVQAQKITIGAIHTYLELYGRYFIDLTPNVALITAHAA Accession: DKRGNLYTGANTEDTPAIVEATTFKSGIVIAQVNEIVD WPO64617969.1 ELPRVDIPSDWVDYYTQSPKHNYIEPLFTRDPAQITEIQ ILMAMMAIKGIYAPYKINRLNHGIGFDTAAIELLLPTY AESLGLKGEICTHWALNPHPTLIPAIESGFIHSVHSFGS EVGMENYVKARSDVFFTGADGSMRSNRAFSQTAGLY ACDLFIGSTLQIDLQGNSSTATADRIAGFGGAPNMGSD PHGRRHASYAYMKAGREAVDGSPIKGRKLVVQMVE TYREHMQSVFVNELDAFKLQQKMGADLPPIMIYGDD VTHIVTEEGIANLLLCRTPDEREQAIRGVAGYTPIGLG RDDTMVARLRERKVIQRPEDLGINPMHATRDLLAAKS VKDLVRWSDRLYEPPSRFRNW MalonateCoA- MNAPQPRQWDSLRQNRARRLERAASLGLAGQNGKEI 85 transferase(MdcA) PVDRIIDLLEAVIQPGDRVCLEGNNQKQADFLSESLAD Dechloromonas CDPARINHLSMVQSVLALPSHVDLFERGLATRLDFSFS aromatica GPQGARLAKLVQEQRIEIGAIHTYLELFGRYFMDLTPN Accession: VALIAAQAADAEGNLYLGPNTEDTPAIVEATAFKGGI WP_011289741.1 VIAQVNERLDKLPRVDVPADWVDFTVLAPKPNYIEPL FTRDPAQITEVQVLMAMMAIKGIYAEYGVTRLNHGIG FDTAAIELLLPTYAADLGLKGKICTHWALNPHPTLIPA IEAGFVESVHCFGSEVGMDDYISARSDIFFTGADGSMR SNRAFSQTAGLYACDMFIGSTLQMDLAGNSSTATLGR ITGFGGAPNMGSDPHGRRHASPAWLKAGREAYGPQA IRGRKLVVQMVETFREHMAPVFVDDLDAWKLQASM GSDLPPIMIYGDDVSHIVTEEGIANLLLCRTPAEREQAI RGVAGFTPVGMARDKGTVENLRDRGIIRRPEDLGIDP RQASRDLLAARSIKDLVRCSGGLYAPPSRFRNW MalonateCoA- MSRQWDTQADSRRQRLQRAAALAPQGRVVAADDVV 86 transferase(MdcA) ALLEAVIEPGDRVCLEGNNQKQADFLARCLTEVDPAR Pseudomonas VHDLHMVQSVLSLAAHLDVFERGIAKRLDFSFSGPQA cissicola ARLAGLVSEGRIEIGAIHTYLELFGRYFIDLTPRIALVT Accession: AQAADRHGNLYTGPNTEDTPVIVEATAFKGGIVIAQV WP_078590875.1 NEILDTLPRVDIPADWVDFVTQAPKPNYIEPLFTRDPA QISEIQVLMAMMAIKGIYAEYGVDRLNHGIGFDTAAIE LLLPTYAQSLGLKGKICRHWALNPHPALIPAIESGFVQ SVHSFGSELGMENYIAARPDIFFTGADGSMRSNRALS QTAGLYACDMFIGSTLQIDLQGNSSTATRDRIAGFGG APNMGSDARGRRHASAAWLKAGREAATPGEMPRGR KLVVQMVETFREHMAPAFVDRLDAWELAERANMPL PPVMIYGDDVSHVLTEEGIANLLLCRTPEEREQAIRGV SGYTAVGLGRDKRMVENLRDRGVIKRPDDLGIRPRD ATRDLLAARTVKDLVRWSGGLYDPPKRFRNW MalonateCoA- MNKIYREKRSWRTRRDRKAKRIEHMKQIAKGKIIPTE 87 transferase(MdcA) KIVEALTALIFPGDRVVIEGNNQKQASFLSKALSQVNP Geobacillus EKVNGLHIIMSSVSRPEHLDLFEKGIARKIDFSYAGPQS subterraneus LRMSQMLEDGKLVIGEIHTYLELYGRLFIDLTPSVALV Accession: AADKADASGNLYTGPNTEETPTLVEATAFRDGIVIAQ WP_184319829.1 VNELADELPRVDIPGSWIDFVVAADHPYELEPLFTRDP RLITEIQILMAMMVIKGIYERHNIQSLNHGIGFNTAAIE LLLPTYGESLGLKGKICKHWALNPHPTLIPAIETGWVE SIHCFGGEVGMEKYIAARPDIFFTGKDGNLRSNRTLSQ VAGQYAVDLFIGSTLQIDRDGNSSTVTNGRLAGFGGA PNMGHDPRGRRHSSPAWLDMITSDHPAAKGRKLVVQ MVETFQKGNRPVFVESLDAIEVGRSARLATTPIMIYGE DVTHIVTEEGIAYLYKASSLEERRQAIAAIAGVTPIGLE RDPRKTEQLRRDGVVAFPEDLGIRRTDAKRSLLAAKSI EELVEWSEGLYEPPARFRSW Pantothenatekinase MLLTIDVGNTHTVLGLFDGEEIVEHWRISTDSRRTADE 88 (CoaX) LAVLLQGLMGTHPLLGMELGEGIDGIAICSTVPAVLH Streptomycessp. ELREVSRRYYGDVPAILVEPGVKTGVPILMDNPKEVG CLI2509 TDRIINAVAAQHLYGGPAIVVDFGTATTFDAVSARGE Accession: YTGGVIAPGIEISVEALGLRGAQLRKIELARPRSVIGKS WP_095682415.1 TVEAMQSGILYGFAGQVDGVVQRMACELAPDPADVT VIATGGLAPMVLGEAAVIDHHEPWLTLIGLRLVYERN AGRR Pantothenatekinase MTKLWLDLGNTRLKYWLTDDSGQVLDHAAEQHLQA 89 (CoaX) PAELLKGLTFRLERLNPDFIGVSSVLGQAVNNHVAESL Streptomyces ERLQKPFEFAQVHAKHALMSSDYNPAQLGVDRWLQ cinereus MLGIIEPSKKQCVIGCGTAVTIDLVDQGHHLGGYIFPSI Accession: YLQRESLFSGTRQISIIDGTFDSIDSGTNTQDAVHHGIM WP_188874884.1 LSIVGAINETIHRYPQFEITMTGGDAHTFEPHLSASVEI RQDLVLAGLQRFFAAKNNTKNQN Pantothenatekinase MLLTIDVGNTQTTLGLFDGEEVVDHWRISTDPRRTAD 90 (CoaX) ELAVLMQGLMGRQPGGAGRERVDGLAICSSVPAVLH Kitasatospora ELREVTRRYYGDLPAVLVAPGVKTGVHVLMDNPKEV kifunensis GADRIVNALAANHLYGGPCIVVDFGTATTFDAINERG Accession: DYVGGAIAPGIEISVEALGVRGAQLRKIELAKPRNVIG WP_184936930.1 KNTVEGMQSGVLYGFAGQVDGLVTRMAKELSPTDPE DVQVIATGGLAPLVLDEASSIDVHEPWLTLIGLRLVYE RNTAS glutamyl-tRNA MTLLALGINHKTAPVSLRERVTFSPDTLDQALDSLQA 91 reductase(hemA) LPMVQGGVVLSTCNRTEIYLSVEEQDNLREALIRWLC Citrobacter EYHNLNEEDLRNSLYWHQDNDAVSHLMRVASGLDS freundii LVLGEPQILGQVKKAFADSQKGHQNASALERMFQKS Accession: FSVAKRVRTETDIGSSAVSVAFAACTLARQIFESLSTV NTY05430.1 TVLLVGAGETIELVARHLREHKVKKMIIANRTRERAQ VLADEVGAEVISLSDIDARLQDADIIISSTASPLPIIGKG MVERALKNRRNQPMLLVDIAVPRDVEPEVGKLSNAY LYSVDDLQSIISHNLAQRKAAAVEAETIVEQEASEFMA WLRAQGASDTIREYRSQSEQIRDELTAKALAALQQGG DAQAIMQDLAWKLTNRLIHAPTKSLQQAARDGDSER LNILRDSLGLE glutamyl-tRNA MTLLALGINHKTAPVSLRERVTFSPETIEQALSSLLQQP 92 reductase(hemA) LVQGGVVLSTCNRTELYLSVEQQENLQEQLVKWLCD Pseudomonas YHHLSADEVRKSLYWHQDNAAVSHLMRVASGLDSL reactans VVGEPQILGQVKKAFAESQHGQAVSGELERLFQKSFS Accession: VAKRVRTETDIGASAVSVAFAACTLARQIFESLSDVSV NWA43040.1 LLVGAGETIELVARHLREHKVRHMMIANRTRERAQV LASEVGAEVITLQDIDARLADADIIISSTASPLPIIGKGM VERALKARRNQPMLMVDIAVPRDIEPEVGKLANAYL YSVDDLHSIIQNNMAQRKAAAVQAESIVEQESSNFMA WLRSQGAVEIIRDYRSRADLVRAEAEAKALAAIAQGA DVSAVIHELAHKLTNRLIHAPTRSLQQAASDGDVERL QILRDSLGLDQQ glutamyl-tRNA MTLLALGINHKTAPVALREKVSFSPDTMGDALNNLLQ 93 reductase(hemA) QPAVRGGVVLSTCNRTELYLSMEDKENSHEQLIRWLC Gamma- QYHQIEPNELQSSIYWHQDNQAVSHLMRVASGLDSL proteobacteria VLGEPQILGQVKKAFADSQNYDSLSSELERLFQKSFSV Accession: AKRVRTETQIGANAVSVAFAACTLARQIFESLSSLTILL WP_193016510.1 VGAGETIELVARHLREHQVKKIIIANRTKERAQRLASE VDAEVITLSEIDECLAQADIVISSTASPLPIIGKGMVER ALKKRRNQPMLLVDIAVPRDIEQDVEKLNNVYLYSV DDLEAIIQHNREQRQAAAVQAEHIVQQESGQFMDWL RAQGAVGAIREYRDSAETLRAEMTEKAITLIQNGADA EKVIQQLSHQLMNRLIHTPTKSLQQAASDGDIERLNLL RESLGITHN 5-aminolevulinic MGPALDVRGKQLAAGYASVAGQADVEKIHQDQGITI 94 acidsynthase PPNATVEMCPHAKAARDAARIAEDLAAAAASKQQPA (ALAS) KKAGGCPFHAAQAQAQAKPAAAPKETVATADKKGK Schizophyllum SPRAAGGFDYEKFYEEELDKKHQDKSYRYFNNINRLA communeH4-8 ARFPTAHTAKVTDEVEVWCSNDYLGMGGNPVVLET Accession: MHRVLDKYGHGAGGTRNIAGNGALHLSLEQELARLH XP_003036856.1 RKEGALVFTSCYVANDATLSTLGSKMPGCVIFSDRMN HASMIQGIRHSGTKKVIFEHNDLADLEKKLAEYPKETP KIIAFESVYSMCGSIGPIKEICDLAEKYGAITFLDEVHA VGLYGPRGAGVAEHLDYDLHKAAGDSPDAIPGTVMD RVDIITGTLGKSYGAIGGYIAGSARFVDMIRSYAPGFIF TTSLPPATVAGAQASVVYQKEYLGDRQLKQVNVREV KRRFAELDIPVVPGPSHIVPVLVGDAALAKQASDKLL AEHDIYVQAINYPTVARGEERLRITVTQRHTLEQMDH LIGAVDQVFNELNINRVQDWKRLGGRASVGVPGGQD FVEPIWTDEQVGLADGSAPLTLRNGQPNEVSHDAVV AARSRFDWLLGPIPSHIQAKRLGQSLEGTPIAPLAPKQ SSGLKLPVEEMTMGQTIAVAA 5-aminolevulinic MDKIARFKQTCPFLGRTKNSTLRNLSTSSSPRFPSLTAL 95 acidsynthase TERATKCPVMGPALNVRSKEIVAGYASVAANSDVALI (ALAS) HKEKGVFPPPGATVEMCPHASAARAAARMADDLAA Crassisporium AAEKKKGHFTSAAPRDEAAQAAAAGCPFHVKAAAD junariophilum AAAARKAAAAPAPVKAKEDGGFNYESFYVNELDKK Accession: HQDKSYRYFNNINRLAAKFPVAHTSNVKDEVEVWCA KAF8165006.1 NDYLGMGNNPVVLETMHRTLDKYGHGAGGTRNIAG NGAMHLSLEQELATLHRKPAALVFSSCYVANDATLST LGAKLPGCIFFSDTMNHASMIQGMRHSGAKRVLFKH NDLEDLENKLKQYPKDTPKVIAFESVYSMCGSIGPIKE ICDLAEQYGALTFLDEVHAVGLYGPRGAGVAEHLDY DAHVAAGESPHPIKGSVMDRVDIITGTLGKAYGAVGG YIAGSDDFVDMIRSYAPGFIFTTSLPPATVAGARASVV YQKHYVGDRQLKQVNVREVKRRFAELDVPVVPGPSH IVPVLVGDAALAKAASDKLLAEHNIYVQSINYPTVAR GEERLRITVTPRHTLEQMDKLVRAVDKIFAELKINRLA DWKALGGRAGVGLTAGAEEAHVDPMWTEEQLGLLD GTSPRTLRNGEAAVVDAMAVGQARAVFDNLLGPISG KLQSERSVLASSTPAAANPARPAARKVVKMKTGGVP MSEDIPLPPPDVSASA 5-aminolevulinic MDKLSSLSRFKASCPFLGRTKTSTLRTLCTSSSPRFPSIS 96 acidsynthase ILTERATKCPVMGPALNVRSKEITAGYASVAGSSEVD (ALAS) QIHKQQGVTVPVNATVEMCPHASAARAAARMADDL Dendrothele AAAAAQKKVGSGASSAKAAAAGCPFHKSVAAGASA bisporaCBS STASKPSAPIHKASVPGGFDYDNFYNNELEKKHKDKS 962.96 YRYFNNINRLASKFPVAHTGDVKDEVQVWCSNDYLG Accession: MGNNPVVLETMHRTLDKYGHGAGGTRNIAGNGALH THV05492.1 LGLEQELAALHRKEAALVFSSCYVANDATLSTLGSKL PGCILFSDKMNHASMIQGMRHSGAKKVIFNHNDLEDL ENKLKQYPKETPKIIAFESVYSMCGSIGPIKEICDLAEK YGALTFLDEVHAVGLYGPHGAGVAEHLDYNAQKAA GKSPEPIPGSVMDRVDIITGTLGKAYGAVGGYIAGSM DFVDTIRSYAPGFIFTTSLPPATVSGAQASVAYQKEYL GDRQLKQVNVREVKRRFAELDIPVIPGPSHILPVLVGD AALAKAASDKLLTDHDIYVQSINYPTVAVGEERLRIT VTPRHTLEQMDKLVRAVNQVFTELNINRISDWKVAG GRAGVGMGVESVEPIWTDEQLGITDGTTPKTLRDGQR FLVDAQGVTAARGRFDTLLGPMSGSLQANPTLPLVD DELKVPLPTLVAAAA 5-aminolevulinic MDYAQFFNTALDRLHTERRYRVFADLERIAGRFPHAL 97 acidsynthase WHSPKGKRDVVIWCSNDYLGMGQHPKVVGAMVETA (ALAS) TRVGTGAGGTRNIAGTHHPLVQLEAELADLHGKEASL Bradyrhizobium LFTSGYVSNQTGIATIAKLIPNCLILSDELNHNSMIEGIR japonicum QSGCERVVFRHNDLADLEEKLKAAGPNRPKLIACESL Accession: YSMDGDVAPLAKICDLAEKYGAMTYVDEVHAVGMY A0A0A3YXD2 GPRGGGIAERDGVMHRIDILEGTLAKAFGCLGGYIAA NGQIIDAVRSYAPGFIFTTALPPAICSAATAAIRHLKTS NWERERHQDRAARVKAILNAAGLPVMSSDTHIVPLFI GDAEKCKQASDLLLEQHGIYIQPINYPTVAKGTERLRI TPSPYHDDGLIDQLAEALLQVWDRLGLPLKQKSLAAE Cytochromeb5 MDKQRVFTLSQVAEHKSKQDCWIIINGRVVDVTKFLE 98 Petuniaxhybrida. EHPGGEEVLIESAGKDATKEFQDIGHSKAAKNLLFKY Accession: QIGYLQGYKASDDSELELNLVTDSIKEPNKAKEMKAY AAD10774.1 VIKEDPKPKYLTFVEYLLPFLAAAFYLYYRYLTGALQ F
(98) TABLE-US-00013 TABLE 12 Glossary of abbreviations Abbreviation Full Name 3GT anthocyanidin-3-O-glycotransferase 4CL 4-coumarate-CoA ligase ACC acetyl-CoA carboxylase ACOT acyl-CoA thioesterase acpP acyl carrier protein ACS acetyl-CoA synthase adhE aldehyde-alcohol dehydrogenase ADP adenosine diphosphate ALA 5-aminolevulinic acid ALAS ALA synthase ANS anthocyanin dioxygenase aroG DAHP synthase aroK shikimate kinase aroL shikimate kinase ATP adenosine triphosphate C3G cyanidin-3-O-glycoside C4H cinnimate-4-hydroxylase CHI chalcone isomerase CHS chalcone synthase CoA coenzyme A CPR cytochrome P450 Reductase DAD diode array detector DAHP deoxy-d-arabino-heptulosonate-7-phosphate DctPQM a malonate transporter DFR dihydroflavonol 4-reductase DHL dihydrokaempferol DHM dihydromyricein DHQ dihydroquercetin DMSO dimethyl sulfoxide E4P erythrose-4-phosphate F3H flavonoid 3 hydroxylase F3H flavanone 3-hydroxylase fabB beta-ketoacyl-ACP synthase I fabD malonyl-coA-ACP transacylase fabF beta-ketoacyl-ACP synthase II FadA 3-ketoacyl-CoA thiolase FadB fatty acid oxidation complex subunit alpha FadE acyl-CoA dehydrogenase GltX glutamyl-tRNA synthetase hemA glutamyl-tRNA reductase hemL glutamate-1-semialdehyde aminotransferase HPLC high performance liquid chromatography ldhA lactate dehydrogenase LAR leucoanthocyanidin reductase matB malonyl-CoA synthase matC malonate transporter mdcA malonate coA-transferase mdcC acyl-carrier protein, subunit of mdc mdcD malonyl-CoA decarboxylase, subunit of mdc mdcE co-decarboxylase, subunit of mdc pABA para-aminobenzoic acid PAL phenylalanine ammonia-lyase PanK pantothenase kinase Pdh pyruvate dehydrogenase PEP phosphoenolpyruvate pHBA para-hydroxybenzoic acid PHE phenylalanine pheA chorismate mutase/prephenate dehydrogenase poxB pyruvate dehydrogenase ppsA phosphoenolpyruvate synthase TAL tyrosine ammonia-lyase TCA tricarboxylic acid cycle tesA thioesterase I tesB thioesterase II tktA transketolase TRP tryptophan TYR tyrosine TyrA chorismate mutase tyrR transcriptional regulator ybgC a thioesterase yciA a thioesterase ydiB QUIN/shikamate dehydrogenase ackA-pta Acetate kinase-phosphate acetyltransferase