WO2021053513A1 - Methods and microorganisms for producing flavonoids - Google Patents

Methods and microorganisms for producing flavonoids Download PDF

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WO2021053513A1
WO2021053513A1 PCT/IB2020/058579 IB2020058579W WO2021053513A1 WO 2021053513 A1 WO2021053513 A1 WO 2021053513A1 IB 2020058579 W IB2020058579 W IB 2020058579W WO 2021053513 A1 WO2021053513 A1 WO 2021053513A1
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flavonoid
coa
chalcone
involved
cell
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PCT/IB2020/058579
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French (fr)
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Lóránd SZABÓ
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Eleszto Genetika, Inc.
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Definitions

  • the subject matter of the present invention relates to microorganisms, such as yeast and bacteria, genetically modified so as to convert carbon substrates into desirable multi-carbon products, such as flavonoids.
  • Phytochemicals are chemicals produced by plants, and include tannins, lignins, and flavonoids.
  • the largest and best studied phytochemicals are the flavonoids, polyphenolic compounds classified into at least six subgroups: flavonols, flavones, flavanones, flavanols (and their oligomers, proanthocyanidins), anthocyanidins, and isoflavonoids.
  • Flavonoids are widely distributed in plants and function as plant pigments, signaling molecules, and defenders against infection and injury. Plants and fungi synthesize flavonoids from aromatic amino acids L-phenylalanine and L-tyrosine.
  • Flavonoids are characterized by two phenolic rings and one heterocyclic ring. Key differences between these compounds rdate to their hydroxylation patters, the position of the second aromatic ring, and the saturation of the heterocydic ring. Grotewold, The Science of Flavonoids, Springer Science Business Media, New York (2006). Consumption of flavonoid-rich foods has been linked to improved health, suggesting that flavonoids may be useful as food or beverage nutritional additives. Shahidi et al., /. Fund. Foods 18:820-897 (2015).
  • a rich source of useful flavonoids is the Cannabis plant.
  • flavonoids in Cannabis (cannflavins), including cannflavin A, cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside, vitexin, isovitexin, apigenin, kaemp ferol, myricetin, quercetin, luteolin, homoorientin and orientin.
  • cannflavins cannflavins
  • cannflavins cannflavin A
  • cannflavin B cannflavin C
  • chrysoeril cosmosiin
  • flavocannabiside vitexin
  • isovitexin apigenin
  • kaemp ferol myricetin
  • quercetin quercetin
  • luteolin homoorientin and orientin.
  • the distribution of these flavonoids in the plant varies depending on the type of flavonoid.
  • the total content of flavonoids in Cannabis leaves and flowers can reach 1-2.5% of its dry weight depending on environment factors and the variety of the plant. [005] Reports have shown that several Cannabis flavonoids have important medicinal and pharmacological properties. ElSohly et al. Life Sciences, 78(5):539-548 (2005). For example, prenylated flavonoids cannflavin and cannflavin B have been shown to inhibit production of PGE2 in human rheumatoid synovial cells and provide anti-inflammatory benefits that were approximately thirty times more effective than aspirin. Barrett et al., Biochem. Pharma, 34(ll):2019-2024 (1985); ML Barrett et al., Experientia, 42:452-453 (1986).
  • cannflavins A and B act to inhibit the in vivo production of two pro-inflammatory mediators, prostaglandin E2 and the leukotrienes. Werz et al., Pham. Nutr., 2:53-60 (2014). Cannflavins A and B have also been shown to have anti-leishmanial activity. Radwan et al., Phytochemistry, 69(14):2627-2633 (2008). The isomeric version of cannflavin B, isocannflavin B, has been shown to induce autophagy in hormone sensitive breast cancer cells. Brunelli et al., Fitorick,
  • Cannabis-based flavonoid extracts were shown to be active against several cancer cell lines, including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, ovarian, pancreatic, colon and prostate cancer.
  • U.S. Patent No. 10,398,674 to Lowe et al. [006] Cannflavins A and B appear to be Cannabis specific and their unique bioactivity appear to be linked to two key modifications of their parent flavone backbone.
  • both cannflavins A and B are modified at the 3’ position of the flavone B-ring with a methoxy group, which also increases lipophilidty and may therefore enhance their cellular retention and access to various cdlular targets.
  • flavonoids can be produced chemically, their efficient organic synthesis has been hindered by the complexity of the molecules and dangerous reaction conditions required to produce them
  • flavanoid biosynthesis begins with the production of flavonoid precursors, such as the (2S)-flavanones, through the phenylpropanoid metabolic pathway (Fig.2). Yan et al., Appl Environ Microbiol ,71:3617-3623 (2005); Koopmann E. et al., Proc Natl Acad Sci USA, 94:14954-14959 (1997); Winkel-Shirley B., Plant Physiol, 126:485—493 (2001). These precursors are essential for the production of other flavonoids such as isoflavonoids, flavonols, dihydroflavonols, and anthocyanidins.
  • flavonoids such as isoflavonoids, flavonols, dihydroflavonols, and anthocyanidins.
  • Those substrates are then converted by 4-coumarate-CoA ligase (4CL) into, respectivdy, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA. Additionally, 4CL can convert trans-cinnamic add into cinnamoyl-CoA (Fig. 2).
  • a polyketide synthase, chalcone synthase sequentially adds three molecules of malonyl-CoA to one molecule of either cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA, yielding, respectivdy, the C15 compounds pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone (Fig.2).
  • chalcone isomerase converts the CIS compounds into thdr respective (2S)-flavanones, namely, (2S)-pinocembrin, (2S)-naringenin, (2S) -eriodictyl, and (2S)- homoeriodictyl (Fig.2).
  • a tyrosine ammonia lyase can convert tyrosine directly to coumaric acid and drcumvent the use of membrane bound P450 rdated enzymes, though this may pose challenges in E. coli.
  • a prenyl moiety must be added to a flavone.
  • researchers have recently reported dght additional full-length cDNA sequences from C. saliva exhibiting 22-53% amino add identity to GuA6DT, which have been putativdy annotated as C.
  • CsPT saliva prenyltransferases 1-8. Rea et al., Phytochemistry, 164:162-171 (2019). These dght CsPTs occupied three of the six groups of known plant prenyltransferases: CsPT2 and CsPT6 reside in a unique dade of prenyltransferases (Group 2), which have been shown to participate in the tocopherol biosynthetic pathway; CsPT5 appears to be orthologous to homogentisate solanesyltransferases (Group V) that function in plastoquinone biosynthesis; and CsPTl, 3, 4, 7, and 8 all formed a third and distantly rdated group (Group VI) that indudes two prenyltransferases from Humulus lupulus (hops), and which are involved in the aromatic prenylation reactions required for terpenophenolic biosynthesis.
  • CsOMT C. saliva O-methyltransferases
  • CsOMT21 a group of enzymes with regiospecificity for 3’-hydroxyl groups on a variety of flavonoid compounds and, further, that at least one such enzyme, CsOMT21, likely catalyzes the penultimate step in cannflavin A and B biosynthesis by converting luteolin to chrysoeriol.
  • expression of enzyme combinations originating from a variety of host organisms has yielded microbial strains capable of producing key flavonoid precursors, such as naringenin.
  • Naringenin is used to produce other flavonoids such as isoflavonoids, flavonols, dihydroflavonols, and anthocyanidins.
  • the pathway for naringenin production consists of five enzymes, phenylalanine/ tyrosine ammonia lyase (PAL), dnnamate 4-h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI).
  • PAL phenylalanine/ tyrosine ammonia lyase
  • C4H dnnamate 4-h droxylase
  • CHS chalcone synthase
  • CHI chalcone isomerase
  • TAL tyrosine ammonia lyase
  • TAL tyrosine ammonia lyase
  • naringenin chalcone is able to convert to naringenin without CHI under certain conditions. This opens up the possibility to bypass CHI and shorten the necessary changes in the host microorganism [0014]
  • a phenylpropanoid precursor such as coumaric add
  • S. cerevisiae have also been reported to convert glucose into naringenin at rdatively high titers. Koopman et al., Microbial CellFactories, 11:155 (2012); Santos et al., Metab Eng 13:392-400 (2011). [0015] S. cerevisiae has several attractive characteristics as a metabolic engineering platform for flavonoid production. In addition to its excellent accessibility to molecular and synthetic biology techniques, its eukaryotic nature may facilitate functional expression of plant-derived flavonoid- biosynthetic genes. For example, S. cerevisiae can functionally express cytochrome P450-containing enzymes and its subcellular compartmentation is comparable to that of plant cells. Jiang et al., Biotechnol
  • the present invention provides a metabolic engineering strategy for microbial production of flavonoids and flavonoid derivatives using a carbon substrate.
  • the flavonoid biosynthetic genes PAL, C4H, 4CL, CHS, TAL, CsPT, and CsOMT, used in the invention may be derived from one or more different prokaryotic or eukaryotic species — including Escherichia coli, Saccharomyces cerevisiae, Cannabis saliva, Arabidopsis thahana, Rhodobacter capsulatus, Petroselinum crispum, and Petunia hybrida — and selected for in planta co-expression profiles.
  • optimization of flavonoid production was explored by engineering of precursor supply to the flavonoid pathway and by reducing the formation of byproducts derived from yeast metabolism.
  • the present invention relates in part to a genetically-modified cell capable of producing a flavonoid or flavonoid derivative.
  • the cell may comprise at least one heterologous enzyme involved in a metabolic pathway that converts a carbon substrate (e.g., sugar) to a flavonoid or flavonoid derivative and/ or at least one heterologous polynucleotide encoding such an enzyme.
  • the invention also relates to a method of making a flavonoid or flavonoid derivative.
  • the method comprises contacting a substrate with the aforementioned genetically-modified cell and growing the cell to produce a flavonoid or flavonoid derivative compound.
  • the invention further relates to the use of a flavonoid or flavonoid derivative for the manufacture of a medicament for the treatment of a disease or a symptom of a disease and to such a medicament.
  • the invention additionally rdates to a method of treating a disease or symptom of a disease comprising administering a flavonoid or flavonoid derivative to a subject in need thereof.
  • Yet another aspect of the invention is a nucleic add encoding at least one enzyme involved in a metabolic pathway that converts a cabon substrate (e.g., sugar) to a flavonoid or flavonoid derivative or a vector encoding such a nucleic add.
  • a cabon substrate e.g., sugar
  • a further aspect of the invention is a method of making a genetically-modified cell capable of synthesizing a flavonoid or flavonoid derivative, the method comprising: contacting a cell with at least one heterologpus polynucleotide encoding an enzyme involved in the flavonoid pathway; and growing the cell so that said polynudeotide is expressed in the microorganism.
  • a yet further aspect of the invention is a composition comprising a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof.
  • FIG. 1A shows the basic skdeton structure of flavonoids and their various classes.
  • FIG. IB shows the flavonoid classes, subdasses and natural sources.
  • FIG. 2 shows an overview of the flavonoid biosynthetic pathway.
  • the enzyme PAL converts phenylalanine into a cinnamic add isomer.
  • the enzyme C4H converts cinnamic acid into dther p-coumaric add, caffeic add, or ferulic acid.
  • the enzyme 4CL converts those substrates into p- coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA, respectivdy. 4CL can also directly convert trans- cinnamic acid into cinnamoyl-CoA.
  • the enzyme CHS sequentially adds three molecules of malonyl-CoA to one molecule of dther dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl- CoA, yielding, respectively, pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone.
  • the enzyme CHI converts these four compounds into thdr respective (2S)-flavanones, namely, (2S)-pinocembrin, (2S)-naringenin, (2S)- eriodictyl, and (2S)-homoeriodictyl.
  • Flavonoid biosynthesis pathways can be assembled and engineered in a tractable host microorganism, such as bacteria or yeast, to create cdl factories for their mass production.
  • FIG. 3 shows the biosynthetic route for the flavonoid precursor naringenin.
  • aspects of the invention include genetically modified microorganisms that can convert carbon substrates into flavonoid compounds or flavonoid derivative.
  • the genetically modified microorganisms include bacteria and yeast, which are capable of produdng such flavonoids utilizing native and/or heterologous enzymes that catalyze various reactions in the flavonoid biosynthesis pathway [0029]
  • the terms “and/or” and “any combination thereof’ and their grammatical equivalents may be used interchangeably. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”
  • the term “about” in relation to a reference numerical value and its grammatical equivalents includes the numerical value itself and a range of values plus or minus 10% from that numerical value.
  • the amount ‘about 10” includes 10 and any amounts from 9 to 11.
  • the term ‘about ”in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value.
  • the numerical disclosed throughout can be “about” that numerical value even without specifically mentioning the term “about.”
  • the term “substrate” refers to any substance or compound that is converted into another compound by the action of an enzyme.
  • the term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures, and other materials that contain at least one substrate, or derivatives thereof.
  • the term “substrate” encompasses not only compounds that provide a carbon source suitable for use as a starting material (e.g., methane), but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein.
  • flavonoids refers to a group of biologically-active plant compounds that provide health benefits through cell signaling pathways and antioxidant effects. Flavonoids can be subdivided into different subgroups depending on the carbon of the C ring on which the B ring is attached and the degree of unsaturation and oxidation of the C ring (Fig. 1A). Flavonoids in which the B ring is linked in position 3 of the C ring are called isoflavones. Those in which the B ring is linked in position 4 are called neoflavonoids, while those in which the B ring is linked in position 2 can be further subdivided into several subgroups on the basis of the structural features of the C ring. These subgroups are: flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins and chalcones (Fig. 1A).
  • Flavones are widely present in leaves, flowers and fruits as glucosides. Celery, parsley, red peppers, chamomile, mint and ginkgo biloba are among the major sources of flavones. Luteolin, apigenin and tangeritin belong to this subclass of flavonoids (Fig. IB). The peels of citrus fruits are rich in the polymethoxylated flavones, tageretin, nobiletin and sinensetin. They have a double bond between positions 2 and 3 and a ketone in position 4 of the C ring.
  • flavones of vegetables and fruits have a hydroxyl group in position 5 of the A ring, while hydroxylation in other positions, for the most part in position 7 of the A ring or 3’ and 4’ of the B ring, may vary according to the taxonomic classification of the particular vegetable or fruit.
  • Flavonols are flavonoids with a ketone group. They are building blocks of proanthocyanins. Flavonols occur abundantly in a variety of fruits and vegetables. The most studied flavonols are kaemp feral, quercetin, myricetin and fisetin (Fig. IB). Onions, kale, lettuce, tomatoes, apples, grapes and berries are rich sources of flavonols. Apart from fruits and vegetables, tea and red wine are also sources of flavonols. Intake of flavonols is found to be associated with a wide range of health benefits which includes antioxidant potential and reduced risk of vascular disease.
  • flavonols Compared with flavones, flavonols have a hydroxyl group in position 3 of the C ring, which may also be glycosylated. Like flavones, flavonols are very diverse in methylation and hydroxylation patters as well and, considering the different glycosylation patterns, they are perhaps the most common and largest subgroup of flavonoids in fruits and vegetables. For example, quercetin is present in many plant foods.
  • Flavanones are another important class of flavonoids, and are generally present in citrus fruits such as oranges, lemons and grapes. Hesperitin, naringenin, eriodictyol, and homo-eriodictyol are examples of this class of flavonoids (Fig. IB). Flavonones are associated with a number of health benefits because of their free radical-scavenging properties. These compounds are responsible for the bitter taste of the juice and peel of citrus fruits. Citrus flavonoids exert interesting pharmacological effects as antioxidant, anti-inflammatory, blood lipid-lowering and cholesterol-lowering agents.
  • Flavanones also called dihydroflavones, have the C ring saturated; therefore, unlike flavones, the double bond between positions 2 and 3 is saturated and this is the only structural difference between the two subgroups of flavonoids. Over the past 15 years, the number of flavanones has significantly increased.
  • Isoflavonoids are a large and very distinctive subgroup of flavonoids. Isoflavonoids enjoy only a limited distribution in the plant kingdom and are predominantly found in soyabeans and other leguminous plants. Some isoflavonoids have also been reported to be present in microbes. They are also found to play an important role as precursors for the development of phytoalexins during plant microbe interactions. Isoflavonoids exhibit tremendous potential to fight a number of diseases. Isoflavones such as genistein and daidzein are commonly regarded to be phyto-oestrogens because of their oestrogenic activity in certain animal models (Fig. IB). [0040] Anthocyanins are a subclass of flavonoids responsible for colors in plants, flowers and fruits.
  • Cyanidin, delphinidin, malvidin, pelargonidin and peonidin are the most commonly studied anthocyanins (Fig. IB). They occur predominantly in the outer cell layers of various fruits such as cranberries, black currants, red grapes, merlot grapes, raspberries, strawberries, blueberries, bilberries and blackberries. Stability coupled with health benefits of these compounds facilitate them to be used in the food industry in a variety of applications. The color of the anthocyanin depends on the pH and also by methylation or acylation at the hydroxyl groups on the A and B rings.
  • Chalcones are a subclass of flavonoids characterized by the absence of ‘ring C’ of the basic flavonoid skeleton structure shown in Fig. 1A. Hence, they can also be referred to as open-chain flavonoids.
  • Major examples of chalcones include phloridzin, arbutin, phloretin and chalconaringenin. Chalcones occur in significant amounts in tomatoes, pears, strawberries, bearberries and certain wheat products. Chalcones and their derivatives have garnered considerable attention because of numerous nutritional and biological benefits. The intake of flavonoids through food sources could be the simplest and safest way to combat diseases as well as modulate activities.
  • cannflavin refers to a flavonoid found in Cannabis.
  • cannflavins indude cannflavin A, cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside, vitexin, isovitexin, apigenin, kaemp feral, myricetin, quercetin, luteolin, homoorientin, orientin, and any natural or unnatural isomers of any of the foregoing.
  • flavonoids pathway and “phenylp ropanoid metabolic pathway” are interchangeable and refer to any metabolic pathway by which flavonoids are biosynthesized.
  • the enzymes phenylalanine/ tyrosine ammonia-lyase (PAL), cinnamate-4-h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) are involved in catalyzing the conversion of phenylalanine to cinnamic add to p-coumaric acid to 4-coumaroyl-CoA to (with the addition of malonyl-CoA) a chalcone to a flavonone, such as naringenin, eriodictyol, or homo-eriodictyol.
  • the basic flavonoid pathway phenylalanine/ tyrosine ammonia-lyase (PAL),
  • flavonoid pathway enzymes refer to any enzyme involved in the flavonoid pathway, inducting PAL, C4H, 4CL, CHS, and CHI.
  • Other enzymes in the flavonoid pathway and encompassed by the present invention indude flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3 ⁇ ), and flavonoid 3’, 5' droxylase (F3’5TI).
  • CsPT prenyltransferase
  • CsPT prenyltransferase
  • CsOMT sativa O- methyltransferase
  • Any other enzyme (or its isoform) involved in the production of flavonoids or flavonoids precursors are also encompassed herein.
  • the enzymes in the flavonoid pathway are often represented by several isoforms, which may differ in substrate preference or kinetic properties. Costa et al., Phytochem, 66:2072-2091 (2005).
  • the different isoforms may be organized into one or more enzyme complexes.
  • the term “fermentation” or “fermentation process” is defined as a process in which a host microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products.
  • feedstock is defined as a raw material or mixture of raw materials supplied to a microorganism, or fermentation process, from which other products can be made.
  • a methane carbon source, a methanol carbon source, or a formaldehyde carbon source are feedstocks for a microorganism that produces a valuable chemical, such as a flavonoid or flavonoid derivative.
  • a feedstock e.g., a methane substrate
  • the fermentation media contains suitable minerals, salts, cofactors, buffers, and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for multi-carbon compound production.
  • genetic modification refers to one or more alterations of a nucleic acid, e.g., the nuddc add within a microorganism’s genome.
  • genetic modification can refer to alterations, additions, and/or deletion of nucldc acid (e.g, whole gpnes or fragments of genes).
  • gene editing and its grammatical equivalents refers to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome.
  • gene editing can be performed using a nuclease (eg, a natural-existing nuclease or an artificially engineered nuclease).
  • promoter refers to a nudeic add sequence capable of controlling the expression of a coding sequence or functional RNA.
  • a coding sequence is located 3’ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different dements derived from different promoters found in nature, or even comprise synthetic nucldc add segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions.
  • Promoters that cause a gene to be expressed in most cdl types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity. Some examples of promoters that can be used indude but are not limited to Gall, GallO, TEF1, TDH3, PGK1, ADH2.
  • operably linked refers to the association of nucleic acid sequences on a single polynucleic add fragment so that the function of one is affected by the other.
  • a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter).
  • Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation.
  • the term “codon optimized” and its grammatical equivalents refers to genes or coding regions of nucleic add molecules (or open reading frames) for transformation of various hosts, can refer to the alteration of codons in the gene or coding regions of the nucldc add molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
  • ORF open reading frame
  • ORF open reading frame
  • a polynucldc add or nudeic add sequence comprising an uninterrupted reading frame consisting of (i) an initiation codon, (ii) a series of two (2) of more codons representing amino adds, and (iii) a termination codon, the ORF being read (or translated) in the 5’ to 3’ direction.
  • the term “operon” and its grammatical equivalents refers to two or more genes, which are transcribed as a single transcriptional unit from a common promoter.
  • the genes, polynudeotides or ORFs comprising the operon are contiguous. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter.
  • any gene, polynudeotide or ORF, or any combination thereof in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase or a decrease in the activity or function of the encoded polypeptide. Further, the modification can inpart new activities on the encoded polypeptide.
  • vector and its grammatical equivalents refers to any means by which a nucldc add can be propagated and/or transferred between organisms, cells, or cellular components.
  • Vectors indude viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artifidal chromosomes), and PLACs (plant artificial chromosomes), and the like, that are “episomes”, that is, that replicate autonomously or can integrate into a chromosome of a host microorganism
  • a vector can also be a naked RNA polynudeotide, a naked DNA polynudeotide, a polynudeotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide
  • the term “host cell” refers to a prokaryotic or eukaryotic cell that can be, or has been, used as a recipients for a nucleic add (e.g., an expression vector that comprises a nucleotide sequence encoding one or more gene products such as mevalonate pathway gene products), and indudes any progeny of the original cdl that has been genetically modified by the nucldc add. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, acddental, or deliberate mutation.
  • a “recombinant host cell” or “genetically modified host cell” is a host cdl into which has been introduced a heterologous nucldc add, e.g., an expression vector.
  • substantially pure and its grammatical equivalents can refer to a particular substance that does not contain a majority of another substance.
  • substantially pure product can mean at least 90% of that product.
  • substantially pure product can mean at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% of that product.
  • substantially pure product can mean at least 70% of the product. In some cases, substantially pure product can mean at least 75% of the product. In some cases, substantially pure product can mean at least 80% of the product. In some cases, substantially pure product can mean at least 85% of the product. In some cases, substantially pure product can mean at least 90% of the product. In some cases, substantially pure product can mean at least 91% of the product. In some cases, substantially pure product can mean at least 92% of the product. In some cases, substantially pure product can mean at least 93% of the product. In some cases, substantially pure product can mean at least 94% of the product. In some cases, substantially pure product can mean at least 95% of the product. In some cases, substantially pure product can mean at least 96% of the product. In some cases, substantially pure product can mean at least 97% of the product. In some cases, substantially pure product can mean at least 98% of the product. In some cases, substantially pure product can mean at least 99% of the product.
  • heterologous and its grammatical equivalents means derived from a different spedes.
  • a “heterologous gene” can mean a gene that is from a spedes different than the reference spedes.
  • a yeast cell comprising a “heterologous gene” comprises a gene that is not from the same species of yeast.
  • the gene can be from a different microorganism altogether, such as bacteria, plant, or algae, from a different species of yeast.
  • nudeic add DNA or RNA
  • RNA nudeic add
  • the term “recombinant” and its grammatical equivalents means that a particular nudeic add (DNA or RNA) is the product of various combinations of doning, restriction, and/or ligation steps resulting in a constmct having a structural coding or non-coding sequence distinguishable from endogenous nudeic adds found in natural systems.
  • DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonudeotide linkers, or from a series of synthetic oligonudeotides, to provide a synthetic nucldc add which is capable of being expressed from a recombinant transcriptional unit contained in a cdl or in a cell-free transcription and translation system.
  • Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes.
  • Genomic DNA comprising the rdevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms.
  • the terms “recombinant polynucleotide” or “recombinant nucleic add” refer to a polynudeotide that is non-naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artifidal combination is often accomplished by dther chemical synthesis means, or by the artifidal manipulation of isolated segments of nucldc adds, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino add, while typically introdudng or removing a sequence recognition site.
  • nucldc acid segments of desired functions it is performed to join together nucldc acid segments of desired functions to generate a desired combination of functions.
  • This artifidal combination is often accomplished by dther chemical synthesis means, or by the artifidal manipulation of isolated segments of nucldc adds, e.g., by genetic engineering techniques.
  • isolated refers to a polynudeotide, polypeptide, or cell that is in an environment different from that in which the polynudeotide, polypeptide, or cdl naturally occurs.
  • An isolated genetically modified host cdl may be present in a mixed population of genetically modified host cells.
  • substantially similar and its grammatical equivalents, when used in reference to the similarity between a sequence and a reference sequence, means that the sequences are at least 50% (but not 100%) identical.
  • the sequences are 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical.
  • the term substantially similar refers to a sequence that is at least 50% identical. In some instances, the term substantially similar refers to a sequence that is 55% identical. In some instances, the term substantially similar refers to a sequence that is 60% identical. In some instances, the term substantially similar refers to a sequence that is 65% identical.
  • the term substantially similar refers to a sequence that is 70% identical. In some instances, the term substantially similar refers to a sequence that is 75% identical. In some instances, the term substantially similar refers to a sequence that is 80% identical. In other instances, the term substantially similar refers to a sequence that is 81% identical. In other instances, the term substantially similar refers to a sequence that is 82% identical. In other instances, the term substantially similar refers to a sequence that is 83% identical. In other instances, the term substantially similar refers to a sequence that is 84% identical. In other instances, the term substantially similar refers to a sequence that is 85% identical. In other instances, the term substantially similar refers to a sequence that is 86% identical.
  • the term substantially similar refers to a sequence that is 87% identical. In other instances, the term substantially similar refers to a sequence that is 88% identical. In other instances, the term substantially similar refers to a sequence that is 89% identical. In some instances, the term substantially similar refers to a sequence that is 90% identical. In some instances, the term substantially similar refers to a sequence that is 91% identical. In some instances, the term substantially similar refers to a sequence that is 92% identical. In some instances, the term substantially similar refers to a sequence that is 93% identical. In some instances, the term substantially similar refers to a sequence that is 94% identical. In some instances, the term substantially similar refers to a sequence that is 95% identical.
  • the term substantially similar refers to a sequence that is 96% identical. In some instances, the term substantially similar refers to a sequence that is 97% identical. In some instances, the term substantially similar refers to a sequence that is 98% identical. In some instances, the term substantially similar refers to a sequence that is 99% identical.
  • the two sequences are aligned, using, for example, the alignment method of Needleman and Wunsch 0. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids/ nucleotides is determined between the two sequences.
  • Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Iipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d.
  • Computer programs will be employed for such calculations.
  • Computer programs that can be used in this regard include, but are not limited to, GCG (Devereux et aL, Nucleic Adds Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et aL, J. Molec. Biol., 1990:215:403).
  • a particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson, ⁇ D, Higgines, D G and Gibson T J, 1994, Nudeic Add Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Nad. Acad. Sci. USA 89: 10915-10919) using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
  • the publications discussed herein are provided solely for thdr disdosure prior to the filing date of the present application. None herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. II. Genetically Modified Microorganisms and Methods of Making the Same [0065] The present disclosure is directed, in part, to genetically modified microorganisms that are able to replicate quickly and produce flavonoid and flavonoid derivative compounds. In some instances, microorganisms that do not naturally produce a specific flavonoid or flavonoid derivative have been genetically modified to do so.
  • molecular switches can allow for direct control of gene expression at a given time. This control of gene expression by using molecular switches can lead to increased cell division compared to microorganism that do not have these molecular switches.
  • the genetically modified microorganisms useful in the present invention include those that can convert a carbon substrates into a desired end product, such as a flavonoid or flavonoid derivative.
  • the microorganisms can be a prokaryote or a eukaryote.
  • the microorganism may, for example, be bacteria, yeast, plant, fungi, or algae.
  • the carbon substrate may be endogenously present in the microorganism in normal culture, or additional amounts of the carbon substrate may be fed to the microorganism exogenously.
  • the carbon substrate is a sugar, such as glucose.
  • the carbon substrate is amino acid, such as phenylalanine or tyrosine.
  • the carbon substrate is a compound derived from a sugar or amino add, and indudes, for example, any compound produced in the flavonoid pathway, such as cinnamic acid, p-coumaric add, caffeic add, ferulic acid, dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, homoeriodictyl chalcone, (2S)-pinocembrin, (2S)-naringenin, (2S) -eriodictyl, (2S)-homoeriodictyl, or any natural or unnatural isomers of the foregoing.
  • any compound produced in the flavonoid pathway such as cinnamic acid, p-coumaric add, caffeic add,
  • the microorganism is yeast, the carbon substrate is coumaric add, cafeic add, or ferulic add, and the desired end product is a cannflavin.
  • the microorganism is Saccharomyces cerevisiae is, the carbon substrate is ferulic acid, and the desired end product is a isocannflavin B.
  • the genetically modified microorganisms useful in the present invention may exhibit increased activity levels of one or more phenylpropanoid metabolic pathway enzymes and/or increased levds of flavonoid or flavonoid derivative production as compared to microorganisms of the same species that have not been genetically modified.
  • a genetically modified host microorganism exhibits increases in flavonoid or flavonoid derivative production, where flavonoid or flavonoid derivative production is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75- fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 103-fold, or more, in the genetically modified host microorganism, compared to the level of flavonoid derivative or flavonoid compound produced in a control host micro
  • Flavonoid or flavonoid derivative production is readily determined using well-known methods, e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ion chromatography- mass spectrometry, pulsed amperometric detection, UV-vis spectrometry, and the like.
  • a genetically modified host microorganism produces a flavonoid or flavonoid derivative in an amount ranging from 1 pg flavonoid compound/ ml to 100,000 pg flavonoid compound/ ml, e.g., from about 1 pg/ml to about 10,000 pg/ml of flavonoid compound, 1 pg/ml to
  • the yeast Saccharomyces cerevisiae is engineered to produce a cannflavin (e.g., cann flavin A, cann flavin B, isocannflavin A, isocann flavin B) from one or more phenylpropanoid metabolic pathway enzymes, such as phenylalanine/ tyrosine ammonia-lyase (PAL), dnnamate-4- h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3 ⁇ ), and flavonoid 3’, 5' droxylase (R3'5 ⁇ ), C. sativa prenyltransferase (CsPT)
  • PAL phen
  • the microorganisms useful in the present invention may be naturally occurring or recombinant.
  • the microorganisms are selectively screened (e.g., using strain adaptation) to have improved properties. Improved properties may include increased growth rate, yield of desired products, and tolerance of likely process contaminants.
  • a high growth variant microorganism is selected, which possesses an exponential phase growth rate that is faster than its parent, reference, or wild-type microorganism.
  • the microorganisms useful in the present invention may be grown as an isolated pure culture, with a heterologous microorganism that may aid with growth, or one or more different strains of microorganisms may be combined to generate a mixed culture.
  • a variety of culture methodologies may be useful in the present invention.
  • the microorganism may be grown by batch culture and/or continuous culture methodologies, both of which are described in the art. See, e.g., Crueger et al., Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989); Deshpande et al .,AppL Biochem. BiotechnoL, 36:227 (1992), the contents of each are herein incorporated by reference.
  • the culture media preferably contains one or more suitable carbon substrates for the microorganisms.
  • the carbon substrate(s) may be added to the culture media initially, intermittently, and/or supplied continuously.
  • a host microorganism contains endogenously, or is transformed with, one or more heterologous polynucleotides that encode an enzyme in the flavonoid pathway.
  • one or more of these genes can be episomally expressed.
  • one of more of these genes can be integrated into the gpnome of the microorganism.
  • one or more of these genes be can be episomally expressed whereas one or more of these gpnes can be integrated into the genome of the microorganism
  • the host microorganism contains (either endogenously or heterologous) one or more polynucleotides encoding the following enzymes (or their isomers): (1) phenylalanine/tyrosine ammonia-lyase (PAL); (2) cinnamate-4-h droxylase (C4H); (3) 4-coumarate- CoA ligase (4CL); (4) chalcone synthase (CHS); and (5) chalcone isomerase (CHI).
  • PAL phenylalanine/tyrosine ammonia-lyase
  • C4H cinnamate-4-h droxylase
  • 4CL 4-coumarate- CoA ligase
  • CHS chalcone synthase
  • CHI chalcone isomerase
  • the microorganism may also contain one or more polynucleotides encoding for other important enzymes in the production of flavonoids or flavonoid derivatives, including flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3T1), and flavonoid 3’, 5’h droxylase (F3’5’H).
  • the microotganism may also contain the enzymes C.
  • PAL phenylalanine/tyrosine ammonia-lyase
  • the PAL enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 1 or SEQ ID NO. 3.
  • the polynucleotide encoding the PAL enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 1 or SEQ ID NO. 3.
  • the PAL enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 2 or SEQ ID NO: 4.
  • the PAL enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
  • the enzyme cinnamate-4-h droxylase (C4H) (EC:1.14.14.91) is capable of catalyzing the conversion of trans-cinnamic acid (C00423) into, e.g., p-coumaric acid (C00811):
  • the C4H enzyme can be encoded by a polynudeotide that is substantially similar to SEQ ID NO. 5.
  • the polynudeotide encoding the C4H enzyme can comprises a polynudeotide 5 sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 5.
  • the C4H enzyme can comprise an amino add sequence that is substantially similar to SEQ ID NO: 6.
  • the C4H enzyme can comprises an amino add sequence that is at least.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 10 or 99.9999% identical to SEQ ID NO: 6.
  • the enzyme 4-Coumarate-CoA ligase (4CL) (EC:6.2.1.12) is capable of catalyzing the conversion of p-coumaric add (C00811) into p-coumaroyl-CoA (C00223), the conversion of caffeate (C01197) into caffeoyl-CoA (C00323), and the conversion of ferulate (C01494) into feruloyl-CoA (C00406).
  • the 4CL enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO:
  • the polynucleotide encoding the 4CL enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 7.
  • the 4CL enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 8.
  • the 4CL enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 8.
  • the enzyme chalcone synthase (EC:2.3.1.74) is capable of catalyzing the conversion of p-coumaroyl-CoA (C00223) into naringenin chalcone (C06561), the conversion of dnnamoyl-CoA (C00540) into pinocembrin chalcone (C16404), the conversion of caffeoyl-CoA (C00323) into eriodictyol chalcone (Cl 5525), and the conversion of feruloyl-CoA (C00406) into homoeriodictyol chalcone (Cl 6405):
  • the polynucleotide encoding the CHS enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 9 or SEQ ID NO. 11.
  • the CHS enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 10 or SEQ ID NO. 12.
  • the CHS enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 10 or SEQ ID NO. 12.
  • the enzyme chalcone isomerase (EC:5.5.1.6) is capable of catalyzing the conversion of a chalcone (Cl 5589) into a flavanone (C00766), inducting the conversion of naringenin chalcone into narigenin, the conversion of pinocembrin chalcone into pinocembrin, the conversion of eriodictyol chalcone into eriodictyol, and the conversion of homoeriodictyol chalcone (C16405) into homoeriodictyol: [0085]
  • the CHI enzyme can be encoded by a polynudeotide that is substantially similar to SEQ
  • the polynudeotide encoding the CHI enzyme can comprises a polynudeotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 13.
  • the CHI enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 14.
  • the CHI enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 14.
  • the enzyme flavanone 3-h droxylase (F3H) (EC:1.14.11.9) is capable of catalyzing the conversion of a flavanone into its corresponding flavanol, such as the conversion of naringenin (C00509) into dihydrokaempferol (C00974):
  • the F3H enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 15.
  • the polynucleotide encoding the F3H enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 15.
  • the F3H enzyme can comprise an amino add sequence that is substantially similar to SEQ ID NO: 16.
  • the F3H enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 16.
  • the enzyme dihydroflavonol 4-reductase (EC:1.1.1.219 and EC:1.1.1.234) is capable of catalyzing the conversion of a dihydroflavonol into its corresponding cis-flavan-3,4-diol, such as the conversion of dihydrokaempferol (C00974) into ds-3,4-leucopelargonidin (C03648):
  • the DFR enzyme can be encoded by a polynudeotide that is substantially similar to SEQ ID NO. 17.
  • the polynucleotide encoding the DFR enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 17.
  • the DFR enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 18.
  • the DFR enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%,
  • SUBSTITUTE SHEET (RULE 26) 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 18.
  • the enzyme flavonoid 3’-h droxylase (F3’H) (EC:1.14.14.82) is capable of catalyzing the conversion of certain flavonoids, such as naringenin (C00509) into, hydroxy flavonoids, such as eriodictyol (C05631):
  • the F3 ⁇ enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 19.
  • the polynucleotide encoding the F3 ⁇ enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 19.
  • the F3 ' H enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 20.
  • the F3TI enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 20.
  • the enzyme flavonoid 3’, 5’ hydroxylase (F3’5’H) (EC:1.14.14.81) is capable of catalyzing acting on naringenin, eriodictyol, dihydroquercetin (taxifolin) and dihydrokaempferol (aromadendrin).
  • the enzyme catalyses, for example, the conversion of naringenin (C00509) and eriodictyol (C05631) into pentahydroxyflavanone (C05911):
  • the F3’5’H enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 21.
  • the polynucleotide encoding the F3’5TI enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 21.
  • the F3’5’H enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 22.
  • the F3’5’H enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 22.
  • the C. saliva prenyltransferase (CsPT) enzyme useful in the present invention may include any enzyme encoded by a polynucleotide that is substantially similar to SEQ ID NOs. 23, 25, 27, 29, 31, 33, 25, 27, or 39.
  • the polynucleotide encoding the CsPT enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 21, 23, 25, 27, 29, 31, 33, 25, 27, or 39.
  • the CsPT enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NOs. 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the CsPT enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
  • the C. saliva O-methyltransferase (CsOMT) enzyme useful in the present invention may include any enzyme encoded by a polynucleotide that is substantially similar to SEQ ID NOs. 41, 43, 45, 47, 49, or 51.
  • the polynucleotide encoding the CsOMT enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 41, 43, 45, 47, 49, or 51.
  • the CsOMT enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NOs. 42, 44, 46, 48, 50, or 52.
  • the CsOMT enzyme can be any enzyme encoded by a polynucleotide that is substantially similar to SEQ ID NOs.
  • SUBSTITUTE SHEET (RULE 26) comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 42, 44, 46, 48, 50, or 52.
  • the genetically modified host microorganism comprises one or more polynucleotides containing a genetic modification (e.g., a mutation, deletion, insertion, or the like) that results in optimized (increased or decreased) activity of the flavonoid pathway enzyme encoded thereby.
  • a genetic modification e.g., a mutation, deletion, insertion, or the like
  • optimized enzymes can indude, for example, optimized forms of PAL, C4H, 4CL, CHS, CHI, F3H, DPR, R3 ⁇ , R3'5 ⁇ , CsPT, and CsOMT.
  • Enzymatic activity may be optimized in a variety of ways, inducting, but not limited to, 1) modulating the strength of the promoter to which the flavonoid pathway enzyme coding region is operably linked; 2) modulating the copy number of the plasmid comprising a nucleotide sequence encoding the flavonoid pathway enzyme; 3) modulating the stability of a flavonoid pathway enzyme mRNA; 4) modifying the sequence of the ribosome binding site of a flavonoid pathway enzyme mRNA such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 5) modifying the sequence between the ribosome binding site of a flavonoid pathway enzyme mRNA and the start codon of the flavonoid pathway enzyme coding sequence such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 6) modifying the entire interdstronic region 5’ of the start codon of the flavonoid pathway enzyme coding region such that translation of the flavonoid pathway enzyme mRNA
  • additional enzymes can be provided to the host microorganism to yield other desired end products.
  • the nucleotide sequence encoding a gpne product is modified such that the nucleotide sequence reflects the codon preference for the particular host cell.
  • conservative amino adds substitutions can be made based on whether the microorganism typically uses a spedfic amino add or how much of that particular amino acid is available for use within the spedfic microorganism
  • the nudeotide sequence will in some embodiments be modified for yeast, bacteria, plant, or algae codon preference.
  • the coding sequence is modified such that the level of translation of the encoded enzyme (e.g., a CsPT, CsOMT, etc.) is modulated, i.e., increased or decreased.
  • Modulating the level of enzyme translation may be achieved by modifying the sequence to indude codons that are rare or not commonly used by the host cell. Codon usage tables for many organisms are available that summarize the percentage of time a spedfic organism uses a specific codon to encode for an amino add. Certain codons are used more often than other codons. The use of less commonly used (so- called “rare”) codons in a sequence generally decreases its rate of translation.
  • a coding sequence may be modified by introducing one or more rare codons, which affect the rate of translation, but not the amino acid sequence of the enzyme translated.
  • one or more the nudeotide sequences encoding for one or more of the enzymes in the flavonoid pathway can be driven by a molecular switch.
  • the molecular switch can be turned on or off by a chemical substance.
  • the gpne encoding the flavonoid pathway enzyme can be disrupted. Any methods used to disrupt gene expression or protein functionality can be used.
  • a CRISPR/ Cas9 system can be used to disrupt gene expression. If using the CRISPR system, the guide
  • RNA can be designed to target a sequence that is substantially similar to at least a portion of the polynucleotide that encodes the flavonoid pathway enzyme.
  • Other systems of disruption include knocking out the gene or knocking down the gpne.
  • a dominant negative can also be used, e.g., a polypeptide that mimics the flavonoid pathway enzyme but is not functional.
  • Prenyl transferases constitute a broad group of enzymes catalyzing that transfer allylic prenyl groups (“prenilate”) to acceptor molecules.
  • the allylic prenyl groups may be, e.g., in the form of a geranyl group (CIO) or a dimethylallyl (C5) group.
  • Prenyl transferases may be divided into two stones, as (or Z) and trans (or E), depending upon the stereochemistry of the resulting products.
  • Suitable prenyl transferases in the present invention indude enzymes that prenilate a flavonoid precursor, such as cinnamic add, p-coumaric acid, cafFdc add, ferulic add, or a natural or unnatural isomer of one of the foregoing.
  • the prenyl transferase prenilates the A ring of ferulic acid at position 8.
  • Suitable prenyl transferases include, but are not limited to, a C. sativa prenyl transferases (CsPTs), geranylgeranyl diphosphate (GGPP) synthase, hexaprenyl diphosphate (HexPP) synthase, heptaprenyl diphosphate (HepPP) synthase, octaprenyl (OPP) diphosphate synthase, solanesyl diphosphate (SPP) synthase, decap renyl diphosphate (DPP) synthase, gorge synthase, and gutta-percha synthase; and a Z-isoprenyl diphosphate synthase, including, but not limited to, nonap renyl diphosphate (NPP) synthase, undecaprenyl diphosphate (UPP) synthase, dehydrodolichyl diphosphate synthase, d
  • nudeotide sequences of numerous prenyl transferases from a variety of spedes are known, and can be used or modified for use in produdng a genetically modified eukaryotic host cdl. See, eg., DNA or RNA encoding: famesyl pyrophosphate synthetase (HFPS) (GenBank Accession No. J05262, Homo sapiens), ⁇ famesyl diphosphate synthetase (FPF) (GenBank Accession No.
  • HFPS famesyl pyrophosphate synthetase
  • FPF famesyl diphosphate synthetase
  • GGPS1 geranylgeranyl pyrophosphate synthase
  • GGPP GGPP synthetase / famesyltranstransferase
  • GenBank Accession No. At4g36810 Arabidopsis thaliana
  • famesyl geranylgeranyl, geranylfamesyl, hexaprenyl, heptaprenyl diphosphate synthase (SelF-HepPS) (GenBank Accession No. AB016095, Synechococcus elongatus); etc.
  • amino add sequences of various prenyl transferase from C. sativa or one its rdatives are known and can be used or modified for use in producing a genetically modified eukaryotic host cell. See, eg., CsPTl (GenBank Accession No. PK28436), CsPT2 (GenBank Accession No. PK02092), CsPT3 (GenBank Accession No. PK17697), CsPT4 (GenBank Accession No. PK15523), CsPT5 (GenBank Accession No. PK11068), CsPT6 (GenBank Accession No. PK13891), CsPT7 (GenBank Accession No. PK29226), CsPT8 (GenBank Accession No.
  • H1PT1 GenBank Accession No. AB543053
  • H1PT2 GenBank Accession No. KM222442
  • AtVTE2-l GeneBank Accession No. AAM10489
  • GmVTE2-l GenBank Accession No. ABB70126
  • TaVTE2-l GenBank Accession No. ABB70123
  • ZmVTE2-l GenBank Accession No. ABB70122
  • expression vectors can be used to express one or more heterologous enzyme within the host microorganism.
  • Vector constructs prepared for introduction into the host microorganisms described throughout can typically, but not always, comprise a replication system (i.e. vector) recognized by the host.
  • the vector includes the intended polynucleotide fragment encoding the desired polypeptide and, optionally, transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment.
  • Expression vectors can include, for example, an origin of replication or autonomously replicating sequence (ARS), expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome- binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, polynucleotides homologous to host chromosomal DNA, and/or a multiple cloning site.
  • Signal peptides can also be included where appropriate, for example from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell.
  • the expression vector may be introduced into the host cell stably or transiently using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, heat shock in the presence of lithium acetate, and the like.
  • a nucleic add will generally further indude a selectable marker, e.g., any of several well-known sdectable markers such as neomycin resistance, ampidllin resistance, tetracycline resistance, chloramphenicol resistance, kanamydn resistance, and the like.
  • the nucldc acid with which the host cdl is genetically modified is an expression vector that indudes a nuddc add comprising a nudeotide sequence that encodes a gene product, e.g., an flavonoid pathway enzyme, a transcription factor, a prenyl transferase, an O- methyltransferases synthase, etc.
  • a gene product e.g., an flavonoid pathway enzyme, a transcription factor, a prenyl transferase, an O- methyltransferases synthase, etc.
  • Suitable expression vectors indude, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artifidal chromosomes, viral vectors (e.g. viral vectors based on vacdnia vims, poliovirus, adenovirus, adeno-associated vims, SV40, herpes simplex vims, and the like), Pl-based artifidal chromosomes, yeast plasmids, yeast artifidal chromosomes, and any other vectors spedfic for specific hosts of interest (such as yeast).
  • viral vectors e.g. viral vectors based on vacdnia vims, poliovirus, adenovirus, adeno-associated vims, SV40, herpes simplex vims, and the like
  • a nudeic add encoding a gene product(s) is included in any one of a variety of expression vectors for expressing the gene produces).
  • Such vectors indude chromosomal, nonchromosomal, and synthetic DNA sequences.
  • the vectors can be constructed using standard methods (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989), and Ausubd et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, (1995).
  • Suitable vectors can be sdected to accommodate a polynudeotide encoding a protdn of a desired size.
  • a suitable host cell eg, the microorganisms described herdn
  • Each vector contains various functional components, which generally indude a cloning site, an origin of replication and at least one selectable marker gene.
  • a vector can additionally possess one or more of the following dements: an enhancer, a promoter, a transcription termination sequence and/or other signal sequences.
  • sequence dements can be optimized for the sdected host spedes.
  • sequence dements can be positioned in the vicinity of the cloning site, such that they are operativdy linked to the gpne encoding a preselected enzyme.
  • Vectors, inducting doning and expression vectors can contain polynudeotides that enable the vector to replicate in one or more selected microorganisms.
  • the sequence can be one that enables the vector to replicate independently of the host chromosomal DNA and can include origins of replication or autonomously replicating sequences.
  • Such sequences are well known for a variety of bacteria, yeast and viruses.
  • the origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria
  • the origin of replication for 2 micron plasmid is suitable for yeast
  • various viral origins of replication e.g. SV40, adenovirus
  • a cloning or expression vector can contain a selection gene, also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed microorganisms in a selective culture medium. Microorganisms not transformed with the vector containing the selection gene will therefore not survive in the culture medium.
  • Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromyrin, kanamyxin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
  • the replication of vectors can be performed in any suitable host, such as E. cob.
  • E. cob An example of an E. coli-selectable marker is the b-lactamase gene, which confers resistance to the antibiotic ampirillin.
  • selectable markers can be obtained from E. cob plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.
  • Vectors can contain a promoter that is recognized by the host microorganism
  • the promoter can be operably linked to a coding sequence of interest.
  • Such a promoter can be inducible, repressible, or constitutive.
  • Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.
  • Different promoters can be used to drive the expression of the genes. For example, if temporary gene expression (i.e., non-constitutively expressed) is desired, expression can be driven by inducible or repressible promoters.
  • the molecular switch can in some cases comprise these inducible or repressible promoters.
  • the desired gene is expressed temporarily.
  • the desired gpne is not constitutively expressed.
  • the expression of the desired gene can be driven by inducible or repressible promoters, which functions as a molecular switch.
  • inducible or repressible switches include, but are not limited to, those promoters inducible or repressible by: (a) sugars such as arabinose and lactose (or non metabolizable analogs, eg., isopropyl b-D-l-thiogalactopyranoside (IPTG)); (b) metals such as copper or calcium (or rare earth metals such as lanthanum or cerium); (c) temperature; (d) Nitrogen-source; (e) oxygen; (f) cell state (growth or stationary); (g) metabolites such as phosphate; (h) CRISPRi; (i) jun; (j) fos, (k) metallothionein and/or (l) heat shock.
  • Inducible or repressible switches that can be particularly useful are switches that are responsive to sugars and rare earth metals.
  • promoters that are sensitive to the sugar arabinose can be used as an inducible switch.
  • arabinose switches can be used to drive expression of one or more gpnes.
  • a desired vector or expression of a gene set can be “tumed-on.” The arabinose switch can turn on the expression of a desired gene.
  • Other particularly useful switches can be rare earth metal switches, such as lanthanum sensitive switches (also simply known as a lanthanum switch).
  • the lanthanum switch can be a repressible switch that can be used to repress expression of one or more genes, until the repressor is removed (e.g., in this case lanthanum), after which the genes are “tumed-on”.
  • the desired gpne set or vector in the presence the rare earth metal lanthanum, can be “tumed-off.”
  • the lanthanum switch can be turned off (and expression of the genes induced) by either removing the lanthanum from the media or diluting the lanthanum in the media to levels where its repressible effects are reduced, minimized, or eliminated.
  • Other rare earth metal switches can be used, such as those disclosed throughout.
  • Constitutively expressed promoters can also be used in the vector systems herein.
  • the expression of one or more desired genes can be controlled by constitutively active promoters. Examples of such promoters include but are not limited to Gal promoters, which are well- known in the art.
  • the promoter is Gall, GallO, TEF1, TDH3, PGK1, or ADH2.
  • the promoter is a Gal80 knock out.
  • the promoter is a synthetic promoter.
  • Promoters suitable for use with prokaryotic hosts can include, for example, the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (tip) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Dalgamo sequence operably linked to the coding sequence. [00121] Generally, a strong promoter can be employed to provide for high level transcription and expression of the desired product. For example, promoters that can be used include but are not limited to Gal promoters, which are well-known in the art.
  • the promoter is Gall, GallO, TEF1, TDH3, PGK1, or ADH2. In one embodiment, the promoter is a Gal80 knock out. In some embodiments, the promoter is a synthetic promoter. In some cases, a mutation can increase the strength of the promoter and therefore result in elevated levels of expression.
  • a weaker promoter is desired. For example, this is the case where too much expression of a certain gene results in a detrimental effect (e.g., the killing of cells).
  • a weak promoter can be used, for example a CYC1 promoter.
  • a weaker promoter can be made by mutation.
  • One or more promoters of a transcription unit can be an inducible promoter.
  • a GFP can be expressed from a constitutive promoter while an inducible promoter is used to drive transcription of a gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.
  • the genes described herein can all have a promoter driving their expression.
  • the methods described herein e.g., genome editing, can be used to edit the polynucleotide of the promoters or used to inhibit the effectiveness of the promoters. Inhibition can be done by blocking the transcription machinery (e.g., transcription factors) from binding to the promoter or by altering the promoter in such a way that the transcription machinery no longer recognizes the promoter sequence.
  • the vectors described herein can also comprise a polynucleotide encoding for one or more of the genes within the flavonoid pathway. These vectors can also contain one or more regulatory elements (inducible and/ or repressible promoters) that control the expression of the genes within the vectors. In some cases, the vectors can include switches, including but not limited to inducible or repressible switches, e.g., an arabinose or lanthanum switches. These genes can be heterologous to the microorganism in which the vector is contacted with (and eventually transformed with).
  • the genes used in the vectors can be any genes described throughout the application, including gpnes encoding enzymes involved in the flavonoid pathway. These enzymes can be encoded by a polynucleotide or by an polypeptide that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to any one ofSEQ ID NOs: 1-24.
  • the genes that are inserted into a host microorganism can be heterologous to the microorganism itself.
  • the inserted genes can, for example, be from plants, fungi, algae, bacteria, or even a different species of yeast.
  • the genes can be endogenously part of the genome of the host microorganism itself.
  • the genetically modified microorganisms disclosed throughout can be made by a variety of ways.
  • the microorganism may be modified (e.g, genetically-engineered) by any method to comprise and/or express one or more polynucleotides encoding for enzymes in a pathway that catalyze a conversion of a carbon source to one or more intermediates in a pathway for the production of flavonoids or flavonoid derivative compounds.
  • Such enzymes can include those discussed herein.
  • one or more of any of the genes discussed throughout can be inserted into a microorganism The genes can be inserted by an expression vector.
  • the genes can also be under the control of one or more different/ same promoters or the one or more genes can be under the control of a switch, such as an inducible or repressible promoter, e.g, an arabinose switch, isopropyl b-D-l- thiogalactopyranoside (IPTG) switch, or a rare earth metal switch.
  • a switch such as an inducible or repressible promoter, e.g, an arabinose switch, isopropyl b-D-l- thiogalactopyranoside (IPTG) switch, or a rare earth metal switch.
  • IPTG isopropyl b-D-l- thiogalactopyranoside
  • the genes can also be stably integrated into the genome of the microorganism In some cases, the genes can be expressed in an episomal vector.
  • An exemplary method of making a genetically modified microorganism disclosed herein is contacting (or transforming) a microorganism with a nucleic acid that expresses at least one heterologous gene encoding 1) PAL, 2) C4H, 3) 4CL, 4) CHS, 5) CHI, 6) F3H, 7) DFR, 8) R3 ⁇ , 9) R3'5 ⁇ , 10) CsPT, 11) CsOMT, and/or 12) a natural or unnatural isomer of any of the foregoing.
  • the microorganism can be any microorganism that is capable of converting a carbon substrate to a desirable end product, such as a flavonoid or flavonoid derivative.
  • the method of the invention further comprises isolating the flavonoid or flavonoid derivative from the host microorganism and/or from the culture medium.
  • the genetically modified host microorganism is cultured in a suitable medium, optionally supplemented with one or more additional agents, such as an inducer (e.g., where one or more nucleotide sequences encoding a gene product is under the control of an inducible promoter).
  • the culture medium is overlaid with an organic solvent, e.g., dodecane, forming an organic layer.
  • the flavonoid confound produced by the genetically modified host microorganism may partition into the organic layer, from which it can be purified.
  • an inducer is added to the culture medium; and, after a suitable time, the flavonoid compound is isolated from the organic layer overlaid on the culture medium.
  • the flavonoid or flavonoid derivative is separated from other products which may be present in the organic layer. Such separation may be achieved using, e.g., standard chromatographic techniques.
  • the flavonoid or flavonoid derivative is pure, e.g., at least about 20% pure, at least about 30% pure, at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure, where the term “pure” in the context of a flavonoid or flavonoid derivative refers to a flavonoid or flavonoid derivative that is free from other flavonoid compounds, contaminants, etc.
  • microorganisms disclosed herein can be genetically engineered by using classic microbiological techniques. Some of these techniques are generally disclosed, for example, in D. Amberg, D. Burke and J. Strathem, Methods in Yeast Genetics, 2005 Edition, Cold Spring Harbor Laboratory Press.
  • the genetically modified microorganisms disclosed herein can include a polynucleotide that has been inserted, deleted or modified (i.e., mutated; eg, by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (eg, over-expression) of one or more enzymes as provided herein within the microorganism
  • Genetic modifications that result in an increase in gpne expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene.
  • Addition of a gpne to increase gene expression can include maintaining the gene(s) on replicating plasmids or integrating the cloned gpne(s) into the genome of the production microorganism
  • increasing the expression of desired genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements.
  • the expression of one or more of the enzymes provided herein is under the control of a regulatory sequence that controls directly or indirectly the enzyme expression in a time- dependent fashion during the fermentation. Inducible promoters can be used to achieve this.
  • a microorganism is transformed or transfected with a genetic vehide, such as an expression vector comprising a heterologous polynudeotide sequence coding for the enzymes are provided herein.
  • the vector(s) can be an episomal vector, or the gene sequence can be integrated into the genome of the microorganism, or any combination thereof.
  • the vectors comprising the heterologous polynudeotide sequence encoding for the enzymes provided herdn are integrated into the genome of the microorganism.
  • the constructs can be designed with at least one cloning site for insertion of any gene coding for any enzyme disclosed herein.
  • the cloning site can be a multiple cloning site, e.g., containing multiple restriction sites.
  • transfection or transformation
  • the exogenous nucldc acid or polynudeotide can be maintained as a non-integrated vector, for example, a plasmid or episomal vector, or alternatively, can be integrated into the host cell genome.
  • transfecting or transfection is intended to encompass all conventional techniques for introducing nudeic acid or polynudeotide into microorganisms.
  • transfection techniques indude, but are not limited to, calcium phosphate predpitation, DEAE-dextran-mediated transfection, lipofection, dectroporation, microinjection, rubidium chloride or polycation mediated transfection, protoplast fusion, and sonication.
  • the transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type is favored.
  • the constmcts are integrated so as to be stably maintained within the host chromosome.
  • the preferred transfection is a stable transfection.
  • the integration of the gene occurs at a specific locus within the genome of the microorganism.
  • Expression vectors or other nucleic adds can be introduced to sdected microorganisms by any of a number of suitable methods.
  • vector constructs can be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors. Standard calcium-chloride- mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria.
  • yeast or other fungal cells For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods can be used (see, e.g., Rose et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990)). Transformed cells can be isolated on selective media appropriate to the selectable marker used. Alteratively, or in addition, plates or filters lifted from plates can be scanned for GFP fluorescence to identify transformed clones.
  • Plasmid vectors can be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., N.Y., N.Y. (1988)).
  • lipofection lipid-mediated transfection
  • DEAE-dextran-mediated transfection DEAE-dextran-mediated transfection
  • electroporation electroporation
  • calcium phosphate precipitation see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., N.Y., N.Y. (1988)).
  • Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. Many companies offer kits and ways for this type of transfection.
  • the host cell can be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation.
  • Microorganisms can be transformed or transfected with the above-described expression vectors or polynucleotides coding for one or more enzymes as disclosed herein and cultured in nutrient media modified as appropriate for the specific microorganism, inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In some cases, electroporation methods can be used to deliver an expression vector.
  • Expression of a vector can be verified by an expression assay, for example, qPCR or by measuring levels of RNA.
  • Expression level can be indicative also of copy number. For example, if expression levels are extremely high, this can indicate that more than one copy of a gene was integrated in a genome. Alteratively, high expression can indicate that a gpne was integrated in a highly transcribed area, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as through Western blotting.
  • CRISPR / cas system The methods disclosed throughout can involve pinpoint insertion of genes or the deletion of genes (or parts of genes). Methods described herein can use a CRISPR/cas system. For example, double-strand breaks (DSBs) can be generated using a CRISPR/ cas system, e.g., a type II CRISPR/ cas system.
  • a Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage.
  • Enzymatic action by Cas9 from Streptococcus pyogenes or any closely related Cas9 can generate double stranded breaks at target site sequences which hybridize to 20 nucleotides of a guide sequence and have a protospacer-adjacent motif (PAM) following the 20 nucleotides of the target sequence.
  • PAM protospacer-adjacent motif
  • a vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Gas protein.
  • Gas proteins that can be used include class 1 and class 2.
  • Non-limiting examples of Gas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, CasSd, CasSt, CasSh, CasSa, Gas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, CseSe, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7,
  • An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9.
  • a CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/ or within a complement of a target sequence.
  • a CRISPR enzyme can direct cleavage of one or both strands within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, or more base pairs from the first or last nucleotide of a target sequence.
  • a vector that encodes a CRISPR enzyme that is mutated to with respect, to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used.
  • a vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences can be used.
  • NLSs nuclear localization sequences
  • a CRISPR enzyme can comprise the NLSs at or near the ammo-terminus (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or at or near the carboxy-terminus (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or any combination of these (eg., one or more NLS at the ammo-terminus and one or more NLS at the carboxy terminus).
  • each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
  • CRISPR enzymes used in the methods can comprise at most 6 NLSs.
  • An NLS is considered near the N- or C-terminus when the nearest amino add to the NLS is within 50 amino acids along a polypeptide chain from the N- or C-terminus, eg, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino adds.
  • guide RNA and its grammatical equivalents refers to an RNA that can be specific for a target DNA and can form a complex with Cas protein.
  • An RNA/Cas complex can assist in “guiding” Cas protein to a target DNA.
  • a method disclosed herein also can comprise introducing into a cell or embryo at least one guide RNA or nucleic acid, eg, DNA encoding at least one guide RNA.
  • a guide RNA can interact with a RNA-guided endonuclease to direct the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in a chromosomal sequence.
  • a guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA).
  • crRNA CRISPR RNA
  • tracrRNA transactivating crRNA
  • a guide RNA can sometimes comprise a single-chain RNA, or single guide RNA (sgRNA) formed by fusion of a portion (eg, a functional portion) of crRNA and tracrRNA.
  • sgRNA single guide RNA
  • a guide RNA can also be a dualRNA comprising a crRNA and a tracrRNA
  • a crRNA can hybridize with a target DNA.
  • a guide RNA can be an expression product.
  • a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA.
  • a guide RNA can be transferred into a cell or microorganism by transfecting the cell or microorganism with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter.
  • a guide RNA can also be transferred into a cell or microorganism in other way, such as using virus-mediated gene delivery.
  • a guide RNA can be isolated.
  • a guide RNA can be transfected in the form of an isolated RNA into a cell or microorganism.
  • a guide RNA can be prepared by in vitro transcription using any in vitro transcription system.
  • a guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
  • a guide RNA can comprise three regions: a first region at the 5’ end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem loop structure, and a third 3’ region that can be single-stranded.
  • a first region of each guide RNA can also be different such that each guide RNA guides a fusion protein to a specific target site.
  • second and third regions of each guide RNA can be identical in all guide RNAs.
  • a first region of a guide RNA can be complementary to sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site.
  • a first region of a guide RNA can comprise from 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to 25 nucleotides; or 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides or more.
  • a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length.
  • a first region of a guide RNA can be 19, 20, or 21 nucleotides in length.
  • a guide RNA can also comprise a second region that forms a secondary structure.
  • a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop.
  • a length of a loop and a stem can vary.
  • a loop can range from 3 to 10 nucleotides in length
  • a stem can range from 6 to 20 base pairs in length.
  • a stem can comprise one or more bulges of 1 to 10 nucleotides.
  • the overall length of a second region can range from 16 to 60 nucleotides in length.
  • a loop can be 4 nucleotides in length and a stem can be 12 base pairs.
  • a guide RNA can also comprise a third region at the 3’ end that can be essentially single- stranded.
  • a third region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA.
  • the length of a third region can vary.
  • a third region can be more than 4 nucleotides in length.
  • the length of a third region can range from 5 to 60 nucleotides in length.
  • a guide RNA can be introduced into a cell or embryo as an RNA molecule.
  • a RNA molecule can be transcribed in vitro and/or can be chemically synthesized.
  • An RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks ® gene fragment.
  • a guide RNA can then be introduced into a cell or embryo as an RNA molecule.
  • a guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule.
  • a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest.
  • RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III).
  • Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors.
  • a plasmid vector eg, px333 vector
  • a DNA sequence encoding a guide RNA can also be part of a vector. Further, a vector can comprise additional expression control sequences (eg, enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc), selectable marker sequences (eg, antibiotic resistance genes), origins of replication, and the like.
  • a DNA molecule encoding a guide RNA can also be linear.
  • a DNA molecule encoding a guide RNA can also be circular.
  • each DNA sequence can be part of a separate molecule (eg., one vector containing an RNA-guided endonuclease coding sequence and a second vector containing a guide RNA coding sequence) or both can be part of a same molecule (eg, one vector containing coding (and regulatory) sequence for both an RNA-guided endonuclease and a guide RNA).
  • Insertion of the gpnes can be site-specific. For example, one or more genes can be inserted adjacent to a promoter.
  • Modification of a targeted locus of a microorganism can be produced by introducing DNA into microorganisms, where the DNA has homology to the target locus.
  • DNA can include a marker gene, allowing for selection of cells comprising the integrated construct.
  • Homologous DNA in a target vector can recombine with DNA at a target locus.
  • a marker gene can be flanked on both sides by homologous DNA sequences, a 3’ recombination arm, and a 5’ recombination arm.
  • a variety of enzymes can catalyze insertion of foreign DNA into a microorganism genome.
  • site-specific recombinases can be clustered into two protein families with distinct biochemical properties, namely tyrosine recombinases (in which DNA is covalently attached to a tyrosine residue) and serine recombinases (where covalent attachment occurs at a serine residue).
  • recombinases can comprise Cre, ⁇ DC31 integrase (a serine recombinase derived from Streptomyces phage FC31), or bacteriophage derived site-spedfic recombinases including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901- 1 integrase).
  • Cre a serine recombinase derived from Streptomyces phage FC31
  • bacteriophage derived site-spedfic recombinases including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901- 1 integrase.
  • the CRISPR/ Cas system can be used to perform site specific insertion.
  • a nick on an insertion site in the genome can be made by CRISPR/ cas to facilitate the insertion of a transgene at the insertion site.
  • the methods described herein can utilize techniques that can be used to allow a DNA or RNA construct entry into a host cell include, but are not limited to, calcium phosphate/DNA coprecipitation, microinjection of DNA into a nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, sperm mediated gene transfer, or any other technique.
  • vectors including the ones described above. Any plasmids and vectors can be used as long as they are replicable and viable in a selected host microorganism. Vectors known in the art and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods.
  • Vectors that can be used include, but not limited to eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBIlOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHllO, and pKK232-8 (Pharmacia, Inc.), pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392, pBlueB
  • vectors can be used to express a gene or portion of a gene of interest.
  • a gene of portion or a gene can be inserted by using known methods, such as restriction enzyme-based techniques.
  • the microorganisms useful in the present invention should be cultured in fermentation conditions that are appropriate to convert a carbon substrate into a flavonoid or flavonoid derivative.
  • Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition.
  • the optimum reaction conditions will depend partly on the particular microorganism used. Fermentation Conditions
  • the pH of the culture media may be optimized based on the microorganism used.
  • the pH used can range from 4 to 10.
  • the pH can be from 5 to 9; 6 to 8; 6.1 to 7.9; 6.2 to 7.8; 6.3 to 7.7; 6.4 to 7.6; or 6.5 to 7.5.
  • the pH can be from 6.6 to 7.4.
  • the pH can be from 5 to 9.
  • the pH can be from 6 to 8.
  • the pH can be from 6.1 to 7.9.
  • the pH can be from 6.2 to 7.8.
  • the pH can be from 6.3 to 7.7.
  • the pH can be from 6.4 to 7.6.
  • the pH can be from 6.5 to 7.5.
  • the pH used can be greater than 6.
  • Temperature can also be adjusted based on the microorganism used.
  • the temperature can range from 25 C° to 48 C°.
  • the temperature of the fermentation can be from 25 C° to 48 C°; 26 C° to 46 C°; 27 C° to 44 C°; 28 C° to 42 C°; 29 C° to 41 C°; 30 C° to 40 C°; 30 C° to 39 C°; 31 C° to 38 C°; 32 C° to 37 C°; 33 C° to 36 C°; 34 C° to 37 C°; 35 C° to 36 C°; 30 C° to 43 C°; 32 C° to 42 C°; 34 C° to 41 C°; 35 C° to 40 C°.
  • the temperature can be 25 C°, 26 C°, 27 C°, 28 C°, 29 C°, 30 C°, 31 C°, 32 C°, 33 C°, 34 C°, 35 C°, 36 C°, 37 C°, 38 C°, 39 C°, 40 C°, 41 C°, 42 C°, 43 C°, 44 C°, or 45 C°.
  • the temperatures can be within one tenth of a degree, such as 25.0 C°, 25.1 C°, 25.2 C°, 25.3 C°, 25.4 C°, 25.5 C°, 25.6 C°, 25.7 C°, 25.8 C°, 25.9 C°, 30.0 C°, 30.1 C°, 30.2 C°, 30.3 C°, 30.4 C°, 30.5 C°, 30.6 C°, 30.7 C°, 30.8 C°, 30.9 C°, 31.0 C°, 31.1 C°, 31.2 C°, 31.3 C°, 31.4 C°, 31.5 C°, 31.6 C°, 31.7 C°, 31.8 C°, 31.9 C°, 32.0 C°, 32.1 C°, 32.2 C°, 32.3 C°, 32.4 C°, 32.5 C°, 32.6 C°, 32.7 C°, 32.8 C°, 32.9 C°, 33.0 C°, 33.1 C°, 33.2 C°, 33.3 C°, 33.4 C°, 33.5 C°, 33.6
  • the temperature of fermentation can be from 37.0 C° to 47.9 C°. In some cases, the temperature of fermentation can be from 37.1 C° to 47.8 C°. In some cases, the temperature of fermentation can be from 37.2 C° to 47.7 C°. In some cases, the temperature of fermentation can be from 37.3 C° to 47.6 C°. In some cases, the temperature of fermentation can be from 37.4 C° to
  • the temperature of fermentation can be from 37.5 C° to 47.4 C°. In some cases, the temperature of fermentation can be from 37.6 C° to 47.3 C°. In some cases, the temperature of fermentation can be from 37.7 C° to 47.2 C°. In some cases, the temperature of fermentation can be from 37.8 C° to 47.1 C°. In some cases, the temperature of fermentation can be from 37.9 C° to 47.0 C°. In some cases, the temperature of fermentation can be from 38.0 C° to 46.9 C°. In some cases, the temperature of fermentation can be from 38.1 C° to 46.8 C°. In some cases, the temperature of fermentation can be from 38.2 C° to 46.7 C°. In some cases, the temperature of fermentation can be from 38.3 C° to 46.6 C°. In some cases, the temperature of fermentation can be from 38.4 C° to
  • the temperature of fermentation can be from 38.5 C° to 46.4 C°. In some cases, the temperature of fermentation can be from 38.6 C° to 46.3 C°. In some cases, the temperature of fermentation can be from 38.7 C° to 46.2 C°. In some cases, the temperature of fermentation can be from 38.8 C° to 46.1 C°. In some cases, the temperature of fermentation can be from 38.9 C° to 46.0 C°. In some cases, the temperature of fermentation can be from 39.0 C° to 45.9 C°. In some cases, the temperature of fermentation can be from 39.1 C° to 45.8 C°. In some cases, the temperature of fermentation can be from 39.2 C° to 45.7 C°. In some cases, the temperature of fermentation can be from 39.3 C° to 45.6 C°. In some cases, the temperature of fermentation can be from 39.4 C° to
  • the temperature of fermentation can be from 39.5 C° to 45.4 C°. In some cases, the temperature of fermentation can be from 39.6 C° to 45.3 C°. In some cases, the temperature of fermentation can be from 39.7 C° to 45.2 C°. In some cases, the temperature of fermentation can be from 39.8 C° to 45.1 C°. In some cases, the temperature of fermentation can be from 39.9 C° to 45.0 C°. In some cases, the temperature of fermentation can be from 40.0 C° to 44.9 C°. In some cases, the temperature of fermentation can be from 40.1 C° to 44.8 C°. In some cases, the temperature of fermentation can be from 40.2 C° to 44.7 C°. In some cases, the temperature of fermentation can be from 40.3 C° to 44.6 C°. In some cases, the temperature of fermentation can be from 40.4 C° to
  • the temperature of fermentation can be from 40.5 C° to 44.4 C°. In some cases, the temperature of fermentation can be from 40.6 C° to 44.3 C°. In some cases, the temperature of fermentation can be from 40.7 C° to 44.2 C°. In some cases, the temperature of fermentation can be from 40.8 C° to 44.1 C°. In some cases, the temperature of fermentation can be from 40.9 C° to 44.0 C°. In some cases, the temperature of fermentation can be from 41.0 C° to 43.9 C°. In some cases, the temperature of fermentation can be from 41.1 C° to 43.8 C°. In some cases, the temperature of fermentation can be from 41.2 C° to 43.7 C°. In some cases, the temperature of fermentation can be from 41.3 C° to 43.6 C°. In some cases, the temperature of fermentation can be from 41.4 C° to
  • the temperature of fermentation can be from 41.5 C° to 43.4 C°. In some cases, the temperature of fermentation can be from 41.6 C° to 43.3 C°. In some cases, the temperature of fermentation can be from 41.7 C° to 43.2 C°. In some cases, the temperature of fermentation can be from 41.8 C° to 43.1 C°. In some cases, the temperature of fermentation can be from 41.9 C° to 43.0 C°. In some cases, the temperature of fermentation can be from 42.0 C° to 42.9 C°. In some cases, the temperature of fermentation can be from 42.1 C° to 42.8 C°. In some cases, the temperature of fermentation can be from 42.2 C° to 42.7 C°. In some cases, the temperature of fermentation can be from 42.3 C° to 42.6 C°. In some cases, the temperature of fermentation can be from 42.4 C° to 42.5 C°.
  • the percent of dissolved oxygen (DO) within the fermentation media can be from 1% to 40%.
  • the DO concentration can be from 1.5% to 35%; 2% to 30%; 2.5% to 25%; 3% to 20%; 4% to 19%; 5% to 18%; 6% to 17%; 7% to 16%; 8% to 15%; 9% to 14%; 10% to 13%; or 11% to 12%.
  • the DO concentration can be from 2% to 30%.
  • the DO can be from 3% to 20%.
  • the DO can be from 4% to 10%.
  • the DO can be from 1.5% to 35%. In some cases, the DO can be from 2.5% to 25%. In some cases, the DO can be from 4% to 19%. In some cases, the DO can be from 5% to 18%. In some cases, the DO can be from 6% to 17%. In some cases, the DO can be from 7% to 16%. In some cases, the DO can be from 8% to 15%. In some cases, the DO can be from 9% to 14%. In some cases, the DO can be from 10% to 13%. In some cases, the DO can be from 11% to 12%.
  • Fermentation reactions can be carried out in any suitable bioreactor.
  • the bioreactor can comprise a first growth reactor in which the microorganisms are cultured, and a second fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product is produced.
  • the fermentation of the microorganisms disclosed herein can produce a broth comprising a desired product (e.g., a flavonoid), one or more by-products, and/or the microorganism itself (e.g., a genetically modified yeast cell) .
  • a desired product e.g., a flavonoid
  • the microorganism itself e.g., a genetically modified yeast cell
  • the microorganisms and the methods herein can produce a desired end product, such as a flavonoid compound or flavonoid derivative, at a relatively high efficiency, more so than other known fermentation processes.
  • the microorganisms and the methods disclosed herein may convert a carbon substrate at a rate of greater than 50%. This means that at least 50% of the carbons of the carbon substrate are converted into the desired product.
  • the conversion of a carbon substrate into desired flavonoid products can be at least 60%, 70%, 80%, 81%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%.
  • the conversion of a carbon substrate into flavonoid products can be at least 60%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 70%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 80%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 81%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 82%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 83%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 84%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 85%.
  • the conversion of a carbon substrate into flavonoid products can be at least 86%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 87%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 88%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 89%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 90%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 91%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 92%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 93%.
  • the conversion of a carbon substrate into flavonoid products can be at least 94%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 95%. In some cases, the conversion of a carbon substrate into mu flavonoid products can be at least 96%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 97%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 98%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 99%.
  • the concentration of flavonoid products in the fermentation broth is at least 0.1 g/L
  • the concentration of flavonoid products produced in the fermentation broth can be from 0.1 g/L to 0.5 g/L, 0.5 g/L to 1 g/L, 1 g/L to 5 g/L, 2 g/L to 6 g/L, 3 g/L to 7 g/L, 4 g/L to 8 g/L, 5 g/L to 9 g/L, or 6 g/L to 10 g/L.
  • the concentration of flavonoid products can be at least 9 g/L.
  • the concentration of flavonoid products can be from 0.1 g/L to to 10 g/L. In some cases, the concentration of flavonoid products can be from 0.5 g/L to 3 g/L. In some cases, the concentration of flavonoid products can be from 1 g/L to 5 g/L. In some cases, the concentration of flavonoid products can be from 2 g/L to 6 g/L. In some cases, the concentration of flavonoid products can be from 3 g/L to 7 g/L. In some cases, the concentration of flavonoid products can be from 4 g/L to 8 g/L. In some cases, the concentration of flavonoid products can be from 5 g/L to 9 g/L. In some cases, the concentration of flavonoid products can be from 6 g/L to 10 g/L.
  • the genetically modified microorganism can produce the same flavonoid product in concentrations that are at least 1.1X the amount that is normally produced by a microorganism that is unmodified and of the same species as the genetically modified microorganism.
  • the genetically modified microorganism can produce at least 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.9X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 60X, 70X, 80X, 90X or 100X the amount that is normally produced by a microorganism that is unmodified and of the same species as the genetically modified microorganism.
  • the genetically modified microorganism can produce at least 2X, 3X, 4X, 5X, 10X, 25X, 50X, and or 100X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 2X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 3X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 4X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 5X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 10X the amount that is normally produced.
  • the genetically modified microorganism can produce at least 25X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 50X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 100X the amount that is normally produced.
  • the flavonoid product produced in the fermentation reaction is converted to a different organic product.
  • the flavonoid product is first recovered from the fermentation broth before conversion to a different organic product.
  • flavonoid product can be continuously removed from a portion of broth and recovered as purified.
  • the recovery of the flavonoid product includes passing the removed portion of the broth containing the flavonoid product through a separation unit to separate the microorganisms (e.g., genetically modified yeast) from the broth, to produce a cell-free flavonoid product permeate, and returning the microorganisms to the bioreactor.
  • the cell-free flavonoid product containing permeate can then can be stored or be used for subsequent conversion to a different desired product.
  • the recovering of the desired flavonoid product and/ or one or more other products or by- products produced in the fermentation reaction can comprise continuously removing a portion of the broth and recovering separately the flavonoid product and one or more other products from the removed portion of the broth.
  • the recovery of the flavonoid product and/or one or more other products includes passing the removed portion of the broth containing the flavonoid product and/or one or more other products through a separation unit to separate microorganisms from the flavonoid product and/or one or more other products, to produce a cell-free flavonoid product and one or more other product-containing permeate, and returning the microorganisms to the bioreactor.
  • the flavonoid product, or a mixed product stream containing the flavonoid product can be recovered from the fermentation broth.
  • methods that can be used can include but are not limited to, fractional distillation or evaporation, pervaporation, and extractive fermentation.
  • indude recovery using steam from whole fermentation broths; reverse osmosis combined with distillation; liquid-liquid extraction techniques involving solvent extraction of the flavonoid product; aqueous two-phase extraction of the flavonoid product in PEG/dextran system; solvent extraction using alcohols or esters, e.g., ethyl acetate, tributylphosphate, diethyl ether, n- butanol, dodecanol, oleyl alcohol, and an ethanol/ phosphate system; aqueous two-phase systems composed of hydrophilic solvents and inorganic salts.
  • alcohols or esters e.g., ethyl acetate, tributylphosphate, diethyl ether, n- butanol, dodecanol, oleyl alcohol, and an ethanol/ phosphate system
  • aqueous two-phase systems composed of hydrophilic solvents and inorganic salts.
  • the flavonoid product and / or other by-products may be recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration, for example), and recovering the flavonoid product and others such as alcohols and adds from the broth.
  • Alcohols can conveniently be recovered for example by distillation, and adds can be recovered for example by adsorption on activated charcoal.
  • the separated microbial cells are returned to the fermentation bioreactor.
  • the cell-free permeate remaining after the alcohol(s) and add(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients can be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor.
  • the pH of the broth is adjusted during recovery of the flavonoid product and/ or by products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
  • compositions and Phamaceutically-Acceptable Derivatives or Prodrugs [00187]
  • the present invention also relates in part to a composition comprising a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof.
  • the composition may further comprise an excipient.
  • the composition may be in the form of a medicament.
  • pharmaceutically acceptable derivative means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative thereof.
  • Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic and organic acids and bases.
  • Salts derived from appropriate bases indude alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4 + salts.
  • the present invention also relates in part to a method of formulating the flavonoid or flavonoid derivative into a pharmaceutical composition.
  • pharmaceutically-acceptable carriers include either solid or liquid carriers.
  • Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules.
  • a solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, including in the latest edition of Remington’s Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
  • the carrier is a finely divided solid, which is in a mixture with the findy divided active component.
  • the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
  • Suitable solid exdpients include carbohydrate or protein fillers, such as lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cdlulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcdlulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen.
  • disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate.
  • Liquid form preparations may also include solutions, suspensions, and emulsions, for example, water or water/ propylene glycol solutions.
  • solutions for example, water or water/ propylene glycol solutions.
  • liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
  • the pharmaceutical preparation can be a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component.
  • the unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules.
  • the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
  • the present invention also relates to a method of making the pharmaceutical composition.
  • a flavonoid or flavonoid derivative is mixed with an excipient to produce a pharmaceutical composition.
  • the flavonoid or flavonoid derivative can be used to treat a disease. This includes treating one or more symptoms of the diseases.
  • the flavonoid or flavonoid derivative can be used to treat one of more of the following diseases: diabetes, heart disease, cardiovascular disease, Parkinson’s disease, malaria, cancer, digestive disorders, autoimmune diseases, chronic inflammation, stroke, obesity, or neurodegenerative disorders.
  • the cancers that the flavonoid or flavonoid derivative may treat include: lung, prostrate, bladder, ovarian, cervical, pancreatic, breast, liver, esophageal, gastric, adenoma, and melanoma.
  • Some of the diseases or symptom of disease can be exclusive to humans, but other diseases or symptom of disease can be shared in more than one animal, such as in all mammals.
  • the present invention relates in part to a method of treating a disease or symptom of a disease, the method comprising administering a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof, to a subject in need of such treatment.
  • Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration.
  • parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.
  • Use of Flavonoid or Flavonoid Derivative includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections.
  • the present invention further relates in part to the use of the flavonoid or flavonoid derivative made using the aforementioned method in the manufacture of a medicament for the treatment or a disease or symptom of a disease.
  • the disease or symptom of a disease may be any disease or symptom capable of being treated by flavonoid or flavonoid derivative, and include those disclosed herein and in the materials incorporated by reference. Examples include diabetes, heart disease, cardiovascular disease, Parkinson’s disease, malaria, cancer (including lung, prostrate, bladder, ovarian, cervical, pancreatic, breast, liver, esophageal, gastric, adenoma, and melanoma), digestive disorders, autoimmune diseases, chronic inflammation, stroke, obesity, or neurodegenerative disorders.
  • a targeted phylogenomics approach combined with in-vitro biochemical assays is employed to explore the biosynthetic pathway towards desirable flavonoids and flavonoid derivatives.
  • a literature and database search of relevant enzymes and substrates is performed to screen for potential enzyme candidates.
  • TSA Transcriptome Shotgun Assembly
  • All previously-characterized enzymes in the rdevant biosynthetic pathway are identified, doned, and tested. If necessary, structural homologs of relevant substrates may be used to locate additional potentially useful enzyme candidates using, e.g., phylogenetic analysis. All such enzyme candidates are then doned and tested.
  • An enzyme library is constructed in yeast to test all the different enzymes identified.
  • the steps involved in building a yeast enzyme library are well known in the art. Briefly, highly expressed copies for each candidate enzyme coding DNA sequence is synthesized (e.g., by GENWIZ) with codon optimization for Saccharomyces cerevisiae and then cloned into a high-copy yeast expression plasmid containing a strong promoter and direct selection marker.
  • the expression construct is then transformed into a suitable strain of Saccharomyces cerevisiae using well-known cloning and transformation techniques. For example, the M2S integration method may used. Li et al., BiotechnoL Biofuels., 9:232-243 (2016).
  • the yeast strain chosen should be already producing a substrate or at least capable of efficiently taking in the substrate.
  • a gpod substrate in this regard is a flavonoid precursor, such as ferulic add, which can be fed to the yeast culture. Positive transformants can be verified, e.g., by sequencing.
  • Transformants are grown in 96-well plate format and, if needed, substrate (e.g., ferulic arid) is fed to the yeast culture. Consumption of the substrate is in the engineered yeast cells is monitored to determine whether it is bring converted to the desired flavonoid.
  • the yeast culture supernatant is also analyzed for the presence of other useful flavonoids or falvonoid derivatives. Products are confirmed by, e.g., high-performance liquid chromatography (HPLC) and mass spectrometry (MS).
  • HPLC high-performance liquid chromatography
  • MS mass spectrometry
  • the yeast cells are collected by centrifugation at 13,000 x g for 10 min and the supernatant is analyzed by HPLC.
  • the yeast cells are washed, disrupted on ice in 30 ml of 50% methanol using a high-pressure homogenizer, and the cell debris is removed by centrifugation at 13,000 x g for 30 min. The supernatant is then collected and analyzed by HPLC.
  • the foregoing flavonoid biosynthesis strategy has several advantages. First, enzymes catalysing the desired flavonoid biosynthesis reactions can be quickly identified and tested and, if necessary, selected for enzyme evolution. Second, this strategy permits for a wide range of non-natural compounds to be quickly produced and tested from a given starting substrate. Third, active library genes can be further combined to catalyze even more complex changes to further increase the diversity and the number and complexity of non-natural products.
  • Isocannflavin B is an unnatural isomer of cannflavin B recently isolated from Cannabis saliva and shown to have statistically significant results against pancreatic cancer in vivo.
  • U.S. Patent No. 10,398,674 to Lowe et al. (incorporated herein by reference).
  • SUBSTITUTE SHEET (RULE 26) accession number X13225; and CHI from Petunia hybnda , GenBank accession number XI 4589.)
  • the modified yeast was fed with different phenylpropanoid acids, including cinnamic add, p-coumaric add, caffdc acid and ferulic acid.
  • 1 mM cinnamic add (148 mg/L) was fed to the yeast as a precursor metabolite, 16.3 mg/L pinocembrin accumulated in the medium Id.
  • ImM p- coumaric add (164 mg/L) was fed to the yeast, 28.3 mg/liter naringenin accumulated in the culture. Id.
  • Koopman and colleagues produced naringenin directly from glucose in modified S. cemdsiae. Koopman et al., Microbial CellFactories, 11:155 (2012) (hereby incorporated by reference). Koopman details the strains, plasmids, and methods used to engineer the naringenin-producing yeast
  • spedfic naringenin biosynthesis genes from Arabidopsis thaliana and Rhodobacter capsulatus; were sdected by comparative expression profiling and overexpressed in S. cemdsiae. These genes encoded the enzymes PALI (phenylalanine/ tyrosine ammonia lyase), C4H (Cinnamate 4- h droxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase), and CHI1 (chalcone isomerase), all obtained from A thaliana, and one gene from PALI (phenylalanine/ tyrosine ammonia lyase), C4H (Cinnamate 4- h droxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synth
  • Rhodobacter capsulatus encoding the enzyme TALI (tyrosine ammonia lyase). Id. The expression of these gpnes yielded relatively low extracellular naringenin concentrations ( ⁇ 5.5 mM). Thus, to optimize naringenin titers, Koopman developed a yeast chassis strain. Synthesis of aromatic amino adds was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (ArolO, Pdc5, Pdc6).
  • isocannflavin may be produced in an engineered yeast strain.
  • the yeast strain may be transformed with, for example, the flavonoid pathway enzymes disdosed in Yan et al .,AppL &Envir. Microbiol, 71:5610-5613 (2005) or Koopman et al., Microbial Cell Factories, 11:155 (2012).
  • the yeast may be transformed with a prenyltransferase and/ or O- methyltransferase, such as those disdosed in Rea et al., Phytochemistiy, 164:162-171 (2019).
  • prenyl transferase that prenilates a flavanone (e.g., naringenin or homoeriodictyl) at the 8 position of the A ring.
  • a flavanone e.g., naringenin or homoeriodictyl
  • Such an enzyme can be identified and cloned, for example, by surveying all the prenyl transferase homologues from Cannabis (or other suitable species) and selecting enzyme candidates that specifically catalyze the A-8 position prenylation, or by generating via enzyme evolution derivatives of prenyl transferase where the A-8 prenylation is favored.
  • Yeast cells successfully transformed with constructs expressing the necessary flavonoid pathway enzymes are grown in 96-well plate format and fed ferulic acid. Ferulic add consumption is then monitored and the reaction lysate is tested for the presence of isocannflavin B, as described above.
  • SEQ ID NO: 27 [Reference: > Cannabis sativa prenyltransferase 3 (PT3) Yeast codon optimized]

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Abstract

Genetically-modified cell comprising at least one heterologous polynucleotide encoding an enzyme involved in the flavonoid pathway. Method of making a flavonoid or flavonoid derivative in such a genetically-modified cell. Use of a flavonoid or flavonoid derivative produced using such a method for the manufacture of a medicament for the treatment of a disease or symptom of a disease. Medicament comprising a flavonoid or flavonoid derivative made using such a method. Method of treating a disease or symptom of a disease comprising administering a flavonoid or flavonoid derivative made using such a method. Isolated nucleic acid encoding at least one enzyme involved in the flavonoid pathway. Vector comprising a nucleic acid encoding at least one enzyme involved in the flavonoid pathway. Method of making a genetically-modified cell capable of synthesizing a flavonoid or flavonoid derivative. Composition comprising a flavonoid or flavonoid derivative.

Description

METHODS AND MICROORGANISMS FOR PRODUCING FLAVONOIDS
BACKGROUND OF THE INVENTION
[001] The subject matter of the present invention relates to microorganisms, such as yeast and bacteria, genetically modified so as to convert carbon substrates into desirable multi-carbon products, such as flavonoids.
[002] Phytochemicals are chemicals produced by plants, and include tannins, lignins, and flavonoids. The largest and best studied phytochemicals are the flavonoids, polyphenolic compounds classified into at least six subgroups: flavonols, flavones, flavanones, flavanols (and their oligomers, proanthocyanidins), anthocyanidins, and isoflavonoids. Flavonoids are widely distributed in plants and function as plant pigments, signaling molecules, and defenders against infection and injury. Plants and fungi synthesize flavonoids from aromatic amino acids L-phenylalanine and L-tyrosine.
[003] Flavonoids are characterized by two phenolic rings and one heterocyclic ring. Key differences between these compounds rdate to their hydroxylation patters, the position of the second aromatic ring, and the saturation of the heterocydic ring. Grotewold, The Science of Flavonoids, Springer Science Business Media, New York (2006). Consumption of flavonoid-rich foods has been linked to improved health, suggesting that flavonoids may be useful as food or beverage nutritional additives. Shahidi et al., /. Fund. Foods 18:820-897 (2015). In addition, the biological activity of different flavonoid compounds has been investigated in rdation to various human diseases and disorders, inducting neurodegenerative and cardiovascular diseases, diabetes, aging, obesity, Parkinson’s disease, and cancer. Moreau et al., Front OncoL 9:660 (2019); Harbome et al., Phytochemistry, 55(6): 481-504 (2000); Vauzour et al., Nutrients, 2(11): 1106-1131 (2010); Dixon, Annu. Rev. Plant Biol. 55: 225-261 (2004). Indeed, the anti-cancer activity of flavonoid compounds has been well documented. Daskiewicz et al.,/. Med. Chem., 48:2790-2804 (2005) (active against human colonic cell lines); Dong et al., Fur. J. Med Chem., 46:5949-5958 (2011) (active against prostate and ovarian cancer, and leukemia); Wang et al.,/. Nat. Proc., 67:757-761 (2004) (active against various cancers, including lung cardnoma, breast adenocarcinoma, and prostate cancer); Yang et al., Fitoterapia, 103:187-191 (2015) (active against various cancer cdl lines, including cervical and liver cancer).
[004] A rich source of useful flavonoids is the Cannabis plant. Studies have identified over twenty flavonoids in Cannabis (cannflavins), including cannflavin A, cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside, vitexin, isovitexin, apigenin, kaemp ferol, myricetin, quercetin, luteolin, homoorientin and orientin. Turner et al., Journal of Natural Products, 43(2), 169-234 (1980). The distribution of these flavonoids in the plant varies depending on the type of flavonoid. The total content of flavonoids in Cannabis leaves and flowers can reach 1-2.5% of its dry weight depending on environment factors and the variety of the plant. [005] Reports have shown that several Cannabis flavonoids have important medicinal and pharmacological properties. ElSohly et al. Life Sciences, 78(5):539-548 (2005). For example, prenylated flavonoids cannflavin and cannflavin B have been shown to inhibit production of PGE2 in human rheumatoid synovial cells and provide anti-inflammatory benefits that were approximately thirty times more effective than aspirin. Barrett et al., Biochem. Pharma, 34(ll):2019-2024 (1985); ML Barrett et al., Experientia, 42:452-453 (1986). It was later reported that the underlying basis for the potent anti inflammatory properties of cannflavins A and B was that they act to inhibit the in vivo production of two pro-inflammatory mediators, prostaglandin E2 and the leukotrienes. Werz et al., Pham. Nutr., 2:53-60 (2014). Cannflavins A and B have also been shown to have anti-leishmanial activity. Radwan et al., Phytochemistry, 69(14):2627-2633 (2008). The isomeric version of cannflavin B, isocannflavin B, has been shown to induce autophagy in hormone sensitive breast cancer cells. Brunelli et al., Fitoterapia,
80(6):327-332 (2009). More recently, various cannabis-based flavonoid extracts were shown to be active against several cancer cell lines, including brain, breast, Kaposi sarcoma, leukemia, lung, melanoma, ovarian, pancreatic, colon and prostate cancer. U.S. Patent No. 10,398,674 to Lowe et al. [006] Cannflavins A and B appear to be Cannabis specific and their unique bioactivity appear to be linked to two key modifications of their parent flavone backbone. First is their distinct prenylation pattern in which a prenyl side-chain, in the form of a geranyl (CIO) or a dimethylallyl (C5) group, are affixed to the 6 position of the flavone A-ring, respectively. Barrett et al., Biochem. Phama, 34(11):2019- 2024 (1985); Choi et al., Phytochem. AnaL, 15:345-354 (2004). These prenyl moieties inpart lipophilidty to the parent flavone, which are believed to enhance uptake and bioaccumulation into cells and promote their interaction with membrane-bound enzymes and receptors that are involved in numerous cell-signaling pathways. Milligan et al.,/. Clin. Endocrinol Metab., 84:2249-2252 (1999); Botta et al., Trends Pharmacol Sd., 26:606-608 (2005); Watjen et al., Food Chem. Toxicol, 45: 119-124 (2007); Werz et al., Pham. Nutr., 2:53-60 (2014); Vochyanova et al., PLoS One, 12 (2017). Second, both cannflavins A and B are modified at the 3’ position of the flavone B-ring with a methoxy group, which also increases lipophilidty and may therefore enhance their cellular retention and access to various cdlular targets. Ibrahim, Can. J. Bot., 83:433-450 (2005); Walle, Mol Pham., 4:826-832 (2007); Berim et al., Phytochemistry Rev., 15:363-390 (2016). [007] Given their potential health-related benefits, it is important to be able to produce sufficient quantities of flavonoids. To date, flavonoid production mostly relies on isolation from plants. However, this has been impeded by low growth rate of some of the producing plants, as well as with the difficulty of extracting and separating flavonoids having highly related structures. Fowler et al., Appl Microbiol Biotechnol 83:799-808 (2009); Wang et al., Appl Microbiol Biotechnol, 91:949-956 (2011).
Moreover, although flavonoids can be produced chemically, their efficient organic synthesis has been hindered by the complexity of the molecules and dangerous reaction conditions required to produce them
[008] In response to the poor production efficiency from plants and chemical synthesis, attention has turned to the heterologous production of flavonoids in microorganisms such as Escherichia coU and Saccharomyces cerevisiae, using metabolic engineering and synthetic biology. Wang et al., Appl Microbiol Biotechnol, 91:949-956 (2011); Leonard et al., Appl Environ Microbiol 73:3877-3886 (2007); Leonard et al., Mol Pharmaceutics, 5(2):257-265 (2008); Santos et al., Metab Eng 13:392-400 (2011); Trantas et al., Metab Eng 11:355-366 (2009); Watts et al., ChemBiochem 5:500-507 (2004). Metabolic engineering alters the function of the microorganism by the insertion of non-native DNA sequences, which produces enzymes that are capable of producing the target compound. This newly established enzymatic system mimics the flavonoid producing pathway in plants and reproduces it, utilizing the already existing precursors in the host microorganism. Ramawat et al., Springer Berlin Heidelberg: Berlin, Heidelberg, pp 1647-1681 (2013). This method saves time, labor, and energy and does not compete with food source. Thus, it could provide a better and more cost-effective alternative sustainable method for the production of flavonoids.
[009] In plants, flavanoid biosynthesis begins with the production of flavonoid precursors, such as the (2S)-flavanones, through the phenylpropanoid metabolic pathway (Fig.2). Yan et al., Appl Environ Microbiol ,71:3617-3623 (2005); Koopmann E. et al., Proc Natl Acad Sci USA, 94:14954-14959 (1997); Winkel-Shirley B., Plant Physiol, 126:485—493 (2001). These precursors are essential for the production of other flavonoids such as isoflavonoids, flavonols, dihydroflavonols, and anthocyanidins. Forkmann et al., Curr. Opin. Biotechnol 12:155-160 (2001). This process begins with conversion of a carbon substrate, such as glucose, into phenylalanine, and the subsequent catalyzation of phenylalanine into trans-cinnamic acid by phenylalanine/ tyrosine ammonia lyase (PAL). Next, cinnamic add is converted by a membrane-bound P450 monooxygenase, cinnamate 4-h droxylase (C4H), into dther p-coumaric add, caffdc add, or ferulic acid (Fig. 2). Those substrates are then converted by 4-coumarate-CoA ligase (4CL) into, respectivdy, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA. Additionally, 4CL can convert trans-cinnamic add into cinnamoyl-CoA (Fig. 2).
[0010] In the next step of the phenylpropanoid metabolic pathway, a polyketide synthase, chalcone synthase (CHS), sequentially adds three molecules of malonyl-CoA to one molecule of either cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA, yielding, respectivdy, the C15 compounds pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone (Fig.2). Finally, chalcone isomerase (CHI) converts the CIS compounds into thdr respective (2S)-flavanones, namely, (2S)-pinocembrin, (2S)-naringenin, (2S) -eriodictyl, and (2S)- homoeriodictyl (Fig.2). Alternatively, a tyrosine ammonia lyase (TAL) can convert tyrosine directly to coumaric acid and drcumvent the use of membrane bound P450 rdated enzymes, though this may pose challenges in E. coli. Wang Y. et al., Appl Microbiol Biotechnol 91:949-956 (2011); Santos et al., MetabEng 13:392-400 (2011).
[0011] To synthesize cannflavins A and B, a prenyl moiety must be added to a flavone. A previously described flavone prenyltrans ferase from Gtycyrrhiza uralensis, GuA6DT (GenBank AIT11912.1) prenylates apigenin, which is a widespread plant flavone that also accumulates in Cannabis saliva. McPartland et al., J. Cannabis Then, 1:103-132 (2001). Researchers have recently reported dght additional full-length cDNA sequences from C. saliva exhibiting 22-53% amino add identity to GuA6DT, which have been putativdy annotated as C. saliva prenyltransferases (CsPT) 1-8. Rea et al., Phytochemistry, 164:162-171 (2019). These dght CsPTs occupied three of the six groups of known plant prenyltransferases: CsPT2 and CsPT6 reside in a unique dade of prenyltransferases (Group 2), which have been shown to participate in the tocopherol biosynthetic pathway; CsPT5 appears to be orthologous to homogentisate solanesyltransferases (Group V) that function in plastoquinone biosynthesis; and CsPTl, 3, 4, 7, and 8 all formed a third and distantly rdated group (Group VI) that indudes two prenyltransferases from Humulus lupulus (hops), and which are involved in the aromatic prenylation reactions required for terpenophenolic biosynthesis. Id. None of the CsPTs were dosdy rdated to any of the flavonoid or coumarin prenyltransferases (Groups I and IV, respectively) that hade been previously identified in various plant species. Id:, Sasaki et al., Plant Physiol., 146: 1075-1084 (2008); Sasaki et al.,/. Biol Chem., 286: 24125-24134 (2011); Akashi et al., Plant Physiol, 149:683-693 (2009); Shen et al., Plant Physiol 159:70-80; Wang et al, J. Biol Chem., 289:35815-35825 (2012); Munakata et al. , New Phytol, 211, 332-344 (2016); Yoneyama et al., Plant Cell Physiol, 57:2497-2509 (2016); Yang et al., J. Biol Chem., 293:28-46 (2018).
[0012] The observation that CsPT3 preferentially prenylates the flavonoid precursor chrysoeriol in vitro and that prenylated luteolin appears to be absent in C. saliva extracts has led researchers to propose that methylation of luteolin to chrysoeriol must occur first in the cannflavin A and/or B pathway. Rea et al., Phytochemistry, 164:162-171 (2019). They reason that the enzyme that methylates luteolin at the 3’-hydroxyl position of the flavone B-ring to yield chrysoeriol would likely fall into the class of S- adenosyl-L-methionine (AdoMet)-dependent O-methyltransferases (OMTs), which are widely distributed throughout the plant kingdom. Id.,· Ibrahim et al., Can. J. Bot., 83:433-450 (2005); Ibrahim et al., Plant MoL BioL, 36:1-10 (1998); Kim et al.,/. Plant BioL, 53:321-329 (2010). Using a previously characterized flavonoid-O-methyltransferase from Oryza saliva that methylates the 3’-hydroxyl group on a variety of flavonoids (OsOMT9) as a query, the researchers identified 24 unique protein sequences annotated as C. saliva O-methyltransferases (CsOMT) 1-24, which were distributed into one of four general groups. Rea et al., Phytochemistry, 164:162-171 (2019). These researchers reported that some of these CsOMTs encompass an evolutionarily conserved group of enzymes with regiospecificity for 3’-hydroxyl groups on a variety of flavonoid compounds and, further, that at least one such enzyme, CsOMT21, likely catalyzes the penultimate step in cannflavin A and B biosynthesis by converting luteolin to chrysoeriol. Id. [0013] Notably, expression of enzyme combinations originating from a variety of host organisms has yielded microbial strains capable of producing key flavonoid precursors, such as naringenin. Naringenin is used to produce other flavonoids such as isoflavonoids, flavonols, dihydroflavonols, and anthocyanidins. The pathway for naringenin production consists of five enzymes, phenylalanine/ tyrosine ammonia lyase (PAL), dnnamate 4-h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI). Alternatively, tyrosine ammonia lyase (TAL) is able to convert tyrosine to p-coumaric acid; in this way, the pathway can bypass C4H. Additionally, naringenin chalcone is able to convert to naringenin without CHI under certain conditions. This opens up the possibility to bypass CHI and shorten the necessary changes in the host microorganism [0014] Several reports describe successful biotransformation processes in which a phenylpropanoid precursor, such as coumaric add, is converted into naringenin by genetically engineered E. cod or S. cerevisiae. Xu et al., Metab Eng 13:578-587 (2011); Wang et al., Appl Microbiol Biotechnol, 91:949-956 (2011); Leonard et al., Appl Environ Microbiol, 73:3877-3886 (2007); Trantas et al., Metab Eng 11:355- 366 (2009); Watts et al., Chem Biochem, 5:500-507 (2004); Jiang et al., Appl Environ Microbiol, 71:2962- 2969 (2005); Yan et al., Appl Environ Microbiol ,71:3617-3623 (2005). Genetically engineered E. cod and
S. cerevisiae have also been reported to convert glucose into naringenin at rdatively high titers. Koopman et al., Microbial CellFactories, 11:155 (2012); Santos et al., Metab Eng 13:392-400 (2011). [0015] S. cerevisiae has several attractive characteristics as a metabolic engineering platform for flavonoid production. In addition to its excellent accessibility to molecular and synthetic biology techniques, its eukaryotic nature may facilitate functional expression of plant-derived flavonoid- biosynthetic genes. For example, S. cerevisiae can functionally express cytochrome P450-containing enzymes and its subcellular compartmentation is comparable to that of plant cells. Jiang et al., Biotechnol
Bioeng, 85:130-137 (2004). Finally, its GRAS (generally recognized as safe) status facilitates subsequent application for the production of pharma- and nutraceuticals.
[0016] The present invention provides a metabolic engineering strategy for microbial production of flavonoids and flavonoid derivatives using a carbon substrate. The flavonoid biosynthetic genes PAL, C4H, 4CL, CHS, TAL, CsPT, and CsOMT, used in the invention may be derived from one or more different prokaryotic or eukaryotic species — including Escherichia coli, Saccharomyces cerevisiae, Cannabis saliva, Arabidopsis thahana, Rhodobacter capsulatus, Petroselinum crispum, and Petunia hybrida — and selected for in planta co-expression profiles. After expression of the plant pathway genes, optimization of flavonoid production was explored by engineering of precursor supply to the flavonoid pathway and by reducing the formation of byproducts derived from yeast metabolism.
SUMMARY OF THE INVENTION
[0017] The present invention relates in part to a genetically-modified cell capable of producing a flavonoid or flavonoid derivative. The cell may comprise at least one heterologous enzyme involved in a metabolic pathway that converts a carbon substrate (e.g., sugar) to a flavonoid or flavonoid derivative and/ or at least one heterologous polynucleotide encoding such an enzyme.
[0018] The invention also relates to a method of making a flavonoid or flavonoid derivative. The method comprises contacting a substrate with the aforementioned genetically-modified cell and growing the cell to produce a flavonoid or flavonoid derivative compound.
[0019] The invention further relates to the use of a flavonoid or flavonoid derivative for the manufacture of a medicament for the treatment of a disease or a symptom of a disease and to such a medicament.
[0020] The invention additionally rdates to a method of treating a disease or symptom of a disease comprising administering a flavonoid or flavonoid derivative to a subject in need thereof.
[0021] Yet another aspect of the invention is a nucleic add encoding at least one enzyme involved in a metabolic pathway that converts a cabon substrate (e.g., sugar) to a flavonoid or flavonoid derivative or a vector encoding such a nucleic add.
[0022] A further aspect of the invention is a method of making a genetically-modified cell capable of synthesizing a flavonoid or flavonoid derivative, the method comprising: contacting a cell with at least one heterologpus polynucleotide encoding an enzyme involved in the flavonoid pathway; and growing the cell so that said polynudeotide is expressed in the microorganism.
[0023] A yet further aspect of the invention is a composition comprising a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof.
BRIEF DESCRIPTION OF THE DRAWINGS
[0024] FIG. 1A shows the basic skdeton structure of flavonoids and their various classes. [0025] FIG. IB shows the flavonoid classes, subdasses and natural sources.
[0026] FIG. 2 shows an overview of the flavonoid biosynthetic pathway. The enzyme PAL converts phenylalanine into a cinnamic add isomer. Next, the enzyme C4H converts cinnamic acid into dther p-coumaric add, caffeic add, or ferulic acid. Next, the enzyme 4CL converts those substrates into p- coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA, respectivdy. 4CL can also directly convert trans- cinnamic acid into cinnamoyl-CoA. Next, the enzyme CHS sequentially adds three molecules of malonyl-CoA to one molecule of dther dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl- CoA, yielding, respectively, pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone. Next, the enzyme CHI converts these four compounds into thdr respective (2S)-flavanones, namely, (2S)-pinocembrin, (2S)-naringenin, (2S)- eriodictyl, and (2S)-homoeriodictyl. Flavonoid biosynthesis pathways can be assembled and engineered in a tractable host microorganism, such as bacteria or yeast, to create cdl factories for their mass production.
[0027] FIG. 3 shows the biosynthetic route for the flavonoid precursor naringenin.
DETAILED DESCRIPTION OF THE INVENTION
[0028] As summarized above, aspects of the invention include genetically modified microorganisms that can convert carbon substrates into flavonoid compounds or flavonoid derivative. The genetically modified microorganisms include bacteria and yeast, which are capable of produdng such flavonoids utilizing native and/or heterologous enzymes that catalyze various reactions in the flavonoid biosynthesis pathway [0029] Before the present invention is described in greater detail, it is to be understood that this invention is not limited to particular cases described. It is also to be understood that the terminology used herein is for the purpose of describing particular embodiments only, and is not intended to be limiting, since the scope of the present invention will be limited only by the appended claims. I. Ranges and Definitions
[0030] Where a range of values is provided, it is understood that each intervening value, to the tenth of the unit of the lower limit unless the context clearly dictates otherwise, between the upper and lower limit of that range and any other stated or intervening value in that stated range, is encompassed within the invention. The upper and lower limits of these smaller ranges may independently be included in the smaller ranges and are also encompassed within the invention, subject to any specifically excluded limit in the stated range. Where the stated range includes one or both of the limits, ranges excluding either or both of those included limits are also included in the invention.
[0031] As used herein and in the appended claims, the singular forms “a”, “an”, and “the” include plural referents unless the context clearly dictates otherwise. It is noted that the claims can be drafted to exclude any optional element. As such, this statement is intended to serve as antecedent basis for use of such exclusive terminology as “solely,” “only” and the like in connection with the recitation of claim elements, or use of a “negative” limitation.
[0032] As used herein, the terms “and/or” and “any combination thereof’ and their grammatical equivalents may be used interchangeably. Solely for illustrative purposes, the following phrases “A, B, and/or C” or “A, B, C, or any combination thereof’ can mean “A individually; B individually; C individually; A and B; B and C; A and C; and A, B, and C.”
[0033] As used herein, the term “about” in relation to a reference numerical value and its grammatical equivalents includes the numerical value itself and a range of values plus or minus 10% from that numerical value. For example, the amount ‘about 10” includes 10 and any amounts from 9 to 11. For example, the term ‘about ”in relation to a reference numerical value can also include a range of values plus or minus 10%, 9%, 8%, 7%, 6%, 5%, 4%, 3%, 2%, or 1% from that value. In some cases, the numerical disclosed throughout can be “about” that numerical value even without specifically mentioning the term “about.”
[0034] As used herein, the term “substrate” refers to any substance or compound that is converted into another compound by the action of an enzyme. The term includes not only a single compound, but also combinations of compounds, such as solutions, mixtures, and other materials that contain at least one substrate, or derivatives thereof. Further, the term “substrate” encompasses not only compounds that provide a carbon source suitable for use as a starting material (e.g., methane), but also intermediate and end product metabolites used in a pathway associated with a metabolically engineered microorganism as described herein.
[0035] As used herein, the term “flavonoids” refers to a group of biologically-active plant compounds that provide health benefits through cell signaling pathways and antioxidant effects. Flavonoids can be subdivided into different subgroups depending on the carbon of the C ring on which the B ring is attached and the degree of unsaturation and oxidation of the C ring (Fig. 1A). Flavonoids in which the B ring is linked in position 3 of the C ring are called isoflavones. Those in which the B ring is linked in position 4 are called neoflavonoids, while those in which the B ring is linked in position 2 can be further subdivided into several subgroups on the basis of the structural features of the C ring. These subgroups are: flavones, flavonols, flavanones, flavanonols, flavanols or catechins, anthocyanins and chalcones (Fig. 1A).
[0036] Flavones are widely present in leaves, flowers and fruits as glucosides. Celery, parsley, red peppers, chamomile, mint and ginkgo biloba are among the major sources of flavones. Luteolin, apigenin and tangeritin belong to this subclass of flavonoids (Fig. IB). The peels of citrus fruits are rich in the polymethoxylated flavones, tageretin, nobiletin and sinensetin. They have a double bond between positions 2 and 3 and a ketone in position 4 of the C ring. Most flavones of vegetables and fruits have a hydroxyl group in position 5 of the A ring, while hydroxylation in other positions, for the most part in position 7 of the A ring or 3’ and 4’ of the B ring, may vary according to the taxonomic classification of the particular vegetable or fruit.
[0037] Flavonols are flavonoids with a ketone group. They are building blocks of proanthocyanins. Flavonols occur abundantly in a variety of fruits and vegetables. The most studied flavonols are kaemp feral, quercetin, myricetin and fisetin (Fig. IB). Onions, kale, lettuce, tomatoes, apples, grapes and berries are rich sources of flavonols. Apart from fruits and vegetables, tea and red wine are also sources of flavonols. Intake of flavonols is found to be associated with a wide range of health benefits which includes antioxidant potential and reduced risk of vascular disease. Compared with flavones, flavonols have a hydroxyl group in position 3 of the C ring, which may also be glycosylated. Like flavones, flavonols are very diverse in methylation and hydroxylation patters as well and, considering the different glycosylation patterns, they are perhaps the most common and largest subgroup of flavonoids in fruits and vegetables. For example, quercetin is present in many plant foods.
[0038] Flavanones are another important class of flavonoids, and are generally present in citrus fruits such as oranges, lemons and grapes. Hesperitin, naringenin, eriodictyol, and homo-eriodictyol are examples of this class of flavonoids (Fig. IB). Flavonones are associated with a number of health benefits because of their free radical-scavenging properties. These compounds are responsible for the bitter taste of the juice and peel of citrus fruits. Citrus flavonoids exert interesting pharmacological effects as antioxidant, anti-inflammatory, blood lipid-lowering and cholesterol-lowering agents. Flavanones, also called dihydroflavones, have the C ring saturated; therefore, unlike flavones, the double bond between positions 2 and 3 is saturated and this is the only structural difference between the two subgroups of flavonoids. Over the past 15 years, the number of flavanones has significantly increased.
[0039] Isoflavonoids are a large and very distinctive subgroup of flavonoids. Isoflavonoids enjoy only a limited distribution in the plant kingdom and are predominantly found in soyabeans and other leguminous plants. Some isoflavonoids have also been reported to be present in microbes. They are also found to play an important role as precursors for the development of phytoalexins during plant microbe interactions. Isoflavonoids exhibit tremendous potential to fight a number of diseases. Isoflavones such as genistein and daidzein are commonly regarded to be phyto-oestrogens because of their oestrogenic activity in certain animal models (Fig. IB). [0040] Anthocyanins are a subclass of flavonoids responsible for colors in plants, flowers and fruits.
Cyanidin, delphinidin, malvidin, pelargonidin and peonidin are the most commonly studied anthocyanins (Fig. IB). They occur predominantly in the outer cell layers of various fruits such as cranberries, black currants, red grapes, merlot grapes, raspberries, strawberries, blueberries, bilberries and blackberries. Stability coupled with health benefits of these compounds facilitate them to be used in the food industry in a variety of applications. The color of the anthocyanin depends on the pH and also by methylation or acylation at the hydroxyl groups on the A and B rings.
[0041] Chalcones are a subclass of flavonoids characterized by the absence of ‘ring C’ of the basic flavonoid skeleton structure shown in Fig. 1A. Hence, they can also be referred to as open-chain flavonoids. Major examples of chalcones include phloridzin, arbutin, phloretin and chalconaringenin. Chalcones occur in significant amounts in tomatoes, pears, strawberries, bearberries and certain wheat products. Chalcones and their derivatives have garnered considerable attention because of numerous nutritional and biological benefits. The intake of flavonoids through food sources could be the simplest and safest way to combat diseases as well as modulate activities.
[0042] As used herein, the term “cannflavin” refers to a flavonoid found in Cannabis. Examples of cannflavins indude cannflavin A, cannflavin B, cannflavin C, chrysoeril, cosmosiin, flavocannabiside, vitexin, isovitexin, apigenin, kaemp feral, myricetin, quercetin, luteolin, homoorientin, orientin, and any natural or unnatural isomers of any of the foregoing.
[0043] As used herein, the terms “flavonoid pathway” and “phenylp ropanoid metabolic pathway” are interchangeable and refer to any metabolic pathway by which flavonoids are biosynthesized. In one common phenylp ropanoid metabolic pathway, the enzymes phenylalanine/ tyrosine ammonia-lyase (PAL), cinnamate-4-h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), and chalcone isomerase (CHI) are involved in catalyzing the conversion of phenylalanine to cinnamic add to p-coumaric acid to 4-coumaroyl-CoA to (with the addition of malonyl-CoA) a chalcone to a flavonone, such as naringenin, eriodictyol, or homo-eriodictyol. The basic flavonoid pathway is illustrated schematically in Fig. 2.
[0044] As used herein, the terms “flavonoid pathway enzymes” refer to any enzyme involved in the flavonoid pathway, inducting PAL, C4H, 4CL, CHS, and CHI. Other enzymes in the flavonoid pathway and encompassed by the present invention indude flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3Ή), and flavonoid 3’, 5' droxylase (F3’5TI). Also encompassed are the enzymes C. sativa prenyltransferase (CsPT) and C. sativa O- methyltransferase (CsOMT), which have been shown to play a role in the biosynthesis of cannflavins A and B. Any other enzyme (or its isoform) involved in the production of flavonoids or flavonoids precursors are also encompassed herein. In plants the enzymes in the flavonoid pathway are often represented by several isoforms, which may differ in substrate preference or kinetic properties. Costa et al., Phytochem, 66:2072-2091 (2005). Moreover, the different isoforms may be organized into one or more enzyme complexes.
[0045] As used herein, the term “fermentation” or “fermentation process” is defined as a process in which a host microorganism is cultivated in a culture medium containing raw materials, such as feedstock and nutrients, wherein the microorganism converts raw materials, such as a feedstock, into products. The term “feedstock” is defined as a raw material or mixture of raw materials supplied to a microorganism, or fermentation process, from which other products can be made. For example, as set forth in the present invention, a methane carbon source, a methanol carbon source, or a formaldehyde carbon source, either alone or in combination, are feedstocks for a microorganism that produces a valuable chemical, such as a flavonoid or flavonoid derivative. However, in addition to a feedstock (e.g., a methane substrate) of the invention, the fermentation media contains suitable minerals, salts, cofactors, buffers, and other components, known to those skilled in the art, suitable for the growth of the cultures and promotion of the enzymatic pathways necessary for multi-carbon compound production.
[0046] As used herein, the term ‘genetic modification” or “genetically modified” and thdr grammatical equivalents refers to one or more alterations of a nucleic acid, e.g., the nuddc add within a microorganism’s genome. For example, genetic modification can refer to alterations, additions, and/or deletion of nucldc acid (e.g, whole gpnes or fragments of genes).
[0047] As used herein, the term gene editing” and its grammatical equivalents refers to genetic engineering in which one or more nucleotides are inserted, replaced, or removed from a genome. For example, gene editing can be performed using a nuclease (eg, a natural-existing nuclease or an artificially engineered nuclease).
[0048] As used herein, the term “promoter” and its grammatical equivalents refers to a nudeic add sequence capable of controlling the expression of a coding sequence or functional RNA. In general, a coding sequence is located 3’ to a promoter sequence. Promoters can be derived in their entirety from a native gene, or be composed of different dements derived from different promoters found in nature, or even comprise synthetic nucldc add segments. It is understood by those skilled in the art that different promoters can direct the expression of a gene in different tissues or cell types, or at different stages of development, or in response to different environmental or physiological conditions. Promoters that cause a gene to be expressed in most cdl types at most times are commonly referred to as “constitutive promoters”. It is further recognized that since in most cases the exact boundaries of regulatory sequences have not been completely defined, DNA fragments of different lengths can have identical promoter activity. Some examples of promoters that can be used indude but are not limited to Gall, GallO, TEF1, TDH3, PGK1, ADH2.
[0049] As used herein, the term “operably linked” and its grammatical equivalents refers to the association of nucleic acid sequences on a single polynucleic add fragment so that the function of one is affected by the other. For example, a promoter is operably linked with a coding sequence when it is capable of effecting the expression of that coding sequence (i.e., that the coding sequence is under the transcriptional control of the promoter). Coding sequences can be operably linked to regulatory sequences in sense or antisense orientation. [0050] As used herein, the term “codon optimized” and its grammatical equivalents refers to genes or coding regions of nucleic add molecules (or open reading frames) for transformation of various hosts, can refer to the alteration of codons in the gene or coding regions of the nucldc add molecules to reflect the typical codon usage of the host organism without altering the polypeptide encoded by the DNA.
[0051] As used herein, the term “open reading frame” (“ORF’) and its grammatical equivalents refers to a polynucldc add or nudeic add sequence (whether naturally occurring, non-naturally occurring, or synthetic) comprising an uninterrupted reading frame consisting of (i) an initiation codon, (ii) a series of two (2) of more codons representing amino adds, and (iii) a termination codon, the ORF being read (or translated) in the 5’ to 3’ direction.
[0052] As used herein, the term “operon” and its grammatical equivalents refers to two or more genes, which are transcribed as a single transcriptional unit from a common promoter. In certain cases, the genes, polynudeotides or ORFs comprising the operon are contiguous. It is understood that transcription of an entire operon can be modified (i.e., increased, decreased, or eliminated) by modifying the common promoter. Altemativdy, any gene, polynudeotide or ORF, or any combination thereof in an operon can be modified to alter the function or activity of the encoded polypeptide. The modification can result in an increase or a decrease in the activity or function of the encoded polypeptide. Further, the modification can inpart new activities on the encoded polypeptide.
[0053] As used herein, the term “vector” and its grammatical equivalents refers to any means by which a nucldc add can be propagated and/or transferred between organisms, cells, or cellular components. Vectors indude viruses, bacteriophage, pro-viruses, plasmids, phagemids, transposons, and artificial chromosomes such as YACs (yeast artificial chromosomes), BACs (bacterial artifidal chromosomes), and PLACs (plant artificial chromosomes), and the like, that are “episomes”, that is, that replicate autonomously or can integrate into a chromosome of a host microorganism A vector can also be a naked RNA polynudeotide, a naked DNA polynudeotide, a polynudeotide composed of both DNA and RNA within the same strand, a poly-lysine-conjugated DNA or RNA, a peptide- conjugated DNA or RNA, a liposome-conjugated DNA, or the like, that are not episomal in nature, or it can be an organism which comprises one or more of the above polynucleotide constructs such as an agrobacterium or a bacterium.
[0054] As used herein, the term “host cell” refers to a prokaryotic or eukaryotic cell that can be, or has been, used as a recipients for a nucleic add (e.g., an expression vector that comprises a nucleotide sequence encoding one or more gene products such as mevalonate pathway gene products), and indudes any progeny of the original cdl that has been genetically modified by the nucldc add. It is understood that the progeny of a single cell may not necessarily be completely identical in morphology or in genomic or total DNA complement as the original parent, due to natural, acddental, or deliberate mutation. A “recombinant host cell” or “genetically modified host cell” is a host cdl into which has been introduced a heterologous nucldc add, e.g., an expression vector.
[0055] As used herein, the term “substantially pure” and its grammatical equivalents can refer to a particular substance that does not contain a majority of another substance. For example, “substantially pure product” can mean at least 90% of that product. In some instances, “substantially pure product” can mean at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.1%, 99.2%, 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, 99.9%, 99.99%, 99.999%, or 99.9999% of that product. For example, substantially pure product can mean at least 70% of the product. In some cases, substantially pure product can mean at least 75% of the product. In some cases, substantially pure product can mean at least 80% of the product. In some cases, substantially pure product can mean at least 85% of the product. In some cases, substantially pure product can mean at least 90% of the product. In some cases, substantially pure product can mean at least 91% of the product. In some cases, substantially pure product can mean at least 92% of the product. In some cases, substantially pure product can mean at least 93% of the product. In some cases, substantially pure product can mean at least 94% of the product. In some cases, substantially pure product can mean at least 95% of the product. In some cases, substantially pure product can mean at least 96% of the product. In some cases, substantially pure product can mean at least 97% of the product. In some cases, substantially pure product can mean at least 98% of the product. In some cases, substantially pure product can mean at least 99% of the product.
[0056] As used herein, the term “heterologous” and its grammatical equivalents means derived from a different spedes. For example, a “heterologous gene” can mean a gene that is from a spedes different than the reference spedes. For example, a yeast cell comprising a “heterologous gene” comprises a gene that is not from the same species of yeast. The gene can be from a different microorganism altogether, such as bacteria, plant, or algae, from a different species of yeast.
[0057] As used herdn, the term “recombinant” and its grammatical equivalents means that a particular nudeic add (DNA or RNA) is the product of various combinations of doning, restriction, and/or ligation steps resulting in a constmct having a structural coding or non-coding sequence distinguishable from endogenous nudeic adds found in natural systems. Generally, DNA sequences encoding the structural coding sequence can be assembled from cDNA fragments and short oligonudeotide linkers, or from a series of synthetic oligonudeotides, to provide a synthetic nucldc add which is capable of being expressed from a recombinant transcriptional unit contained in a cdl or in a cell-free transcription and translation system. Such sequences can be provided in the form of an open reading frame uninterrupted by internal non-translated sequences, or introns, which are typically present in eukaryotic genes. Genomic DNA comprising the rdevant sequences can also be used in the formation of a recombinant gene or transcriptional unit. Sequences of non-translated DNA may be present 5’ or 3’ from the open reading frame, where such sequences do not interfere with manipulation or expression of the coding regions, and may indeed act to modulate production of a desired product by various mechanisms.
[0058] Thus, as used herein, the terms “recombinant polynucleotide” or “recombinant nucleic add” refer to a polynudeotide that is non-naturally occurring, e.g., is made by the artificial combination of two otherwise separated segments of sequence through human intervention. This artifidal combination is often accomplished by dther chemical synthesis means, or by the artifidal manipulation of isolated segments of nucldc adds, e.g., by genetic engineering techniques. Such is usually done to replace a codon with a redundant codon encoding the same or a conservative amino add, while typically introdudng or removing a sequence recognition site. Altemativdy, it is performed to join together nucldc acid segments of desired functions to generate a desired combination of functions. This artifidal combination is often accomplished by dther chemical synthesis means, or by the artifidal manipulation of isolated segments of nucldc adds, e.g., by genetic engineering techniques.
[0059] As used herein the term “isolated” refers to a polynudeotide, polypeptide, or cell that is in an environment different from that in which the polynudeotide, polypeptide, or cdl naturally occurs. An isolated genetically modified host cdl may be present in a mixed population of genetically modified host cells. [0060] As used herein, the term “substantially similar” and its grammatical equivalents, when used in reference to the similarity between a sequence and a reference sequence, means that the sequences are at least 50% (but not 100%) identical. In some cases, the sequences are 55%, 60%, 65%, 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical. In some cases, the term substantially similar refers to a sequence that is at least 50% identical. In some instances, the term substantially similar refers to a sequence that is 55% identical. In some instances, the term substantially similar refers to a sequence that is 60% identical. In some instances, the term substantially similar refers to a sequence that is 65% identical. In some instances, the term substantially similar refers to a sequence that is 70% identical. In some instances, the term substantially similar refers to a sequence that is 75% identical. In some instances, the term substantially similar refers to a sequence that is 80% identical. In other instances, the term substantially similar refers to a sequence that is 81% identical. In other instances, the term substantially similar refers to a sequence that is 82% identical. In other instances, the term substantially similar refers to a sequence that is 83% identical. In other instances, the term substantially similar refers to a sequence that is 84% identical. In other instances, the term substantially similar refers to a sequence that is 85% identical. In other instances, the term substantially similar refers to a sequence that is 86% identical. In other instances, the term substantially similar refers to a sequence that is 87% identical. In other instances, the term substantially similar refers to a sequence that is 88% identical. In other instances, the term substantially similar refers to a sequence that is 89% identical. In some instances, the term substantially similar refers to a sequence that is 90% identical. In some instances, the term substantially similar refers to a sequence that is 91% identical. In some instances, the term substantially similar refers to a sequence that is 92% identical. In some instances, the term substantially similar refers to a sequence that is 93% identical. In some instances, the term substantially similar refers to a sequence that is 94% identical. In some instances, the term substantially similar refers to a sequence that is 95% identical. In some instances, the term substantially similar refers to a sequence that is 96% identical. In some instances, the term substantially similar refers to a sequence that is 97% identical. In some instances, the term substantially similar refers to a sequence that is 98% identical. In some instances, the term substantially similar refers to a sequence that is 99% identical.
[0061] To determine the percentage of identity between two sequences, the two sequences are aligned, using, for example, the alignment method of Needleman and Wunsch 0. Mol. Biol., 1970, 48: 443), as revised by Smith and Waterman (Adv. Appl. Math., 1981, 2: 482) so that the highest order match is obtained between the two sequences and the number of identical amino acids/ nucleotides is determined between the two sequences. Methods to calculate the percentage identity between two amino acid sequences are generally art recognized and include, for example, those described by Carillo and Iipton (SIAM J. Applied Math., 1988, 48:1073) and those described in Computational Molecular Biology, Lesk, e.d. Oxford University Press, New York, 1988, Biocomputing: Informatics and Genomics Projects. Generally, computer programs will be employed for such calculations. Computer programs that can be used in this regard include, but are not limited to, GCG (Devereux et aL, Nucleic Adds Res., 1984, 12: 387) BLASTP, BLASTN and FASTA (Altschul et aL, J. Molec. Biol., 1990:215:403). A particularly preferred method for determining the percentage identity between two polypeptides involves the Clustal W algorithm (Thompson,} D, Higgines, D G and Gibson T J, 1994, Nudeic Add Res 22(22): 4673-4680 together with the BLOSUM 62 scoring matrix (Henikoff S & Henikoff, J G, 1992, Proc. Nad. Acad. Sci. USA 89: 10915-10919) using a gap opening penalty of 10 and a gap extension penalty of 0.1, so that the highest order match obtained between two sequences wherein at least 50% of the total length of one of the two sequences is involved in the alignment.
[0062] As will be apparent to those of skill in the art upon reading this disdosure, each of the individual embodiments described and illustrated herein has discrete components and features, which can be readily separated from or combined with the features of any of the other several cases without departing from the scope or spirit of the present invention. Any recited method can be carried out in the order of events redted or in any other order that is logically possible.
[0063] Unless defined otherwise herein, all technical and sdentific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention bdongs. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present invention, representative illustrative methods and materials are now described.
[0064] The publications discussed herein are provided solely for thdr disdosure prior to the filing date of the present application. Nothing herein is to be construed as an admission that the present invention is not entitled to antedate such publication by virtue of prior invention. Further, the dates of publication provided may be different from the actual publication dates, which may need to be independently confirmed. II. Genetically Modified Microorganisms and Methods of Making the Same [0065] The present disclosure is directed, in part, to genetically modified microorganisms that are able to replicate quickly and produce flavonoid and flavonoid derivative compounds. In some instances, microorganisms that do not naturally produce a specific flavonoid or flavonoid derivative have been genetically modified to do so.
[0066] In some cases, the use of molecular switches can allow for direct control of gene expression at a given time. This control of gene expression by using molecular switches can lead to increased cell division compared to microorganism that do not have these molecular switches.
Microorganisms [0067] The genetically modified microorganisms useful in the present invention include those that can convert a carbon substrates into a desired end product, such as a flavonoid or flavonoid derivative. The microorganisms can be a prokaryote or a eukaryote. The microorganism may, for example, be bacteria, yeast, plant, fungi, or algae. The carbon substrate may be endogenously present in the microorganism in normal culture, or additional amounts of the carbon substrate may be fed to the microorganism exogenously. In some embodiments, the carbon substrate is a sugar, such as glucose. In some embodiments, the carbon substrate is amino acid, such as phenylalanine or tyrosine. In some embodiments, the carbon substrate is a compound derived from a sugar or amino add, and indudes, for example, any compound produced in the flavonoid pathway, such as cinnamic acid, p-coumaric add, caffeic add, ferulic acid, dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA, pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, homoeriodictyl chalcone, (2S)-pinocembrin, (2S)-naringenin, (2S) -eriodictyl, (2S)-homoeriodictyl, or any natural or unnatural isomers of the foregoing. In one embodiment, the microorganism is yeast, the carbon substrate is coumaric add, cafeic add, or ferulic add, and the desired end product is a cannflavin. In another embodiment, the microorganism is Saccharomyces cerevisiae is, the carbon substrate is ferulic acid, and the desired end product is a isocannflavin B.
[0068] The genetically modified microorganisms useful in the present invention may exhibit increased activity levels of one or more phenylpropanoid metabolic pathway enzymes and/or increased levds of flavonoid or flavonoid derivative production as compared to microorganisms of the same species that have not been genetically modified. In some embodiments, a genetically modified host microorganism exhibits increases in flavonoid or flavonoid derivative production, where flavonoid or flavonoid derivative production is increased by at least about 10%, at least about 15%, at least about 20%, at least about 25%, at least about 30%, at least about 35%, at least about 40%, at least about 45%, at least about 50%, at least about 60%, at least about 70%, at least about 80%, at least about 90%, at least about 2-fold, at least about 2.5-fold, at least about 5-fold, at least about 10-fold, at least about 20-fold, at least about 30-fold, at least about 40-fold, at least about 50-fold, at least about 75- fold, at least about 100-fold, at least about 200-fold, at least about 300-fold, at least about 400-fold, at least about 500-fold, or at least about 103-fold, or more, in the genetically modified host microorganism, compared to the level of flavonoid derivative or flavonoid compound produced in a control host microorganism that is not genetically modified as described herein. Flavonoid or flavonoid derivative production is readily determined using well-known methods, e.g., gas chromatography-mass spectrometry, liquid chromatography-mass spectrometry, ion chromatography- mass spectrometry, pulsed amperometric detection, UV-vis spectrometry, and the like.
[0069] In some embodiments, a genetically modified host microorganism produces a flavonoid or flavonoid derivative in an amount ranging from 1 pg flavonoid compound/ ml to 100,000 pg flavonoid compound/ ml, e.g., from about 1 pg/ml to about 10,000 pg/ml of flavonoid compound, 1 pg/ml to
5000 pg/ml of flavonoid compound, 1 pg/ml to 4500 pg/ml of flavonoid compound, 1 pg/ml to
4000 pg/ml of flavonoid compound, 1 pg/ml to 3500 pg/ml of flavonoid compound, 1 pg/ml to
3000 pg/ml of flavonoid compound, 1 pg/ml to 2500 pg/ml of flavonoid compound, 1 pg/ml to
2000 pg/ml of flavonoid compound, 1 pg/ml to 1500 pg/ml of flavonoid compound, 1 pg/ml to 1000 pg/ml of flavonoid compound, 5 pg/ml to 5000 pg/ml of flavonoid compound, 10 pg/ml to
5000 pg/ml of flavonoid compound, 20 pg/ml to 5000 pg/ml of flavonoid compound, 30 pg/ml to 1000 pg/ml of flavonoid compound, 40 pg/ml to 500 pg/ml of flavonoid compound, 50 pg/ml to 300 pg/ml of flavonoid compound, 60 pg/ml to 100 pg/ml of flavonoid compound, 70 pg/ml to 80 pg/ml of flavonoid compound, from about 1 pg/ml to about 1,000 pg/ml, from about 1,000 pg/ml to about 2,000 pg/ml, from about 2,000 pg/ml to about 3,000 pg/ml, from about 3,000 pg/ml to about 4,000 pg/ml, from about 4,000 pg/ml to about 5,000 pg/ml, from about 5,000 pg/ml to about 7,500 pg/ml, or from about 7,500 pg/ml to about 10,000 pg/ml, or greater than 10,000 pg/ml flavonoid compound, e.g., from about 10 mg flavonoid compound/ ml to about 20 mg flavonoid compound/ ml, from about 20 mg flavonoid compound/ ml to about 50 mg flavonoid compound/ ml, from about 50 mg flavonoid compound/ ml to about 100 mg flavonoid compound/ ml, or more.
[0070] In one embodiment, the yeast Saccharomyces cerevisiae is engineered to produce a cannflavin (e.g., cann flavin A, cann flavin B, isocannflavin A, isocann flavin B) from one or more phenylpropanoid metabolic pathway enzymes, such as phenylalanine/ tyrosine ammonia-lyase (PAL), dnnamate-4- h droxylase (C4H), 4-coumarate-CoA ligase (4CL), chalcone synthase (CHS), chalcone isomerase (CHI), flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3Ή), and flavonoid 3’, 5' droxylase (R3'5Ή), C. sativa prenyltransferase (CsPT), and C. sativa O- methyltransferase (CsOMT).
[0071] The microorganisms useful in the present invention may be naturally occurring or recombinant. In some embodiments, the microorganisms are selectively screened (e.g., using strain adaptation) to have improved properties. Improved properties may include increased growth rate, yield of desired products, and tolerance of likely process contaminants. In a particular embodiment, a high growth variant microorganism is selected, which possesses an exponential phase growth rate that is faster than its parent, reference, or wild-type microorganism.
[0072] The microorganisms useful in the present invention may be grown as an isolated pure culture, with a heterologous microorganism that may aid with growth, or one or more different strains of microorganisms may be combined to generate a mixed culture. A variety of culture methodologies may be useful in the present invention. For example, the microorganism may be grown by batch culture and/or continuous culture methodologies, both of which are described in the art. See, e.g., Crueger et al., Biotechnology: A Textbook of Industrial Microbiology, Second Edition (1989); Deshpande et al .,AppL Biochem. BiotechnoL, 36:227 (1992), the contents of each are herein incorporated by reference. The culture media preferably contains one or more suitable carbon substrates for the microorganisms. The carbon substrate(s) may be added to the culture media initially, intermittently, and/or supplied continuously.
Flavonoid Pathway Polynucleotides and Enzymes
[0073] In some embodiments, a host microorganism contains endogenously, or is transformed with, one or more heterologous polynucleotides that encode an enzyme in the flavonoid pathway. In some cases, one or more of these genes can be episomally expressed. In some cases, one of more of these genes can be integrated into the gpnome of the microorganism. In some cases, one or more of these genes be can be episomally expressed whereas one or more of these gpnes can be integrated into the genome of the microorganism
[0074] In some embodiments, the host microorganism contains (either endogenously or heterologous) one or more polynucleotides encoding the following enzymes (or their isomers): (1) phenylalanine/tyrosine ammonia-lyase (PAL); (2) cinnamate-4-h droxylase (C4H); (3) 4-coumarate- CoA ligase (4CL); (4) chalcone synthase (CHS); and (5) chalcone isomerase (CHI). The microorganism may also contain one or more polynucleotides encoding for other important enzymes in the production of flavonoids or flavonoid derivatives, including flavanone 3-h droxylase (F3H), dihydroflavonol 4-reductase (DFR), flavonoid 3’-h droxylase (F3T1), and flavonoid 3’, 5’h droxylase (F3’5’H). The microotganism may also contain the enzymes C. sativa prenyltransferase (CsPT) and C sativa O-methyltransferase (CsOMT), which have been reported to play a role in the biosynthesis of cannflavins A and B. [0075] The enzyme phenylalanine/tyrosine ammonia-lyase (PAL) (EC:4.3.1.24 and EC:4.3.1.25) is capable of catalyzing the conversion of phenylalanine (C00079) into trans-cinnamic acid (C00423) and ammonia (C00014):
Figure imgf000023_0001
[0076] The PAL enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 1 or SEQ ID NO. 3. For example, the polynucleotide encoding the PAL enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 1 or SEQ ID NO. 3. In some cases, the PAL enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 2 or SEQ ID NO: 4. For example, the PAL enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%,
93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 3 or SEQ ID NO: 4.
[0077] The enzyme cinnamate-4-h droxylase (C4H) (EC:1.14.14.91) is capable of catalyzing the conversion of trans-cinnamic acid (C00423) into, e.g., p-coumaric acid (C00811):
SUBSTITUTE SHEET (RULE 26)
Figure imgf000024_0001
[0078] The C4H enzyme can be encoded by a polynudeotide that is substantially similar to SEQ ID NO. 5. For example, the polynudeotide encoding the C4H enzyme can comprises a polynudeotide 5 sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 5. In some cases, the C4H enzyme can comprise an amino add sequence that is substantially similar to SEQ ID NO: 6. For example, the C4H enzyme can comprises an amino add sequence that is at least.60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, 10 or 99.9999% identical to SEQ ID NO: 6.
[0079] The enzyme 4-Coumarate-CoA ligase (4CL) (EC:6.2.1.12) is capable of catalyzing the conversion of p-coumaric add (C00811) into p-coumaroyl-CoA (C00223), the conversion of caffeate (C01197) into caffeoyl-CoA (C00323), and the conversion of ferulate (C01494) into feruloyl-CoA (C00406).
1
Figure imgf000024_0002
SUBSTITUTE SHEET (RULE 26)
Figure imgf000025_0001
[0081] The 4CL enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID
NO. 7. For example, the polynucleotide encoding the 4CL enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 7. In some cases, the 4CL enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 8. For example, the 4CL enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 8.
[0082] The enzyme chalcone synthase (CHS) (EC:2.3.1.74) is capable of catalyzing the conversion of p-coumaroyl-CoA (C00223) into naringenin chalcone (C06561), the conversion of dnnamoyl-CoA (C00540) into pinocembrin chalcone (C16404), the conversion of caffeoyl-CoA (C00323) into eriodictyol chalcone (Cl 5525), and the conversion of feruloyl-CoA (C00406) into homoeriodictyol chalcone (Cl 6405):
SUBSTITUTE SHEET (RULE 26)
Figure imgf000026_0001
SUBSTITUTE SHEET (RULE 26) ID NO. 9. or SEQ ID NO. 11. For example, the polynucleotide encoding the CHS enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 9 or SEQ ID NO. 11. In some cases, the CHS enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 10 or SEQ ID NO. 12. For example, the CHS enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 10 or SEQ ID NO. 12.
[0084] The enzyme chalcone isomerase (CHI) (EC:5.5.1.6) is capable of catalyzing the conversion of a chalcone (Cl 5589) into a flavanone (C00766), inducting the conversion of naringenin chalcone into narigenin, the conversion of pinocembrin chalcone into pinocembrin, the conversion of eriodictyol chalcone into eriodictyol, and the conversion of homoeriodictyol chalcone (C16405) into homoeriodictyol:
Figure imgf000027_0001
Figure imgf000027_0002
[0085] The CHI enzyme can be encoded by a polynudeotide that is substantially similar to SEQ
ID NO. 13. For example, the polynudeotide encoding the CHI enzyme can comprises a polynudeotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 13. In some cases, the CHI enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 14. For example, the CHI enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 14.
[0086] The enzyme flavanone 3-h droxylase (F3H) (EC:1.14.11.9) is capable of catalyzing the conversion of a flavanone into its corresponding flavanol, such as the conversion of naringenin (C00509) into dihydrokaempferol (C00974):
SUBSTITUTE SHEET (RULE 26)
Figure imgf000028_0001
[0087] The F3H enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 15. For example, the polynucleotide encoding the F3H enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 15. In some cases, the F3H enzyme can comprise an amino add sequence that is substantially similar to SEQ ID NO: 16. For example, the F3H enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 16. [0088] The enzyme dihydroflavonol 4-reductase (DFR) (EC:1.1.1.219 and EC:1.1.1.234) is capable of catalyzing the conversion of a dihydroflavonol into its corresponding cis-flavan-3,4-diol, such as the conversion of dihydrokaempferol (C00974) into ds-3,4-leucopelargonidin (C03648):
Figure imgf000028_0002
[0089] The DFR enzyme can be encoded by a polynudeotide that is substantially similar to SEQ ID NO. 17. For example, the polynucleotide encoding the DFR enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 17. In some cases, the DFR enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 18. For example, the DFR enzyme can comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%,
SUBSTITUTE SHEET (RULE 26) 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 18.
[0090] The enzyme flavonoid 3’-h droxylase (F3’H) (EC:1.14.14.82) is capable of catalyzing the conversion of certain flavonoids, such as naringenin (C00509) into, hydroxy flavonoids, such as eriodictyol (C05631):
Figure imgf000029_0001
[0091] The F3Ή enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 19. For example, the polynucleotide encoding the F3Ή enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 19. In some cases, the F3'H enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 20. For example, the F3TI enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 20.
[0092] The enzyme flavonoid 3’, 5’ hydroxylase (F3’5’H) (EC:1.14.14.81) is capable of catalyzing acting on naringenin, eriodictyol, dihydroquercetin (taxifolin) and dihydrokaempferol (aromadendrin). The enzyme catalyses, for example, the conversion of naringenin (C00509) and eriodictyol (C05631) into pentahydroxyflavanone (C05911):
Figure imgf000029_0002
SUBSTITUTE SHEET (RULE 26)
Figure imgf000030_0001
[0093] The F3’5’H enzyme can be encoded by a polynucleotide that is substantially similar to SEQ ID NO. 21. For example, the polynucleotide encoding the F3’5TI enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 21. In some cases, the F3’5’H enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NO: 22. For example, the F3’5’H enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NO: 22. [0094] The C. saliva prenyltransferase (CsPT) enzyme useful in the present invention may include any enzyme encoded by a polynucleotide that is substantially similar to SEQ ID NOs. 23, 25, 27, 29, 31, 33, 25, 27, or 39. For example, the polynucleotide encoding the CsPT enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 21, 23, 25, 27, 29, 31, 33, 25, 27, or 39. In some cases, the CsPT enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NOs. 24, 26, 28, 30, 32, 34, 36, 38, or 40. For example, the CsPT enzyme can comprises an amino acid sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 22, 24, 26, 28, 30, 32, 34, 36, 38, or 40.
[0095] The C. saliva O-methyltransferase (CsOMT) enzyme useful in the present invention may include any enzyme encoded by a polynucleotide that is substantially similar to SEQ ID NOs. 41, 43, 45, 47, 49, or 51. For example, the polynucleotide encoding the CsOMT enzyme can comprises a polynucleotide sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 41, 43, 45, 47, 49, or 51. In some cases, the CsOMT enzyme can comprise an amino acid sequence that is substantially similar to SEQ ID NOs. 42, 44, 46, 48, 50, or 52. For example, the CsOMT enzyme can
SUBSTITUTE SHEET (RULE 26) comprises an amino add sequence that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to SEQ ID NOs. 42, 44, 46, 48, 50, or 52.
[0096] In some cases, the genetically modified host microorganism comprises one or more polynucleotides containing a genetic modification (e.g., a mutation, deletion, insertion, or the like) that results in optimized (increased or decreased) activity of the flavonoid pathway enzyme encoded thereby. Such optimized enzymes can indude, for example, optimized forms of PAL, C4H, 4CL, CHS, CHI, F3H, DPR, R3Ή, R3'5Ή, CsPT, and CsOMT. Enzymatic activity may be optimized in a variety of ways, inducting, but not limited to, 1) modulating the strength of the promoter to which the flavonoid pathway enzyme coding region is operably linked; 2) modulating the copy number of the plasmid comprising a nucleotide sequence encoding the flavonoid pathway enzyme; 3) modulating the stability of a flavonoid pathway enzyme mRNA; 4) modifying the sequence of the ribosome binding site of a flavonoid pathway enzyme mRNA such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 5) modifying the sequence between the ribosome binding site of a flavonoid pathway enzyme mRNA and the start codon of the flavonoid pathway enzyme coding sequence such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 6) modifying the entire interdstronic region 5’ of the start codon of the flavonoid pathway enzyme coding region such that translation of the flavonoid pathway enzyme mRNA is modulated; 7) modifying the codon usage of flavonoid pathway enzyme such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 8) expressing rare codon tRNAs used in the flavonoid pathway enzyme such that the level of translation of the flavonoid pathway enzyme mRNA is modulated; 9) modulating the enzyme stability of flavonoid pathway enzyme; 10) modulating the specific activity (units activity per unit protein) of the flavonoid pathway enzyme; 11) expressing a modified form of a flavonoid pathway enzyme such that the modified enzyme exhibits increased or decreased solubility in the host cell; or 12) expressing a modified form of a flavonoid pathway enzyme such that the modified enzyme lacks or contains a domain through which regulation occurs. The foregoing modifications may be made singly or in combination; that is, two or more of the foregoing modifications may be made to provide for a modulated level of flavonoid pathway enzyme activity.
[0097] In some cases, additional enzymes can be provided to the host microorganism to yield other desired end products.
[0098] In some cases, the nucleotide sequence encoding a gpne product (e.g., a CsPT, CsOMT, etc.) is modified such that the nucleotide sequence reflects the codon preference for the particular host cell. In such cases, conservative amino adds substitutions can be made based on whether the microorganism typically uses a spedfic amino add or how much of that particular amino acid is available for use within the spedfic microorganism For example, the nudeotide sequence will in some embodiments be modified for yeast, bacteria, plant, or algae codon preference.
[0099] In some cases, the coding sequence is modified such that the level of translation of the encoded enzyme (e.g., a CsPT, CsOMT, etc.) is modulated, i.e., increased or decreased. Modulating the level of enzyme translation may be achieved by modifying the sequence to indude codons that are rare or not commonly used by the host cell. Codon usage tables for many organisms are available that summarize the percentage of time a spedfic organism uses a specific codon to encode for an amino add. Certain codons are used more often than other codons. The use of less commonly used (so- called “rare”) codons in a sequence generally decreases its rate of translation. Thus, a coding sequence may be modified by introducing one or more rare codons, which affect the rate of translation, but not the amino acid sequence of the enzyme translated.
[00100] In some cases, one or more the nudeotide sequences encoding for one or more of the enzymes in the flavonoid pathway can be driven by a molecular switch. In some cases, the molecular switch can be turned on or off by a chemical substance.
[00101] In some cases, the gpne encoding the flavonoid pathway enzyme can be disrupted. Any methods used to disrupt gene expression or protein functionality can be used. For example, a CRISPR/ Cas9 system can be used to disrupt gene expression. If using the CRISPR system, the guide
RNA can be designed to target a sequence that is substantially similar to at least a portion of the polynucleotide that encodes the flavonoid pathway enzyme. Other systems of disruption include knocking out the gene or knocking down the gpne. Additionally, a dominant negative can also be used, e.g., a polypeptide that mimics the flavonoid pathway enzyme but is not functional. Prenyltransferases
[00102] Prenyl transferases constitute a broad group of enzymes catalyzing that transfer allylic prenyl groups (“prenilate”) to acceptor molecules. The allylic prenyl groups may be, e.g., in the form of a geranyl group (CIO) or a dimethylallyl (C5) group. Prenyl transferases may be divided into two dasses, as (or Z) and trans (or E), depending upon the stereochemistry of the resulting products. Suitable prenyl transferases in the present invention indude enzymes that prenilate a flavonoid precursor, such as cinnamic add, p-coumaric acid, cafFdc add, ferulic add, or a natural or unnatural isomer of one of the foregoing. In one embodiment, the prenyl transferase prenilates the A ring of ferulic acid at position 8.
[00103] Suitable prenyl transferases include, but are not limited to, a C. sativa prenyl transferases (CsPTs), geranylgeranyl diphosphate (GGPP) synthase, hexaprenyl diphosphate (HexPP) synthase, heptaprenyl diphosphate (HepPP) synthase, octaprenyl (OPP) diphosphate synthase, solanesyl diphosphate (SPP) synthase, decap renyl diphosphate (DPP) synthase, chide synthase, and gutta-percha synthase; and a Z-isoprenyl diphosphate synthase, including, but not limited to, nonap renyl diphosphate (NPP) synthase, undecaprenyl diphosphate (UPP) synthase, dehydrodolichyl diphosphate synthase, dcosaprenyl diphosphate synthase, natural mbber synthase, and other Z- isoprenyl diphosphate synthases.
[00104] The nudeotide sequences of numerous prenyl transferases from a variety of spedes are known, and can be used or modified for use in produdng a genetically modified eukaryotic host cdl. See, eg., DNA or RNA encoding: famesyl pyrophosphate synthetase (HFPS) (GenBank Accession No. J05262, Homo sapiens),· famesyl diphosphate synthetase (FPF) (GenBank Accession No. J05091, Saccharomyces cerevisiae); isopentenyl diphosphate:dimethylallyl diphosphate isomerase J 05090, Saccharomyces cerevisiae); famesyl pyrophosphate synthetase 2 (FPS2) /FPP synthetase 2 / famesyl diphosphate synthase 2 (GenBank Accession No. At4gl7190, Arabidopsis thaliana ); geranylgeranyl diphosphate synthase (GGPPS) (GenBank Accession No. AY371321, Ginkgo biloba ); geranylgeranyl pyrophosphate synthase (GGPS1) / GGPP synthetase / famesyltranstransferase (GenBank Accession No. At4g36810, Arabidopsis thaliana); famesyl, geranylgeranyl, geranylfamesyl, hexaprenyl, heptaprenyl diphosphate synthase (SelF-HepPS) (GenBank Accession No. AB016095, Synechococcus elongatus); etc.
[00105] The amino add sequences of various prenyl transferase from C. sativa or one its rdatives are known and can be used or modified for use in producing a genetically modified eukaryotic host cell. See, eg., CsPTl (GenBank Accession No. PK28436), CsPT2 (GenBank Accession No. PK02092), CsPT3 (GenBank Accession No. PK17697), CsPT4 (GenBank Accession No. PK15523), CsPT5 (GenBank Accession No. PK11068), CsPT6 (GenBank Accession No. PK13891), CsPT7 (GenBank Accession No. PK29226), CsPT8 (GenBank Accession No. PK07278), H1PT1 (GenBank Accession No. AB543053), H1PT2 (GenBank Accession No. KM222442), AtVTE2-l (GenBank Accession No. AAM10489), GmVTE2-l (GenBank Accession No. ABB70126), TaVTE2-l (GenBank Accession No. ABB70123), ZmVTE2-l (GenBank Accession No. ABB70122), ApVTE2-
1 (GenBank Accession No. ABB70124), CpVTE2-l (GenBank Accession No. ABB70125), AtVTE2-
2 (GenBank Accession No. ABB70127), GmVTE2-2 (GenBank Accession No. ABB70128), OsVTE2-2 (GenBank Accession No. XP_015646905), OsHGGT (GenBank Accession No. AAP43913), HvHGGT (GenBank Accession No. AAP43911), TaHGGT (GenBank Accession No.
AAP43912), S£N8DT-1 (GenBank Accession No. BAG 12671), GuA6DT (GenBank Accession No. KJ123716), LaPTl (GenBank Accession No. AER35706), SfiLDT (GenBank Accession No. BAK52290), SfG6DT (GenBank Accession No. BAK52291), GmG4DT (GenBank Accession No. BAH22520), AHR4DT-1 (GenBank Accession No. AQM74172), AhR3T)T-l (GenBank Accession No. AQM74173), AhR3T)T-2 (GenBank Accession No. AQM74174), AhR3T)T-4 (GenBank Accession No. AQM74176), C1PT1 (GenBank Accession No. BAP27988), PcPT (GenBank Accession No. BA031627), PsPTl (GenBank Accession No. AJW31563), PsPT2 (GenBank Accession No. AJW31564), MalDT (GenBank Accession No. AJD80982), CtIDT (GenBank Accession No. AJD80983); etc. Vectors
[00106] Since some of the enzymes described throughout may not be native to the host microorganism, expression vectors can be used to express one or more heterologous enzyme within the host microorganism. Vector constructs prepared for introduction into the host microorganisms described throughout can typically, but not always, comprise a replication system (i.e. vector) recognized by the host. In some cases, the vector includes the intended polynucleotide fragment encoding the desired polypeptide and, optionally, transcription and translational initiation regulatory sequences operably linked to the polypeptide-encoding segment. Expression vectors can include, for example, an origin of replication or autonomously replicating sequence (ARS), expression control sequences, a promoter, an enhancer and necessary processing information sites, such as ribosome- binding sites, RNA splice sites, polyadenylation sites, transcriptional terminator sequences, mRNA stabilizing sequences, polynucleotides homologous to host chromosomal DNA, and/or a multiple cloning site. Signal peptides can also be included where appropriate, for example from secreted polypeptides of the same or related species, which allow the protein to cross and/or lodge in cell membranes or be secreted from the cell. [00107] The expression vector may be introduced into the host cell stably or transiently using established techniques, including, but not limited to, electroporation, calcium phosphate precipitation, DEAE-dextran mediated transfection, liposome-mediated transfection, heat shock in the presence of lithium acetate, and the like. For stable transformation, a nucleic add will generally further indude a selectable marker, e.g., any of several well-known sdectable markers such as neomycin resistance, ampidllin resistance, tetracycline resistance, chloramphenicol resistance, kanamydn resistance, and the like. In some embodiments, the nucldc acid with which the host cdl is genetically modified is an expression vector that indudes a nuddc add comprising a nudeotide sequence that encodes a gene product, e.g., an flavonoid pathway enzyme, a transcription factor, a prenyl transferase, an O- methyltransferases synthase, etc.
[00108] Suitable expression vectors indude, but are not limited to, baculovirus vectors, bacteriophage vectors, plasmids, phagemids, cosmids, fosmids, bacterial artifidal chromosomes, viral vectors (e.g. viral vectors based on vacdnia vims, poliovirus, adenovirus, adeno-associated vims, SV40, herpes simplex vims, and the like), Pl-based artifidal chromosomes, yeast plasmids, yeast artifidal chromosomes, and any other vectors spedfic for specific hosts of interest (such as yeast). Thus, for example, a nudeic add encoding a gene product(s) is included in any one of a variety of expression vectors for expressing the gene produces). Such vectors indude chromosomal, nonchromosomal, and synthetic DNA sequences.
[00109] The vectors can be constructed using standard methods (see, eg, Sambrook et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Labs Press (1989), and Ausubd et al., Current Protocols in Molecular Biology, Greene Publishing, Co. N.Y, (1995). [00110] Suitable vectors can be sdected to accommodate a polynudeotide encoding a protdn of a desired size. Following production of a sdected vector, a suitable host cell (eg, the microorganisms described herdn) is transfected or transformed with the vector. Each vector contains various functional components, which generally indude a cloning site, an origin of replication and at least one selectable marker gene. A vector can additionally possess one or more of the following dements: an enhancer, a promoter, a transcription termination sequence and/or other signal sequences. Such sequence dements can be optimized for the sdected host spedes. Such sequence dements can be positioned in the vicinity of the cloning site, such that they are operativdy linked to the gpne encoding a preselected enzyme.
[00111] Vectors, inducting doning and expression vectors, can contain polynudeotides that enable the vector to replicate in one or more selected microorganisms. For example, the sequence can be one that enables the vector to replicate independently of the host chromosomal DNA and can include origins of replication or autonomously replicating sequences. Such sequences are well known for a variety of bacteria, yeast and viruses. For example, the origin of replication from the plasmid pBR322 is suitable for most gram-negative bacteria, the origin of replication for 2 micron plasmid is suitable for yeast, and various viral origins of replication (e.g. SV40, adenovirus) are useful for cloning vectors.
[00112] A cloning or expression vector can contain a selection gene, also referred to as a selectable marker. This gene encodes a protein necessary for the survival or growth of transformed microorganisms in a selective culture medium. Microorganisms not transformed with the vector containing the selection gene will therefore not survive in the culture medium. Typical selection genes encode proteins that confer resistance to antibiotics and other toxins, e.g. ampicillin, neomycin, methotrexate, hygromyrin, kanamyxin, thiostrepton, apramycin or tetracycline, complement auxotrophic deficiencies, or supply critical nutrients not available in the growth media.
[00113] The replication of vectors can be performed in any suitable host, such as E. cob. An example of an E. coli-selectable marker is the b-lactamase gene, which confers resistance to the antibiotic ampirillin. These selectable markers can be obtained from E. cob plasmids, such as pBR322 or a pUC plasmid such as pUC18 or pUC19, or pUC119.
Promoters
[00114] Vectors can contain a promoter that is recognized by the host microorganism The promoter can be operably linked to a coding sequence of interest. Such a promoter can be inducible, repressible, or constitutive. Polynucleotides are operably linked when the polynucleotides are in a relationship permitting them to function in their intended manner.
[00115] Different promoters can be used to drive the expression of the genes. For example, if temporary gene expression (i.e., non-constitutively expressed) is desired, expression can be driven by inducible or repressible promoters. The molecular switch can in some cases comprise these inducible or repressible promoters.
[00116] In some cases, the desired gene is expressed temporarily. In other words, the desired gpne is not constitutively expressed. The expression of the desired gene can be driven by inducible or repressible promoters, which functions as a molecular switch. Examples of inducible or repressible switches include, but are not limited to, those promoters inducible or repressible by: (a) sugars such as arabinose and lactose (or non metabolizable analogs, eg., isopropyl b-D-l-thiogalactopyranoside (IPTG)); (b) metals such as copper or calcium (or rare earth metals such as lanthanum or cerium); (c) temperature; (d) Nitrogen-source; (e) oxygen; (f) cell state (growth or stationary); (g) metabolites such as phosphate; (h) CRISPRi; (i) jun; (j) fos, (k) metallothionein and/or (l) heat shock. These switches can be used in a yeast or bacterial system. [00117] Inducible or repressible switches that can be particularly useful are switches that are responsive to sugars and rare earth metals. For example, promoters that are sensitive to the sugar arabinose can be used as an inducible switch. In some cases, arabinose switches can be used to drive expression of one or more gpnes. For example, in the presence arabinose, a desired vector or expression of a gene set can be “tumed-on.” The arabinose switch can turn on the expression of a desired gene.
[00118] Other particularly useful switches can be rare earth metal switches, such as lanthanum sensitive switches (also simply known as a lanthanum switch). In some cases, the lanthanum switch can be a repressible switch that can be used to repress expression of one or more genes, until the repressor is removed (e.g., in this case lanthanum), after which the genes are “tumed-on”. For example, in the presence the rare earth metal lanthanum, the desired gpne set or vector can be “tumed-off.” The lanthanum switch can be turned off (and expression of the genes induced) by either removing the lanthanum from the media or diluting the lanthanum in the media to levels where its repressible effects are reduced, minimized, or eliminated. Other rare earth metal switches can be used, such as those disclosed throughout. [00119] Constitutively expressed promoters can also be used in the vector systems herein. For example, the expression of one or more desired genes can be controlled by constitutively active promoters. Examples of such promoters include but are not limited to Gal promoters, which are well- known in the art. In some embodiments the promoter is Gall, GallO, TEF1, TDH3, PGK1, or ADH2. In one embodiment, the promoter is a Gal80 knock out. In some embodiments, the promoter is a synthetic promoter.
[00120] Promoters suitable for use with prokaryotic hosts can include, for example, the a-lactamase and lactose promoter systems, alkaline phosphatase, the tryptophan (tip) promoter system, the erythromycin promoter, apramycin promoter, hygromycin promoter, methylenomycin promoter and hybrid promoters such as the tac promoter. Promoters for use in bacterial systems will also generally contain a Shine-Dalgamo sequence operably linked to the coding sequence. [00121] Generally, a strong promoter can be employed to provide for high level transcription and expression of the desired product. For example, promoters that can be used include but are not limited to Gal promoters, which are well-known in the art. In some embodiments, the promoter is Gall, GallO, TEF1, TDH3, PGK1, or ADH2. In one embodiment, the promoter is a Gal80 knock out. In some embodiments, the promoter is a synthetic promoter. In some cases, a mutation can increase the strength of the promoter and therefore result in elevated levels of expression.
[00122] In some cases however, a weaker promoter is desired. For example, this is the case where too much expression of a certain gene results in a detrimental effect (e.g., the killing of cells). A weak promoter can be used, for example a CYC1 promoter. However, in some cases, a weaker promoter can be made by mutation.
[00123] One or more promoters of a transcription unit can be an inducible promoter. For example, a GFP can be expressed from a constitutive promoter while an inducible promoter is used to drive transcription of a gene coding for one or more enzymes as disclosed herein and/or the amplifiable selectable marker.
Genes
[00124] The genes described herein can all have a promoter driving their expression. The methods described herein, e.g., genome editing, can be used to edit the polynucleotide of the promoters or used to inhibit the effectiveness of the promoters. Inhibition can be done by blocking the transcription machinery (e.g., transcription factors) from binding to the promoter or by altering the promoter in such a way that the transcription machinery no longer recognizes the promoter sequence.
[00125] The vectors described herein can also comprise a polynucleotide encoding for one or more of the genes within the flavonoid pathway. These vectors can also contain one or more regulatory elements (inducible and/ or repressible promoters) that control the expression of the genes within the vectors. In some cases, the vectors can include switches, including but not limited to inducible or repressible switches, e.g., an arabinose or lanthanum switches. These genes can be heterologous to the microorganism in which the vector is contacted with (and eventually transformed with).
[00126] The genes used in the vectors can be any genes described throughout the application, including gpnes encoding enzymes involved in the flavonoid pathway. These enzymes can be encoded by a polynucleotide or by an polypeptide that is at least 60%, 65%, 70%, 75%, 80%, 85%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 99.9%, 99.99%, 99.999%, or 99.9999% identical to any one ofSEQ ID NOs: 1-24.
[00127] The genes that are inserted into a host microorganism can be heterologous to the microorganism itself. For example, if the host microorganism is a yeast cell, the inserted genes can, for example, be from plants, fungi, algae, bacteria, or even a different species of yeast. Alternatively, the genes can be endogenously part of the genome of the host microorganism itself.
III. Methods for Making the Genetically Modified Microorganisms
[00128] The genetically modified microorganisms disclosed throughout can be made by a variety of ways. For example, the microorganism may be modified (e.g, genetically-engineered) by any method to comprise and/or express one or more polynucleotides encoding for enzymes in a pathway that catalyze a conversion of a carbon source to one or more intermediates in a pathway for the production of flavonoids or flavonoid derivative compounds. Such enzymes can include those discussed herein. For example, one or more of any of the genes discussed throughout can be inserted into a microorganism The genes can be inserted by an expression vector. The genes can also be under the control of one or more different/ same promoters or the one or more genes can be under the control of a switch, such as an inducible or repressible promoter, e.g, an arabinose switch, isopropyl b-D-l- thiogalactopyranoside (IPTG) switch, or a rare earth metal switch. The genes can also be stably integrated into the genome of the microorganism In some cases, the genes can be expressed in an episomal vector.
[00129] An exemplary method of making a genetically modified microorganism disclosed herein is contacting (or transforming) a microorganism with a nucleic acid that expresses at least one heterologous gene encoding 1) PAL, 2) C4H, 3) 4CL, 4) CHS, 5) CHI, 6) F3H, 7) DFR, 8) R3Ή, 9) R3'5Ή, 10) CsPT, 11) CsOMT, and/or 12) a natural or unnatural isomer of any of the foregoing. The microorganism can be any microorganism that is capable of converting a carbon substrate to a desirable end product, such as a flavonoid or flavonoid derivative.
[00130] In some embodiments, the method of the invention further comprises isolating the flavonoid or flavonoid derivative from the host microorganism and/or from the culture medium.
[00131] In general, the genetically modified host microorganism is cultured in a suitable medium, optionally supplemented with one or more additional agents, such as an inducer (e.g., where one or more nucleotide sequences encoding a gene product is under the control of an inducible promoter). In some embodiments, the culture medium is overlaid with an organic solvent, e.g., dodecane, forming an organic layer. In such cases, the flavonoid confound produced by the genetically modified host microorganism may partition into the organic layer, from which it can be purified. In some embodiments, where one or more gene product-encoding nucleotide sequence is operably linked to an inducible promoter, an inducer is added to the culture medium; and, after a suitable time, the flavonoid compound is isolated from the organic layer overlaid on the culture medium.
[00132] In some embodiments, the flavonoid or flavonoid derivative is separated from other products which may be present in the organic layer. Such separation may be achieved using, e.g., standard chromatographic techniques.
[00133] In some embodiments, the flavonoid or flavonoid derivative is pure, e.g., at least about 20% pure, at least about 30% pure, at least about 40% pure, at least about 50% pure, at least about 60% pure, at least about 70% pure, at least about 80% pure, at least about 90% pure, at least about 95% pure, at least about 98% pure, at least about 99% pure, or more than 99% pure, where the term “pure” in the context of a flavonoid or flavonoid derivative refers to a flavonoid or flavonoid derivative that is free from other flavonoid compounds, contaminants, etc.
Techniques for Genetic Modification
[00134] The microorganisms disclosed herein can be genetically engineered by using classic microbiological techniques. Some of these techniques are generally disclosed, for example, in D. Amberg, D. Burke and J. Strathem, Methods in Yeast Genetics, 2005 Edition, Cold Spring Harbor Laboratory Press. [00135] The genetically modified microorganisms disclosed herein can include a polynucleotide that has been inserted, deleted or modified (i.e., mutated; eg, by insertion, deletion, substitution, and/or inversion of nucleotides), in such a manner that such modifications provide the desired effect of expression (eg, over-expression) of one or more enzymes as provided herein within the microorganism Genetic modifications that result in an increase in gpne expression or function can be referred to as amplification, overproduction, overexpression, activation, enhancement, addition, or up-regulation of a gene. Addition of a gpne to increase gene expression can include maintaining the gene(s) on replicating plasmids or integrating the cloned gpne(s) into the genome of the production microorganism Furthermore, increasing the expression of desired genes can include operatively linking the cloned gene(s) to native or heterologous transcriptional control elements. [00136] Where desired, the expression of one or more of the enzymes provided herein is under the control of a regulatory sequence that controls directly or indirectly the enzyme expression in a time- dependent fashion during the fermentation. Inducible promoters can be used to achieve this.
[00137] In some cases, a microorganism is transformed or transfected with a genetic vehide, such as an expression vector comprising a heterologous polynudeotide sequence coding for the enzymes are provided herein. In some cases, the vector(s) can be an episomal vector, or the gene sequence can be integrated into the genome of the microorganism, or any combination thereof. In some cases, the vectors comprising the heterologous polynudeotide sequence encoding for the enzymes provided herdn are integrated into the genome of the microorganism.
[00138] To facilitate insertion and expression of different genes coding for the enzymes as disclosed herein from the constructs and expression vectors, the constructs can be designed with at least one cloning site for insertion of any gene coding for any enzyme disclosed herein. The cloning site can be a multiple cloning site, e.g., containing multiple restriction sites.
Transfection
[00139] Standard transfection techniques can be used to insert genes into a microorganism. As used herdn, the term “transfection” or “transformation” can refer to the insertion of an exogenous nuddc add or polynucleotide into a host cell. The exogenous nucldc acid or polynudeotide can be maintained as a non-integrated vector, for example, a plasmid or episomal vector, or alternatively, can be integrated into the host cell genome. The term transfecting or transfection is intended to encompass all conventional techniques for introducing nudeic acid or polynudeotide into microorganisms. Examples of transfection techniques indude, but are not limited to, calcium phosphate predpitation, DEAE-dextran-mediated transfection, lipofection, dectroporation, microinjection, rubidium chloride or polycation mediated transfection, protoplast fusion, and sonication. The transfection method that provides optimal transfection frequency and expression of the construct in the particular host cell line and type is favored. For stable transfectants, the constmcts are integrated so as to be stably maintained within the host chromosome. In some cases, the preferred transfection is a stable transfection. In some cases, the integration of the gene occurs at a specific locus within the genome of the microorganism.
Transformation
[00140] Expression vectors or other nucleic adds can be introduced to sdected microorganisms by any of a number of suitable methods. For example, vector constructs can be introduced to appropriate cells by any of a number of transformation methods for plasmid vectors. Standard calcium-chloride- mediated bacterial transformation is still commonly used to introduce naked DNA to bacteria.
[00141] For the introduction of vector constructs to yeast or other fungal cells, chemical transformation methods can be used (see, e.g., Rose et al., Methods in Yeast Genetics, Cold Spring Harbor Laboratory Press, Cold Spring Harbor, N.Y. (1990)). Transformed cells can be isolated on selective media appropriate to the selectable marker used. Alteratively, or in addition, plates or filters lifted from plates can be scanned for GFP fluorescence to identify transformed clones.
[00142] For the introduction of vectors comprising differentially expressed sequences to certain types of cells, the method used can depend on the form of the vector. Plasmid vectors can be introduced by any of a number of transfection methods, including, for example, lipid-mediated transfection (“lipofection”), DEAE-dextran-mediated transfection, electroporation or calcium phosphate precipitation (see, e.g., Ausubel et al., Current Protocols in Molecular Biology, John Wiley & Sons, Inc., N.Y., N.Y. (1988)).
[00143] Lipofection reagents and methods suitable for transient transfection of a wide variety of transformed and non-transformed or primary cells are widely available, making lipofection an attractive method of introducing constructs to eukaryotic, and particularly mammalian cells in culture. Many companies offer kits and ways for this type of transfection.
[00144] The host cell can be capable of expressing the construct encoding the desired protein, processing the protein and transporting a secreted protein to the cell surface for secretion. Processing includes co- and post-translational modification such as leader peptide cleavage, GPI attachment, glycosylation, ubiquitination, and disulfide bond formation.
[00145] Microorganisms can be transformed or transfected with the above-described expression vectors or polynucleotides coding for one or more enzymes as disclosed herein and cultured in nutrient media modified as appropriate for the specific microorganism, inducing promoters, selecting transformants, or amplifying the genes encoding the desired sequences. In some cases, electroporation methods can be used to deliver an expression vector.
[00146] Expression of a vector (and the gene contained in the vector) can be verified by an expression assay, for example, qPCR or by measuring levels of RNA. Expression level can be indicative also of copy number. For example, if expression levels are extremely high, this can indicate that more than one copy of a gene was integrated in a genome. Alteratively, high expression can indicate that a gpne was integrated in a highly transcribed area, for example, near a highly expressed promoter. Expression can also be verified by measuring protein levels, such as through Western blotting.
CRISPR / cas system [00147] The methods disclosed throughout can involve pinpoint insertion of genes or the deletion of genes (or parts of genes). Methods described herein can use a CRISPR/cas system. For example, double-strand breaks (DSBs) can be generated using a CRISPR/ cas system, e.g., a type II CRISPR/ cas system. A Cas enzyme used in the methods disclosed herein can be Cas9, which catalyzes DNA cleavage. Enzymatic action by Cas9 from Streptococcus pyogenes or any closely related Cas9 can generate double stranded breaks at target site sequences which hybridize to 20 nucleotides of a guide sequence and have a protospacer-adjacent motif (PAM) following the 20 nucleotides of the target sequence.
[00148] A vector can be operably linked to an enzyme-coding sequence encoding a CRISPR enzyme, such as a Gas protein. Gas proteins that can be used include class 1 and class 2. Non-limiting examples of Gas proteins include Casl, CaslB, Cas2, Cas3, Cas4, Cas5, CasSd, CasSt, CasSh, CasSa, Gas6, Cas7, Cas8, Cas9 (also known as Csnl or Csxl2), CaslO, Csyl , Csy2, Csy3, Csy4, Csel, Cse2, Cse3, Cse4, CseSe, Cscl, Csc2, Csa5, Csnl, Csn2, Csml, Csm2, Csm3, Csm4, Csm5, Csm6, Cmrl, Cmr3, Cmr4, Cmr5, Cmr6, Csbl, Csb2, Csb3, Csxl7, Csxl4, CsxlO, Csxl6, CsaX, Csx3, Csxl, CsxlS, Csfl, Cs£2, CsO, Csf4, Csdl, Csd2, Cstl, Cst2, Cshl, Csh2, Csal, Csa2, Csa3, Csa4, Csa5, C2cl, C2c2, C2c3, Cpfl, CARE, DinG, homologues thereof, or modified versions thereof. An unmodified CRISPR enzyme can have DNA cleavage activity, such as Cas9. A CRISPR enzyme can direct cleavage of one or both strands at a target sequence, such as within a target sequence and/ or within a complement of a target sequence. For example, a CRISPR enzyme can direct cleavage of one or both strands within 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 15, 20, 25, 30, 35, 40, 45, 50, 60, 70, 80, 90, 100, 125, 150, 175, 200, 300, 400, 500, or more base pairs from the first or last nucleotide of a target sequence. A vector that encodes a CRISPR enzyme that is mutated to with respect, to a corresponding wild-type enzyme such that the mutated CRISPR enzyme lacks the ability to cleave one or both strands of a target polynucleotide containing a target sequence can be used.
[00149] A vector that encodes a CRISPR enzyme comprising one or more nuclear localization sequences (NLSs) can be used. For example, there can be 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs used. A CRISPR enzyme can comprise the NLSs at or near the ammo-terminus (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or at or near the carboxy-terminus (eg, 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 NLSs), or any combination of these (eg., one or more NLS at the ammo-terminus and one or more NLS at the carboxy terminus). When more than one NLS is present, each can be selected independently of others, such that a single NLS can be present in more than one copy and/or in combination with one or more other NLSs present in one or more copies.
[00150] CRISPR enzymes used in the methods can comprise at most 6 NLSs. An NLS is considered near the N- or C-terminus when the nearest amino add to the NLS is within 50 amino acids along a polypeptide chain from the N- or C-terminus, eg, within 1, 2, 3, 4, 5, 10, 15, 20, 25, 30, 40, or 50 amino adds.
Guide RNA
[00151] As used herein, the term ‘guide RNA” and its grammatical equivalents refers to an RNA that can be specific for a target DNA and can form a complex with Cas protein. An RNA/Cas complex can assist in “guiding” Cas protein to a target DNA.
[00152] A method disclosed herein also can comprise introducing into a cell or embryo at least one guide RNA or nucleic acid, eg, DNA encoding at least one guide RNA. A guide RNA can interact with a RNA-guided endonuclease to direct the endonuclease to a specific target site, at which site the 5’ end of the guide RNA base pairs with a specific protospacer sequence in a chromosomal sequence.
[00153] A guide RNA can comprise two RNAs, e.g., CRISPR RNA (crRNA) and transactivating crRNA (tracrRNA). A guide RNA can sometimes comprise a single-chain RNA, or single guide RNA (sgRNA) formed by fusion of a portion (eg, a functional portion) of crRNA and tracrRNA. A guide RNA can also be a dualRNA comprising a crRNA and a tracrRNA Furthermore, a crRNA can hybridize with a target DNA.
[00154] As discussed above, a guide RNA can be an expression product. For example, a DNA that encodes a guide RNA can be a vector comprising a sequence coding for the guide RNA. A guide RNA can be transferred into a cell or microorganism by transfecting the cell or microorganism with an isolated guide RNA or plasmid DNA comprising a sequence coding for the guide RNA and a promoter. A guide RNA can also be transferred into a cell or microorganism in other way, such as using virus-mediated gene delivery.
[00155] A guide RNA can be isolated. For example, a guide RNA can be transfected in the form of an isolated RNA into a cell or microorganism. A guide RNA can be prepared by in vitro transcription using any in vitro transcription system. A guide RNA can be transferred to a cell in the form of isolated RNA rather than in the form of plasmid comprising encoding sequence for a guide RNA.
[00156] A guide RNA can comprise three regions: a first region at the 5’ end that can be complementary to a target site in a chromosomal sequence, a second internal region that can form a stem loop structure, and a third 3’ region that can be single-stranded. A first region of each guide RNA can also be different such that each guide RNA guides a fusion protein to a specific target site. Further, second and third regions of each guide RNA can be identical in all guide RNAs.
[00157] A first region of a guide RNA can be complementary to sequence at a target site in a chromosomal sequence such that the first region of the guide RNA can base pair with the target site. In some cases, a first region of a guide RNA can comprise from 10 nucleotides to 25 nucleotides (i.e., from 10 nucleotides to 25 nucleotides; or 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides; or from 10 nucleotides to 25 nucleotides or more. For example, a region of base pairing between a first region of a guide RNA and a target site in a chromosomal sequence can be 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 22, 23, 24, 25, or more nucleotides in length. Sometimes, a first region of a guide RNA can be 19, 20, or 21 nucleotides in length.
[00158] A guide RNA can also comprise a second region that forms a secondary structure. For example, a secondary structure formed by a guide RNA can comprise a stem (or hairpin) and a loop. A length of a loop and a stem can vary. For example, a loop can range from 3 to 10 nucleotides in length, and a stem can range from 6 to 20 base pairs in length. A stem can comprise one or more bulges of 1 to 10 nucleotides. The overall length of a second region can range from 16 to 60 nucleotides in length. For example, a loop can be 4 nucleotides in length and a stem can be 12 base pairs.
[00159] A guide RNA can also comprise a third region at the 3’ end that can be essentially single- stranded. For example, a third region is sometimes not complementarity to any chromosomal sequence in a cell of interest and is sometimes not complementarity to the rest of a guide RNA. Further, the length of a third region can vary. A third region can be more than 4 nucleotides in length. For example, the length of a third region can range from 5 to 60 nucleotides in length.
[00160] A guide RNA can be introduced into a cell or embryo as an RNA molecule. For example, a RNA molecule can be transcribed in vitro and/or can be chemically synthesized. An RNA can be transcribed from a synthetic DNA molecule, e.g., a gBlocks® gene fragment. A guide RNA can then be introduced into a cell or embryo as an RNA molecule. A guide RNA can also be introduced into a cell or embryo in the form of a non-RNA nucleic acid molecule, e.g., DNA molecule. For example, a DNA encoding a guide RNA can be operably linked to promoter control sequence for expression of the guide RNA in a cell or embryo of interest. A RNA coding sequence can be operably linked to a promoter sequence that is recognized by RNA polymerase III (Pol III). Plasmid vectors that can be used to express guide RNA include, but are not limited to, px330 vectors and px333 vectors. In some cases, a plasmid vector (eg, px333 vector) can comprise two guide RNA-encoding DNA sequences.
[00161] A DNA sequence encoding a guide RNA can also be part of a vector. Further, a vector can comprise additional expression control sequences (eg, enhancer sequences, Kozak sequences, polyadenylation sequences, transcriptional termination sequences, etc), selectable marker sequences (eg, antibiotic resistance genes), origins of replication, and the like. A DNA molecule encoding a guide RNA can also be linear. A DNA molecule encoding a guide RNA can also be circular.
[00162] When DNA sequences encoding an RNA-guided endonuclease and a guide RNA are introduced into a cell, each DNA sequence can be part of a separate molecule (eg., one vector containing an RNA-guided endonuclease coding sequence and a second vector containing a guide RNA coding sequence) or both can be part of a same molecule (eg, one vector containing coding (and regulatory) sequence for both an RNA-guided endonuclease and a guide RNA).
Site-specie insertion
[00163] Insertion of the gpnes can be site-specific. For example, one or more genes can be inserted adjacent to a promoter.
[00164] Modification of a targeted locus of a microorganism can be produced by introducing DNA into microorganisms, where the DNA has homology to the target locus. DNA can include a marker gene, allowing for selection of cells comprising the integrated construct. Homologous DNA in a target vector can recombine with DNA at a target locus. A marker gene can be flanked on both sides by homologous DNA sequences, a 3’ recombination arm, and a 5’ recombination arm.
[00165] A variety of enzymes can catalyze insertion of foreign DNA into a microorganism genome. For example, site-specific recombinases can be clustered into two protein families with distinct biochemical properties, namely tyrosine recombinases (in which DNA is covalently attached to a tyrosine residue) and serine recombinases (where covalent attachment occurs at a serine residue). In some cases, recombinases can comprise Cre, <DC31 integrase (a serine recombinase derived from Streptomyces phage FC31), or bacteriophage derived site-spedfic recombinases including Flp, lambda integrase, bacteriophage HK022 recombinase, bacteriophage R4 integrase and phage TP901- 1 integrase).
[00166] The CRISPR/ Cas system can be used to perform site specific insertion. For example, a nick on an insertion site in the genome can be made by CRISPR/ cas to facilitate the insertion of a transgene at the insertion site.
[00167] The methods described herein, can utilize techniques that can be used to allow a DNA or RNA construct entry into a host cell include, but are not limited to, calcium phosphate/DNA coprecipitation, microinjection of DNA into a nucleus, electroporation, bacterial protoplast fusion with intact cells, transfection, lipofection, infection, particle bombardment, sperm mediated gene transfer, or any other technique.
[00168] Certain aspects disclosed herein can utilize vectors (including the ones described above). Any plasmids and vectors can be used as long as they are replicable and viable in a selected host microorganism. Vectors known in the art and those commercially available (and variants or derivatives thereof) can be engineered to include one or more recombination sites for use in the methods. Vectors that can be used include, but not limited to eukaryotic expression vectors such as pFastBac, pFastBacHT, pFastBacDUAL, pSFV, and pTet-Splice (Invitrogen), pEUK-Cl, pPUR, pMAM, pMAMneo, pBIlOl, pBI121, pDR2, pCMVEBNA, and pYACneo (Clontech), pSVK3, pSVL, pMSG, pCHllO, and pKK232-8 (Pharmacia, Inc.), pXTl, pSG5, pPbac, pMbac, pMClneo, and pOG44 (Stratagene, Inc.), and pYES2, pAC360, pBlueBa-cHis A, B, and C, pVL1392, pBlueBaclll, pCDM8, pcDNAl, pZeoSV, pcDNA3, pREP4, pCEP4, and pEBVHis (Invitrogen, Corp.), and variants or derivatives thereof.
[00169] These vectors can be used to express a gene or portion of a gene of interest. A gene of portion or a gene can be inserted by using known methods, such as restriction enzyme-based techniques.
III. Fermentation
[00170] In some embodiments, the microorganisms useful in the present invention should be cultured in fermentation conditions that are appropriate to convert a carbon substrate into a flavonoid or flavonoid derivative. Reaction conditions that should be considered include temperature, media flow rate, pH, media redox potential, agitation rate (if using a continuous stirred tank reactor), inoculum level, maximum substrate concentrations and rates of introduction of the substrate to the bioreactor to ensure that substrate level does not become limiting, and maximum product concentrations to avoid product inhibition. The optimum reaction conditions will depend partly on the particular microorganism used. Fermentation Conditions
[00171] In those embodiments in which the microorganism is cultured in fermentation conditions, the pH of the culture media may be optimized based on the microorganism used. For example, the pH used can range from 4 to 10. In other instances, the pH can be from 5 to 9; 6 to 8; 6.1 to 7.9; 6.2 to 7.8; 6.3 to 7.7; 6.4 to 7.6; or 6.5 to 7.5. For example, the pH can be from 6.6 to 7.4. In some cases, the pH can be from 5 to 9. In some cases, the pH can be from 6 to 8. In some cases, the pH can be from 6.1 to 7.9. In some cases, the pH can be from 6.2 to 7.8. In some cases, the pH can be from 6.3 to 7.7. In some cases, the pH can be from 6.4 to 7.6. In some cases, the pH can be from 6.5 to 7.5. In some cases, the pH used can be greater than 6.
[00172] Temperature can also be adjusted based on the microorganism used. For example, the temperature can range from 25 C° to 48 C°. In other instances, the temperature of the fermentation can be from 25 C° to 48 C°; 26 C° to 46 C°; 27 C° to 44 C°; 28 C° to 42 C°; 29 C° to 41 C°; 30 C° to 40 C°; 30 C° to 39 C°; 31 C° to 38 C°; 32 C° to 37 C°; 33 C° to 36 C°; 34 C° to 37 C°; 35 C° to 36 C°; 30 C° to 43 C°; 32 C° to 42 C°; 34 C° to 41 C°; 35 C° to 40 C°. For example, the temperature can be 25 C°, 26 C°, 27 C°, 28 C°, 29 C°, 30 C°, 31 C°, 32 C°, 33 C°, 34 C°, 35 C°, 36 C°, 37 C°, 38 C°, 39 C°, 40 C°, 41 C°, 42 C°, 43 C°, 44 C°, or 45 C°. In some cases, the temperatures can be within one tenth of a degree, such as 25.0 C°, 25.1 C°, 25.2 C°, 25.3 C°, 25.4 C°, 25.5 C°, 25.6 C°, 25.7 C°, 25.8 C°, 25.9 C°, 30.0 C°, 30.1 C°, 30.2 C°, 30.3 C°, 30.4 C°, 30.5 C°, 30.6 C°, 30.7 C°, 30.8 C°, 30.9 C°, 31.0 C°, 31.1 C°, 31.2 C°, 31.3 C°, 31.4 C°, 31.5 C°, 31.6 C°, 31.7 C°, 31.8 C°, 31.9 C°, 32.0 C°, 32.1 C°, 32.2 C°, 32.3 C°, 32.4 C°, 32.5 C°, 32.6 C°, 32.7 C°, 32.8 C°, 32.9 C°, 33.0 C°, 33.1 C°, 33.2 C°, 33.3 C°, 33.4 C°, 33.5 C°, 33.6 C°, 33.7 C°, 33.8 C°, 33.9 C°, 34.0 C°, 34.1 C°, 34.2 C°, 34.3 C°, 34.4 C°, 34.5 C°, 34.6 C°, 34.7 C°, 34.8 C°, 34.9 C°, 35.0 C°, 35.1 C°, 35.2 C°, 35.3 C°, 35.4 C°, 35.5 C°, 35.6 C°, 35.7 C°, 35.8 C°, 35.9 C°, 36.0 C°, 36.1 C°, 36.2 C°, 36.3 C°, 36.4 C°, 36.5 C°, 36.6 C°, 36.7 C°, 36.8 C°, 36.9 C°, 37.0 C°, 37.1 C°, 37.2 C°, 37.3 C°, 37.4 C°, 37.5 C°, 37.6 C°, 37.7 C°, 37.8 C°, 37.9 C°, 38.0 C°, 38.1 C°, 38.2 C°, 38.3 C°, 38.4 C°, 38.5 C°, 38.6 C°, 38.7 C°, 38.8 C°, 38.9 C°, 39.0 C°, 39.1 C°, 39.2 C°, 39.3 C°, 39.4 C°, 39.5 C°, 39.6 C°, 39.7 C°, 39.8 C°, 39.9 C°, 40.0 C°, 40.1 C°, 40.2 C°, 40.3 C°, 40.4 C°, 40.5 C°, 40.6 C°, 40.7 C°, 40.8 C°, 40.9 C°, 41.0 C°, 41.1 C°, 41.2 C°, 41.3 C°, 41.4 C°, 41.5 C°, 41.6 C°, 41.7 C°, 41.8 C°, 41.9 C°, 42.0 C°, 42.1 C°, 42.2 C°, 42.3 C°, 42.4 C°,
42.5 C°, 42.6 C°, 42.7 C°, 42.8 C°, 42.9 C°, 43.0 C°, 43.1 C°, 43.2 C°, 43.3 C°, 43.4 C°, 43.5 C°, 43.6 C°, 43.7 C°, 43.8 C°, 43.9 C°, 44.0 C°, 44.1 C°, 44.2 C°, 44.3 C°, 44.4 C°, 44.5 C°, 44.6 C°, 44.7 C°, 44.8 C°, 44.9 C°, 45.0 C°, 45.1 C°, 45.2 C°, 45.3 C°, 45.4 C°, 45.5 C°, 45.6 C°, 45.7 C°, 45.8 C°, 45.9 C°, 46.0 C°, 46.1 C°, 46.2 C°, 46.3 C°, 46.4 C°, 46.5 C°, 46.6 C°, 46.7 C°, 46.8 C°, 46.9 C°, 47.0 C°,
47.1 C°, 47.2 C°, 47.3 C°, 47.4 C°, 47.5 C°, 47.6 C°, 47.7 C°, 47.8 C°, or 47.9 C°.
[00173] In some cases, the temperature of fermentation can be from 37.0 C° to 47.9 C°. In some cases, the temperature of fermentation can be from 37.1 C° to 47.8 C°. In some cases, the temperature of fermentation can be from 37.2 C° to 47.7 C°. In some cases, the temperature of fermentation can be from 37.3 C° to 47.6 C°. In some cases, the temperature of fermentation can be from 37.4 C° to
47.5 C°. In some cases, the temperature of fermentation can be from 37.5 C° to 47.4 C°. In some cases, the temperature of fermentation can be from 37.6 C° to 47.3 C°. In some cases, the temperature of fermentation can be from 37.7 C° to 47.2 C°. In some cases, the temperature of fermentation can be from 37.8 C° to 47.1 C°. In some cases, the temperature of fermentation can be from 37.9 C° to 47.0 C°. In some cases, the temperature of fermentation can be from 38.0 C° to 46.9 C°. In some cases, the temperature of fermentation can be from 38.1 C° to 46.8 C°. In some cases, the temperature of fermentation can be from 38.2 C° to 46.7 C°. In some cases, the temperature of fermentation can be from 38.3 C° to 46.6 C°. In some cases, the temperature of fermentation can be from 38.4 C° to
46.5 C°. In some cases, the temperature of fermentation can be from 38.5 C° to 46.4 C°. In some cases, the temperature of fermentation can be from 38.6 C° to 46.3 C°. In some cases, the temperature of fermentation can be from 38.7 C° to 46.2 C°. In some cases, the temperature of fermentation can be from 38.8 C° to 46.1 C°. In some cases, the temperature of fermentation can be from 38.9 C° to 46.0 C°. In some cases, the temperature of fermentation can be from 39.0 C° to 45.9 C°. In some cases, the temperature of fermentation can be from 39.1 C° to 45.8 C°. In some cases, the temperature of fermentation can be from 39.2 C° to 45.7 C°. In some cases, the temperature of fermentation can be from 39.3 C° to 45.6 C°. In some cases, the temperature of fermentation can be from 39.4 C° to
45.5 C°. In some cases, the temperature of fermentation can be from 39.5 C° to 45.4 C°. In some cases, the temperature of fermentation can be from 39.6 C° to 45.3 C°. In some cases, the temperature of fermentation can be from 39.7 C° to 45.2 C°. In some cases, the temperature of fermentation can be from 39.8 C° to 45.1 C°. In some cases, the temperature of fermentation can be from 39.9 C° to 45.0 C°. In some cases, the temperature of fermentation can be from 40.0 C° to 44.9 C°. In some cases, the temperature of fermentation can be from 40.1 C° to 44.8 C°. In some cases, the temperature of fermentation can be from 40.2 C° to 44.7 C°. In some cases, the temperature of fermentation can be from 40.3 C° to 44.6 C°. In some cases, the temperature of fermentation can be from 40.4 C° to
44.5 C°. In some cases, the temperature of fermentation can be from 40.5 C° to 44.4 C°. In some cases, the temperature of fermentation can be from 40.6 C° to 44.3 C°. In some cases, the temperature of fermentation can be from 40.7 C° to 44.2 C°. In some cases, the temperature of fermentation can be from 40.8 C° to 44.1 C°. In some cases, the temperature of fermentation can be from 40.9 C° to 44.0 C°. In some cases, the temperature of fermentation can be from 41.0 C° to 43.9 C°. In some cases, the temperature of fermentation can be from 41.1 C° to 43.8 C°. In some cases, the temperature of fermentation can be from 41.2 C° to 43.7 C°. In some cases, the temperature of fermentation can be from 41.3 C° to 43.6 C°. In some cases, the temperature of fermentation can be from 41.4 C° to
43.5 C°. In some cases, the temperature of fermentation can be from 41.5 C° to 43.4 C°. In some cases, the temperature of fermentation can be from 41.6 C° to 43.3 C°. In some cases, the temperature of fermentation can be from 41.7 C° to 43.2 C°. In some cases, the temperature of fermentation can be from 41.8 C° to 43.1 C°. In some cases, the temperature of fermentation can be from 41.9 C° to 43.0 C°. In some cases, the temperature of fermentation can be from 42.0 C° to 42.9 C°. In some cases, the temperature of fermentation can be from 42.1 C° to 42.8 C°. In some cases, the temperature of fermentation can be from 42.2 C° to 42.7 C°. In some cases, the temperature of fermentation can be from 42.3 C° to 42.6 C°. In some cases, the temperature of fermentation can be from 42.4 C° to 42.5 C°.
[00174] Availability of oxygpn and other gases may affect yield and fermentation rate. For example, when considering oxygpn availability, the percent of dissolved oxygen (DO) within the fermentation media can be from 1% to 40%. In certain instances, the DO concentration can be from 1.5% to 35%; 2% to 30%; 2.5% to 25%; 3% to 20%; 4% to 19%; 5% to 18%; 6% to 17%; 7% to 16%; 8% to 15%; 9% to 14%; 10% to 13%; or 11% to 12%. For example, in some cases the DO concentration can be from 2% to 30%. In other cases, the DO can be from 3% to 20%. In some cases, the DO can be from 4% to 10%. In some cases, the DO can be from 1.5% to 35%. In some cases, the DO can be from 2.5% to 25%. In some cases, the DO can be from 4% to 19%. In some cases, the DO can be from 5% to 18%. In some cases, the DO can be from 6% to 17%. In some cases, the DO can be from 7% to 16%. In some cases, the DO can be from 8% to 15%. In some cases, the DO can be from 9% to 14%. In some cases, the DO can be from 10% to 13%. In some cases, the DO can be from 11% to 12%.
Bioreactor
[00175] Fermentation reactions can be carried out in any suitable bioreactor. In some cases, the bioreactor can comprise a first growth reactor in which the microorganisms are cultured, and a second fermentation reactor, to which broth from the growth reactor is fed and in which most of the fermentation product is produced.
Product Recovery
[00176] The fermentation of the microorganisms disclosed herein can produce a broth comprising a desired product (e.g., a flavonoid), one or more by-products, and/or the microorganism itself (e.g., a genetically modified yeast cell) .
[00177] The microorganisms and the methods herein can produce a desired end product, such as a flavonoid compound or flavonoid derivative, at a relatively high efficiency, more so than other known fermentation processes. For example, the microorganisms and the methods disclosed herein may convert a carbon substrate at a rate of greater than 50%. This means that at least 50% of the carbons of the carbon substrate are converted into the desired product. In some cases, the conversion of a carbon substrate into desired flavonoid products can be at least 60%, 70%, 80%, 81%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 93%, 94%, 95%, 96%, 97%, 98%, or 99%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 60%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 70%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 80%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 81%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 82%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 83%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 84%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 85%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 86%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 87%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 88%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 89%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 90%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 91%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 92%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 93%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 94%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 95%. In some cases, the conversion of a carbon substrate into mu flavonoid products can be at least 96%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 97%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 98%. In some cases, the conversion of a carbon substrate into flavonoid products can be at least 99%.
[00178] In certain methods of producing flavonoid products, the concentration of flavonoid products in the fermentation broth is at least 0.1 g/L For example, the concentration of flavonoid products produced in the fermentation broth can be from 0.1 g/L to 0.5 g/L, 0.5 g/L to 1 g/L, 1 g/L to 5 g/L, 2 g/L to 6 g/L, 3 g/L to 7 g/L, 4 g/L to 8 g/L, 5 g/L to 9 g/L, or 6 g/L to 10 g/L. In some cases, the concentration of flavonoid products can be at least 9 g/L. In some cases, the concentration of flavonoid products can be from 0.1 g/L to to 10 g/L. In some cases, the concentration of flavonoid products can be from 0.5 g/L to 3 g/L. In some cases, the concentration of flavonoid products can be from 1 g/L to 5 g/L. In some cases, the concentration of flavonoid products can be from 2 g/L to 6 g/L. In some cases, the concentration of flavonoid products can be from 3 g/L to 7 g/L. In some cases, the concentration of flavonoid products can be from 4 g/L to 8 g/L. In some cases, the concentration of flavonoid products can be from 5 g/L to 9 g/L. In some cases, the concentration of flavonoid products can be from 6 g/L to 10 g/L.
[00179] In other cases, when microorganisms are used that normally produce at least some of the same flavonoid products, the genetically modified microorganism can produce the same flavonoid product in concentrations that are at least 1.1X the amount that is normally produced by a microorganism that is unmodified and of the same species as the genetically modified microorganism. In some cases, the genetically modified microorganism can produce at least 1.2X, 1.3X, 1.4X, 1.5X, 1.6X, 1.7X, 1.8X, 1.9X, 2X, 3X, 4X, 5X, 6X, 7X, 8X, 9X, 10X, 15X, 20X, 25X, 30X, 35X, 40X, 45X, 50X, 60X, 70X, 80X, 90X or 100X the amount that is normally produced by a microorganism that is unmodified and of the same species as the genetically modified microorganism. In some cases, the genetically modified microorganism can produce at least 2X, 3X, 4X, 5X, 10X, 25X, 50X, and or 100X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 2X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 3X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 4X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 5X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 10X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 25X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 50X the amount that is normally produced. In some cases, the genetically modified microorganism can produce at least 100X the amount that is normally produced.
[00180] As discussed above, in certain cases the flavonoid product produced in the fermentation reaction is converted to a different organic product. In other cases, the flavonoid product is first recovered from the fermentation broth before conversion to a different organic product.
[00181] In some cases, flavonoid product can be continuously removed from a portion of broth and recovered as purified. In particular cases, the recovery of the flavonoid product includes passing the removed portion of the broth containing the flavonoid product through a separation unit to separate the microorganisms (e.g., genetically modified yeast) from the broth, to produce a cell-free flavonoid product permeate, and returning the microorganisms to the bioreactor. The cell-free flavonoid product containing permeate can then can be stored or be used for subsequent conversion to a different desired product.
[00182] The recovering of the desired flavonoid product and/ or one or more other products or by- products produced in the fermentation reaction can comprise continuously removing a portion of the broth and recovering separately the flavonoid product and one or more other products from the removed portion of the broth. In some cases, the recovery of the flavonoid product and/or one or more other products includes passing the removed portion of the broth containing the flavonoid product and/or one or more other products through a separation unit to separate microorganisms from the flavonoid product and/or one or more other products, to produce a cell-free flavonoid product and one or more other product-containing permeate, and returning the microorganisms to the bioreactor.
[00183] The flavonoid product, or a mixed product stream containing the flavonoid product, can be recovered from the fermentation broth. For example, methods that can be used can include but are not limited to, fractional distillation or evaporation, pervaporation, and extractive fermentation. Further examples indude: recovery using steam from whole fermentation broths; reverse osmosis combined with distillation; liquid-liquid extraction techniques involving solvent extraction of the flavonoid product; aqueous two-phase extraction of the flavonoid product in PEG/dextran system; solvent extraction using alcohols or esters, e.g., ethyl acetate, tributylphosphate, diethyl ether, n- butanol, dodecanol, oleyl alcohol, and an ethanol/ phosphate system; aqueous two-phase systems composed of hydrophilic solvents and inorganic salts. U.S. Pat. Pub. Appl. No. 2012/0045807 to Simpson.
[00184] In some cases, the flavonoid product and / or other by-products may be recovered from the fermentation broth by continuously removing a portion of the broth from the bioreactor, separating microbial cells from the broth (conveniently by filtration, for example), and recovering the flavonoid product and others such as alcohols and adds from the broth. Alcohols can conveniently be recovered for example by distillation, and adds can be recovered for example by adsorption on activated charcoal. The separated microbial cells are returned to the fermentation bioreactor. The cell-free permeate remaining after the alcohol(s) and add(s) have been removed is also preferably returned to the fermentation bioreactor. Additional nutrients can be added to the cell-free permeate to replenish the nutrient medium before it is returned to the bioreactor.
[00185] Also, if the pH of the broth is adjusted during recovery of the flavonoid product and/ or by products, the pH should be re-adjusted to a similar pH to that of the broth in the fermentation bioreactor, before being returned to the bioreactor.
[00186] While some cases and embodiments have been shown and described herein, such are provided by way of example only. Numerous variations, changes, and substitutions will now occur to those skilled in the art without departing from the invention. It should be understood that various alternatives to the cases of the invention described herein will be employed in practicing the invention.
Compositions and Phamaceutically-Acceptable Derivatives or Prodrugs [00187] The present invention also relates in part to a composition comprising a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof. The composition may further comprise an excipient. The composition may be in the form of a medicament. A “pharmaceutically acceptable derivative” means any pharmaceutically acceptable salt, ester, salt of an ester, pro-drug or other derivative thereof. Pharmaceutically acceptable salts include those derived from pharmaceutically acceptable inorganic and organic acids and bases. Examples of suitable acid salts indude acetate, adipate, benzoate, benzenesulfonate, butyrate, dtrate, digluconate, dodecylsulfate, formate, fumarate, glycolate, hemisulfate, heptanoate, hexanoate, hydrochloride, hydrobromide, hydroiodide, lactate, maleate, malonate, methanesulfonate, 2-naphthalenesulfonate, nicotinate, nitrate, palmoate, phosphate, picrate, pivalate, propionate, salicylate, sucdnate, sulfate, tartrate, tosylate and undecanoate. Salts derived from appropriate bases indude alkali metal (e.g., sodium), alkaline earth metal (e.g., magnesium), ammonium and N-(alkyl)4+ salts.
[00188] The present invention also relates in part to a method of formulating the flavonoid or flavonoid derivative into a pharmaceutical composition. For preparing pharmaceutical compositions from the compounds of the present invention, pharmaceutically-acceptable carriers include either solid or liquid carriers. Solid form preparations include powders, tablets, pills, capsules, cachets, suppositories, and dispersible granules. A solid carrier can be one or more substances, which also acts as diluents, flavoring agents, binders, preservatives, tablet disintegrating agents, or an encapsulating material. Details on techniques for formulation and administration are well described in the scientific and patent literature, including in the latest edition of Remington’s Pharmaceutical Sciences, Maack Publishing Co, Easton PA.
[00189] In powders, the carrier is a finely divided solid, which is in a mixture with the findy divided active component. In tablets, the active component is mixed with the carrier having the necessary binding properties in suitable proportions and compacted in the shape and size desired.
[00190] Suitable solid exdpients include carbohydrate or protein fillers, such as lactose, sucrose, mannitol, or sorbitol; starch from com, wheat, rice, potato, or other plants; cellulose such as methyl cdlulose, hydroxypropylmethyl-cellulose, or sodium carboxymethylcdlulose; and gums including arabic and tragacanth; as well as proteins such as gelatin and collagen. If desired, disintegrating or solubilizing agents may be added, such as the cross-linked polyvinyl pyrrolidone, agar, alginic acid, or a salt thereof, such as sodium alginate. [00191] Liquid form preparations may also include solutions, suspensions, and emulsions, for example, water or water/ propylene glycol solutions. For parenteral injection, liquid preparations can be formulated in solution in aqueous polyethylene glycol solution.
[00192] The pharmaceutical preparation can be a unit dosage form. In such form the preparation is subdivided into unit doses containing appropriate quantities of the active component. The unit dosage form can be a packaged preparation, the package containing discrete quantities of preparation, such as packeted tablets, capsules, and powders in vials or ampoules. Also, the unit dosage form can be a capsule, tablet, cachet, or lozenge itself, or it can be the appropriate number of any of these in packaged form.
[00193] The present invention also relates to a method of making the pharmaceutical composition. In some cases, a flavonoid or flavonoid derivative is mixed with an excipient to produce a pharmaceutical composition.
Treatment of Disease and Symptoms of Disease
[00194] The flavonoid or flavonoid derivative can be used to treat a disease. This includes treating one or more symptoms of the diseases. For example, the flavonoid or flavonoid derivative can be used to treat one of more of the following diseases: diabetes, heart disease, cardiovascular disease, Parkinson’s disease, malaria, cancer, digestive disorders, autoimmune diseases, chronic inflammation, stroke, obesity, or neurodegenerative disorders. Among the cancers that the flavonoid or flavonoid derivative may treat include: lung, prostrate, bladder, ovarian, cervical, pancreatic, breast, liver, esophageal, gastric, adenoma, and melanoma. [00195] Some of the diseases or symptom of disease can be exclusive to humans, but other diseases or symptom of disease can be shared in more than one animal, such as in all mammals.
[00196] The present invention relates in part to a method of treating a disease or symptom of a disease, the method comprising administering a flavonoid or flavonoid derivative, or a pharmaceutically-acceptable derivative or prodrug thereof, to a subject in need of such treatment. Suitable routes of administration include, but are not limited to, oral, intravenous, rectal, aerosol, parenteral, ophthalmic, pulmonary, transmucosal, transdermal, vaginal, otic, nasal, and topical administration. In addition, by way of example only, parenteral delivery includes intramuscular, subcutaneous, intravenous, intramedullary injections, as well as intrathecal, direct intraventricular, intraperitoneal, intralymphatic, and intranasal injections. Use of Flavonoid or Flavonoid Derivative
[00197] The present invention further relates in part to the use of the flavonoid or flavonoid derivative made using the aforementioned method in the manufacture of a medicament for the treatment or a disease or symptom of a disease. The disease or symptom of a disease may be any disease or symptom capable of being treated by flavonoid or flavonoid derivative, and include those disclosed herein and in the materials incorporated by reference. Examples include diabetes, heart disease, cardiovascular disease, Parkinson’s disease, malaria, cancer (including lung, prostrate, bladder, ovarian, cervical, pancreatic, breast, liver, esophageal, gastric, adenoma, and melanoma), digestive disorders, autoimmune diseases, chronic inflammation, stroke, obesity, or neurodegenerative disorders.
EXAMPLES
Example 1. Biosynthesis of flavonoids and flavonoid derivatives
[00198] Guided by previously published metabolomic data, a targeted phylogenomics approach combined with in-vitro biochemical assays is employed to explore the biosynthetic pathway towards desirable flavonoids and flavonoid derivatives. [00199] In order to decipher the biosynthetic pathway of a candidate flavonoid, a literature and database search of relevant enzymes and substrates is performed to screen for potential enzyme candidates. For example, the Transcriptome Shotgun Assembly (TSA) database, which is accessible through NCBI, may be searched. All previously-characterized enzymes in the rdevant biosynthetic pathway are identified, doned, and tested. If necessary, structural homologs of relevant substrates may be used to locate additional potentially useful enzyme candidates using, e.g., phylogenetic analysis. All such enzyme candidates are then doned and tested.
[00200] An enzyme library is constructed in yeast to test all the different enzymes identified. The steps involved in building a yeast enzyme library are well known in the art. Briefly, highly expressed copies for each candidate enzyme coding DNA sequence is synthesized (e.g., by GENWIZ) with codon optimization for Saccharomyces cerevisiae and then cloned into a high-copy yeast expression plasmid containing a strong promoter and direct selection marker. The expression construct is then transformed into a suitable strain of Saccharomyces cerevisiae using well-known cloning and transformation techniques. For example, the M2S integration method may used. Li et al., BiotechnoL Biofuels., 9:232-243 (2016). The yeast strain chosen should be already producing a substrate or at least capable of efficiently taking in the substrate. A gpod substrate in this regard is a flavonoid precursor, such as ferulic add, which can be fed to the yeast culture. Positive transformants can be verified, e.g., by sequencing.
[00201] Transformants are grown in 96-well plate format and, if needed, substrate (e.g., ferulic arid) is fed to the yeast culture. Consumption of the substrate is in the engineered yeast cells is monitored to determine whether it is bring converted to the desired flavonoid. The yeast culture supernatant is also analyzed for the presence of other useful flavonoids or falvonoid derivatives. Products are confirmed by, e.g., high-performance liquid chromatography (HPLC) and mass spectrometry (MS). For example, to detect flavonoids in the yeast supernatant, the yeast cells are collected by centrifugation at 13,000 x g for 10 min and the supernatant is analyzed by HPLC. To detect flavonoids of flavonoid precursors within the cells, the yeast cells are washed, disrupted on ice in 30 ml of 50% methanol using a high-pressure homogenizer, and the cell debris is removed by centrifugation at 13,000 x g for 30 min. The supernatant is then collected and analyzed by HPLC.
[00202] The foregoing flavonoid biosynthesis strategy has several advantages. First, enzymes catalysing the desired flavonoid biosynthesis reactions can be quickly identified and tested and, if necessary, selected for enzyme evolution. Second, this strategy permits for a wide range of non-natural compounds to be quickly produced and tested from a given starting substrate. Third, active library genes can be further combined to catalyze even more complex changes to further increase the diversity and the number and complexity of non-natural products.
Example 2. Biosynthesis of isocannflavin B through a flavonoid intermediate
[00203] Isocannflavin B is an unnatural isomer of cannflavin B recently isolated from Cannabis saliva and shown to have statistically significant results against pancreatic cancer in vivo. U.S. Patent No. 10,398,674 to Lowe et al. (incorporated herein by reference).
Figure imgf000058_0001
[00204] As such, it is desirable to construct a biosynthetic route to produce isocannflavin B. To do so, the strategy outlined in Example 1 is used. Notably, some of the investigatory research has already been performed. For example, flavanone biosynthesis has been demonstrated in modified S. cerevisiae. In 2005, Yan and colleagues cloned and expressed gene sequences encoding the four flavonoid pathway enzymes C4H, 4CL, CHS, and CHI in yeast cells. Yan et al., Appl. & Envir. Microbiol., 71:5610-5613 (2005) (hereby incorporated by reference) (C4H from Arabidopsis thaliana, isolated from expressed sequence tag clone RAFL06- 11-J16, available from RIKEN BioResource Center; 4CL from Petroselinum crispum, GenBank accession number AF233638; CHS from Petunia hybrid, GenBank
SUBSTITUTE SHEET (RULE 26) accession number X13225; and CHI from Petunia hybnda , GenBank accession number XI 4589.) The modified yeast was fed with different phenylpropanoid acids, including cinnamic add, p-coumaric add, caffdc acid and ferulic acid. Id. When 1 mM cinnamic add (148 mg/L) was fed to the yeast as a precursor metabolite, 16.3 mg/L pinocembrin accumulated in the medium Id. When ImM p- coumaric add (164 mg/L) was fed to the yeast, 28.3 mg/liter naringenin accumulated in the culture. Id. When 1 mM caffdc add (180 mg/L) was fed to the yeast, (2S)-eriodictyol was produced at 6.5 mg/liter. Id. However, when ferulic add was fed to the yeast, no homoeriodictyol was detected. Id.
[00205] In 2012, Koopman and colleagues produced naringenin directly from glucose in modified S. cemdsiae. Koopman et al., Microbial CellFactories, 11:155 (2012) (hereby incorporated by reference). Koopman details the strains, plasmids, and methods used to engineer the naringenin-producing yeast
Id. Briefly, spedfic naringenin biosynthesis genes from Arabidopsis thaliana and Rhodobacter capsulatus; were sdected by comparative expression profiling and overexpressed in S. cemdsiae. These genes encoded the enzymes PALI (phenylalanine/ tyrosine ammonia lyase), C4H (Cinnamate 4- h droxylase), CPR1 (cytochrome P450 reductase), 4CL3 (4-coumaric acid-CoA ligase), CHS3 (chalcone synthase), and CHI1 (chalcone isomerase), all obtained from A thaliana, and one gene from
Rhodobacter capsulatus encoding the enzyme TALI (tyrosine ammonia lyase). Id. The expression of these gpnes yielded relatively low extracellular naringenin concentrations (<5.5 mM). Thus, to optimize naringenin titers, Koopman developed a yeast chassis strain. Synthesis of aromatic amino adds was deregulated by alleviating feedback inhibition of 3-deoxy-d-arabinose-heptulosonate-7-phosphate synthase (Aro3, Aro4) and byproduct formation was reduced by eliminating phenylpyruvate decarboxylase (ArolO, Pdc5, Pdc6). Together with an increased copy number of the chalcone synthase gene and expression of a heterologous tyrosine ammonia lyase, these modifications resulted in a 40- fold increase of extracellular naringenin titers (to approximately 200 mM) in glucose-grown shake-flask cultures. In aerated, pH controlled batch reactors, extracellular naringenin concentrations of over 400 mM (about 100 mg/L) were reached. Figure 1 from Koopman is reproduced bdow. It shows a schematic representation of the engineered naringenin production pathway in S. cemdsiae. Dashed lines indicate feedback inhibition. Grey arrows indicate the S. cemdsiae pathway for phenylethanol production. Bold dark grey arrows indicate the naringenin production pathway as described for A thaliana. (Aro3/Aro4: 3-deoxy-D-arabino-heptulosonate-7-phosphate (DAHP) synthase, Pdcl, 5, 6; pyruvate decarboxylases, ArolO; phenylpyruvate decarboxylase.)
Figure imgf000060_0001
[00206] In 2019, Rea and colleagues studied the compounds cannflavin A and cannflavin B, two prenylated flavonoids that specifically accumulate in C. sativa. Rea et al., Phytochemistry, 164:162-171 (2019) (hereby incorporated by reference). Using a combination of phylogenomic and biochemical approaches, they identified an aromatic prenyltransferase from C. sativa (CsPT3) which catalyzes the regiospecific addition of either geranyl diphosphate (GPP) or dimethylallyl diphosphate (DMAPP) to the methylated flavone, chrysoeriol, to produce cannflavins A and B, respectively. Id. They also showed that an O-methyltransferase (CsOMT21) encoded within the C. sativa genome may convert
SUBSTITUTE SHEET (RULE 26) the widespread plant flavone known as luteolin into chrysoeriol, both of which accumulate in C. sativa Id. The researchers suggest that these results imply the following reaction sequence for cannflavins A and B biosynthesis: luteolin to chrysoeriol to cannflavin A and cannflavin B. Id. Taken together, the identification of these two enzymes represent a branch point from the general flavonoid pathway in C sativa and offer a potentially tractable route towards metabolic engineering strategies designed to produce these two medicinally relevant Cannabis compounds. Id.
[00207] Building upon the foregoing studies, isocannflavin may be produced in an engineered yeast strain. The yeast strain may be transformed with, for example, the flavonoid pathway enzymes disdosed in Yan et al .,AppL &Envir. Microbiol, 71:5610-5613 (2005) or Koopman et al., Microbial Cell Factories, 11:155 (2012). Optionally, the yeast may be transformed with a prenyltransferase and/ or O- methyltransferase, such as those disdosed in Rea et al., Phytochemistiy, 164:162-171 (2019). Additionally, to optimize isocannflavin B production, it may be necessary to transform the yeast with a prenyl transferase that prenilates a flavanone (e.g., naringenin or homoeriodictyl) at the 8 position of the A ring. Such an enzyme can be identified and cloned, for example, by surveying all the prenyl transferase homologues from Cannabis (or other suitable species) and selecting enzyme candidates that specifically catalyze the A-8 position prenylation, or by generating via enzyme evolution derivatives of prenyl transferase where the A-8 prenylation is favored.
[00208] Yeast cells successfully transformed with constructs expressing the necessary flavonoid pathway enzymes are grown in 96-well plate format and fed ferulic acid. Ferulic add consumption is then monitored and the reaction lysate is tested for the presence of isocannflavin B, as described above.
SEQUENCE LISTING
SEQ ID NO: 1
[Reference: >osa: 4330034 K13064 phenylalanine/tyrosine ammonia-lyase [EC:4.3.1.25] | (RefSeq) phenylalanine ammonia-lyase (N)]
Figure imgf000062_0001
SEQ ID NO: 2
[Reference: osa:4330034 K13064 phenylalanine/ tyrosine ammonia-lyase [EC:4.3.1.25] | (RefSeq) phenylalanine ammonia-lyase (A)]
Figure imgf000063_0001
SEQ ID NO: 3
[Reference: >athAT2G37040 K10775 phenylalanine ammonia-lyase [EC:4.3.1.24] | (RefSeq) PALI; PHE ammonia lyase 1 (N)]
Figure imgf000063_0002
Figure imgf000064_0001
SEQ ID NO: 4
[Reference: >athAT2G37040 K10775 phenylalanine ammonia-lyase [EC:4.3.1.24] | (RefSeq) PALI; PHE ammonia lyase 1 (A)]
Figure imgf000064_0002
SEQ ID NO: S
[Reference: >athAT2G30490 K00487 trans-dnnamate 4-monooxygenase [EC:1.14.14.91] | (RefSeq) C4H; cinnamate-4-h droxylase (N)]
Figure imgf000065_0001
SEQ ID NO: 6
[Reference: >athAT2G30490 K00487 trans-dnnamate 4-monooxygenase [EC:1.14.14.91] | (RefSeq) C4H; cinnamate-4-h droxylase (A)]
Figure imgf000065_0002
Figure imgf000066_0001
SEQ ID NO: 7
[Reference: >athATlG51680 K019044-coumarate— CoA ligase [EC:6.2.1.12] | (RefSeq) 4CL1; 4- coumarate:CoA ligase 1 (N)]
Figure imgf000066_0002
SEQ ID NO: 8 [Reference: >athATlG51680 K019044-coumarate— CoA ligase [EC:6.2.1.12] | 4CL1; 4- coumarate:CoA ligase 1 (A)]
Figure imgf000067_0001
SEQ ID NO: 9
[Reference: >athAT5G 13930 K00660 chalcone synthase [EC:2.3.1.74] | (RefSeq) TT4; Chalcone and stilbene synthase family protein (N)]
Figure imgf000067_0002
Figure imgf000068_0002
SEQ ID NO: 10
[Reference: >athAT5G 13930 K00660 chalcone synthase [EC:2.3.1.74] | (RefSeq) TT4; Chalcone and stilbene synthase family protein (A)]
Figure imgf000068_0001
SEQ ID NO: 11
[Reference: >gmx:547911 K08243 polyketide reductase | (RefSeq) GMCHR; NAD(P)H-dependent 6'-deoxychalcone synthase (N)]
Figure imgf000068_0003
SEQ ID NO: 12
[Reference: >gmx:547911 K08243 polyketide reductase | (RefSeq) GMCHR; NAD(P)H-dependent 6'-deoxychalcone synthase (A)]
Figure imgf000069_0001
SEQ ID NO: 13
[Reference: >zma:100276821 KOI 859 chalcone isomerase [EC:5.5.1.6] | (RefSeq) chil; chalcone— flavonone isomerase (N)]
Figure imgf000069_0002
SEQ ID NO: 14
[Reference: >zma:100276821 KOI 859 chalcone isomerase [EC:5.5.1.6] | (RefSeq) chil; chalcone— flavonone isomerase (A)]
Figure imgf000069_0003
Figure imgf000070_0001
SEQ ID NO: 15
[Reference: >gmx: 100780057 K00475 naringenin 3-dioxygenase [EC:1.14.11.9] | (RefSeq) naringenin,2-oxoglutarate 3-dioxygenase (N)]
Figure imgf000070_0002
SEQ ID NO: 16
[Reference: >gmx: 100780057 K00475 naringenin 3-dioxygenase [EC:1.14.11.9] | (RefSeq) naringenin,2-oxoglutarate 3-dioxygenase (A)]
Figure imgf000070_0003
SEQ ID NO: 17
[Reference: >RG001:011060262 K13082 bifunctional dihydroflavonol 4-reductase/ flavanone 4- reductase [EC:1.1.1.219 1.1.1.234] | OM-RGC.vl.Ol 1060262; TARA_099_SRF_0.22- 3_C20245996_1_gene416651 (N)]
Figure imgf000071_0001
SEQ ID NO: 18
[Reference: >RG001:011060262 K13082 bifunctional dihydroflavonol 4-reductase/ flavanone 4- reductase [EC:1.1.1.219 1.1.1.234] | OM-RGC.vl.Ol 1060262; TARA_099_SRF_0.22- 3_C20245996_1_gene416651 (A)]
Figure imgf000071_0002
SEQ ID NO: 19
[Reference: >nta:107795677 K05280 flavonoid 3' -monooxygenase [EC:1.14.14.82] | (RefSeq) F3'H1; flavonoid 3'-monooxygenase (N)]
Figure imgf000072_0001
SEQ ID NO: 20
[Reference: >nta:107795677 K05280 flavonoid 3' -monooxygenase [EC: 1.14.14.82] | (RefSeq) F3'H1; flavonoid 3'-monooxygenase (A)]
Figure imgf000072_0002
SEQ ID NO: 21
[Reference: >ats:109731523 K13083 flavonoid 3',5'-h droxylase [EC:1.14.14.81] | (RefSeq) LOC109731523; flavonoid 3',5'-h droxylase 2-like (N)]
Figure imgf000073_0001
Figure imgf000073_0002
SEQ ID NO: 22
[Reference: >ats:109731523 K13083 flavonoid 3',5'-h droxylase [EC:1.14.14.81] | (RefSeq) LOC109731523; flavonoid 3',5'-h droxylase 2-like (A)]
Figure imgf000073_0003
Figure imgf000074_0001
SEQ ID NO: 23
[Reference: >Cannabis sativa prenyltransferase 1 (PT1) mRNA, complete cds
Figure imgf000074_0002
SEQ ID NO: 24
[Reference: >CsPTl (CsPTl) [PK28436]]
Figure imgf000074_0003
Figure imgf000075_0001
SEQ ID NO: 25
[Reference: >Cannabis sativa prenyltransferase 2 (PT2) Yeast codon optimized]
Figure imgf000075_0002
SEQ ID NO: 26
Figure imgf000075_0003
SEQ ID NO: 27 [Reference: > Cannabis sativa prenyltransferase 3 (PT3) Yeast codon optimized]
Figure imgf000076_0002
Figure imgf000076_0001
SEQ ID NO: 28 [Reference: >CsPT3 [PK17697]]
Figure imgf000076_0003
SEQ ID NO: 29
[Reference: >Cannabis sativa prenyltransferase 3 (PT4) Yeast codon optimized]
Figure imgf000076_0004
Figure imgf000077_0001
SRO ID NO: 30 [Reference: >CsPT4 [PK15523]]
Figure imgf000077_0002
SRO ID NO: 31
[Reference: >Cannabis sativa prenyltransferase Yeast codon optimized]
Figure imgf000077_0003
Figure imgf000078_0001
SEQ ID NO: 32 [Reference: >CsPT5 [PK11068]]
Figure imgf000078_0002
SEQ ID NO: 33
[Reference: >Cannabis sativa prenyltransferase 6 (PT6) Yeast codon optimized]
Figure imgf000078_0003
Figure imgf000079_0001
SEQ ID NO: 34
[Reference: >DAC76715.1 TPA_exp: prenyltransferase 6, partial [Cannabis sativa]] FQPSSL
Figure imgf000079_0002
Figure imgf000079_0003
SEQ ID NO: 35
[Reference: >Cannabis sativa prenyltransferase 7 (PT7) Yeast ccxlon optimized]
Figure imgf000079_0004
SEQ ID NO: 36 [Reference: >CsPT7 [PK29226]]
Figure imgf000080_0001
SEQ ID NO: 37
[Reference: >Cannabis sativa 2-acylphloroglucinol 4-p renyltrans ferase-like (LOC115707809) Yeast codon optimized]
Figure imgf000080_0002
SEQ ID NO: 38 [Reference: >CsPT8 [PK07278]]
Figure imgf000080_0003
Figure imgf000081_0001
SEQ ID NO: 39
[Reference: >GuA6DT [KJ123716] Yeast codon optimized]
Figure imgf000081_0002
SEQ ID NO: 40
[Reference: >GuA6DT [KJ123716]]
Figure imgf000081_0003
Figure imgf000082_0001
SEQ ID NO: 41
[Reference: >XM_030628986.1:103-1209 PREDICTED: Cannabis sativa xanthohumol 4-O- methyltransferase-like (LOCI 15701238), mRNA]
Figure imgf000082_0002
SEQ ID NO: 42
[Reference: >XP_030484846.1 xanthohumol 4-O-methyltransferase-like [Cannabis sativa]]
Figure imgf000082_0003
SEQ ID NO: 43
[Reference: >lcl | XM_002517911.3_cds_XP_002517957.1_1 |gene=LOC8262105] [db_xref=GeneID:8262105] [protein=xanthohumol 4-O-methyltransferase] [protein_id=XP_002517957.1] [location=32..1090] |gbkey=CDS]]
Figure imgf000083_0001
SEQ ID NO: 44
[Reference: >lcl | XM_002517911.3_prot_XP_002517957.1_1 [gene=LOC8262105] [db_xref=GeneID:8262105] [protein=xanthohumol 4-O-methyltransferase] [protein_id=XP_002517957.1] [location=32..1090] |gbkey=CDS]]
Figure imgf000083_0002
SEQ ID NO: 4S
[Reference: >lcl | MG996010.l_cds_AWH62810.1_1 [protein=0-methyltransferase] [protein_id=AWH62810.1] [location=1..1065] |gbkey=CDS]]
Figure imgf000084_0001
SEQ ID NO: 46
[Reference: >lcl | MG996010.l_prot_AWH62810.1_1 [protein=0-methyltransferase] [protein_id=AWH62810.1] [location=1..1065] |gbkey=CDS]]
Figure imgf000084_0002
SEQ ID NO: 47 [Reference: >lcl | XM_016025088.2_cds_XP_015880574.1_1 |gene=LOC107416580] [db_xref=GeneID:107416580] [protein=xanthohumol 4-O-methyltransferase-like] [protein_id=XP_015880574.1] [location= 134..1201] |gbkey=CDS]]
Figure imgf000085_0001
SEQ ID NO: 48
[Reference: >lcl |XM_016025088.2_prot_XP_015880574.1_l [gene=LPC107416580] [db_xref=GeneID:107416580] [protein=xanthohumol 4-O-methyltransferase-like] [protein_id=XP_015880574.1] [location= 134..1201] |gbkey=CDS]]
Figure imgf000085_0002
SEQ ID NO: 49 [Reference: >lcl | XM_010095992.l_cds_XP_010094294.1_1 [gene=LOC21387018] [db_xref=GeneID:21387018] [protein=xanthohumol 4'-0-methyltransferase] [protein_id=XP_010094294.1] [location=1..1071] [g)bkey=CDS]]
Figure imgf000086_0001
SEQ ID NO: 50
[Reference: >lcl | XM_010095992.l_prot_XP_010094294.1_1 [gene=LOC21387018] [db_xref=GeneID:21387018] [protein=xanthohumol 4'-0-methyltransferase] [protein_id=XP_010094294.1] [location=1..1071] [g)bkey=CDS]]
Figure imgf000086_0002
SEQ ID NO: 51 [Reference: >lcl | XM_024164200.l_cds_XP_024019968.1_1 |gene=LOC21387021] [db_xref=GeneID:21387021] [protein=xanthohumol 4'-0-methyltransferase] [protein_id=XP_024019968.1] [location= 150..1226] |gbkey=CDS]]
Figure imgf000087_0001
SEQ ID NO: 52
[Reference: >lcl | XM_024164200.l_prot_XP_024019968.1_1 [gene=LOC21387021] [db_xref=GeneID:21387021] [protein=xanthohumol 4'-0-methyltransferase] [protein_id=XP_024019968.1] [location= 150..1226] |gbkey=CDS]]
Figure imgf000087_0002

Claims

1. A genetically-modified cell capable of producing a flavonoid or flavonoid derivative comprising at least one heterologous polynudeotide encoding an enzyme involved in a metabolic pathway that converts sugar to a flavonoid or flavonoid derivative.
2. The cell of daim 1, comprising at least two heterologous polynucleotides, each encoding an enzyme involved in a metabolic pathway that converts sugar to a flavonoid or flavonoid derivative, wherein the encoded enzymes are operably connected along the metabolic pathway.
3. The cell of daim 1 or 2, wherein the flavonoid or flavonoid derivative is a flavone, flavanone, isoflavonoid, flavonol, dihydroflavonols, anthocyanidin.
4. The cell of claim 1 or 2, wherein the flavonoid or flavonoid derivative is pinocembrin, naringenin, eriodictyl, homoeriodictyl, or a functional isomer of one of the foregoing.
5. The cell of claim 1 or 2, wherein the flavonoid or flavonoid derivative is cinnamic acid, p- coumaric add, caffdc acid, ferulic add, p-coumaroyl-CoA, caffeoyl-CoA, feruloyl-CoA., dnnamoyl- CoA, pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, homoeriodictyl chalcone, or a functional isomer of one of the foregoing.
6. The cell of claim 1 or 2, wherein the flavonoid or flavonoid derivative is a cannflavin or functional isomer thereof.
7. The cell of claim of 6, wherein the cannflavin is cannflavin A, cannflavin B, isocannflavin A, or isocannflavin B.
8. The cdl of claim of 7, wherein the cannflavin is isocannflavin B.
9. The cell of any one of claims 1—8, wherein the encoded enzyme is phenylalanine/ tyrosine ammonia-lyase (PAL); dnnamate-4-h droxylase (C4H); 4-coumarate-CoA ligase (4CL); chalcone synthase (CHS); chalcone isomerase (CHI); flavanone 3-h droxylase (F3H); dihydroflavonol 4- reductase (DFR); flavonoid 3’-h droxylase (R3Ή); flavonoid 3’, 5' droxylase (R3'5Ή); a C. sativa prenyltransferase (CsPT); or a C sativa O-methyltransferase (CsOMI).
10. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that converts glucose into phenylalanine.
11. The cell of any one of claims 1-9, wherein the encoded enzyme is involved in the metabolic pathway that converts phenylalanine into cinnamic acid.
12. The cell of any one of claims 1-9, wherein the encoded enzyme is involved in the metabolic pathway that converts cinnamic add into p-coumaric add, caffeic add, or ferulic add.
13. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that converts: p-coumaric add into p-coumaroyl-CoA; caffeic add into caffeoyl-CoA; or ferulic acid into feruloyl-CoA.
14. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that converts cinnamic add into cinnamoyl-CoA.
15. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that catalyzes the addition of three molecules of malonyl-CoA to one molecule of dther dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA to yield, respectively, either pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone.
16. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that converts: pinocembrin chalcone into pinocembrin or a functional isomer thereof; naringen chalcone (tetrahydroxychalcone) into naringenin or a functional isomer thereof; eriodictyl chalcone into eriodictyl or a functional isomer thereof; or homoeriodictyl chalcone into homoeriodictyl or a functional isomer thereof.
17. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that adds a prenyl moiety to a flavonoid precursor.
18. The cell of claim 17, wherdn the encoded enzyme is a phenyl transferase obtained or derived from C. saliva.
19. The cell of any one of claims 1-9, wherdn the encoded enzyme is involved in the metabolic pathway that adds a methyl group to a flavonoid precursor.
20. The cell of claim 17, wherein the encoded enzyme is a methyl transferase obtained or derived from C. sativa.
21. The cdl of any one of claims 1-20, wherein the cell is a bacterium or a yeast cell.
22. The cell of any one of claims 1—21, wherein the cell is Saccharomyces cerevisiae.
23. A method of making a c, the method comprising:
(a) contacting a substrate with the genetically-modified cell of any one of claims 1-22; and
(b) growing the cell to make a flavonoid or flavonoid derivative.
24. The method of claim 23, further comprising isolating the flavonoid or flavonoid derivative from the cell.
25. The use of a flavonoid or flavonoid derivative made using the method of claim 23 or 24 for the manufacture of a medicament for the treatment of a disease or a symptom of a disease.
26. The use of claim 25, wherein the disease or symptom of a disease is cancer.
27. The use of claim 26, wherein the cancer is pancreatic cancer.
28. A medicament comprising a flavonoid or flavonoid derivative made using the method of claim 23 or 24.
29. A method of treating a disease or symptom of a disease comprising administering a flavonoid or flavonoid derivative made using the method of claim 23 or 24 to a subject in need thereof.
30. The method of claim 29, wherein the disease or symptom of a disease is cancer.
31. The method of claim 30, wherein the cancer is pancreatic cancer.
32. An isolated polynucleotide encoding at least one enzyme involved in a metabolic pathway that converts sugar to a flavonoid or flavonoid derivative.
33. The polynucleotide of claim 32, wherein the encoded enzyme is phenylalanine/ tyrosine ammonia-lyase (PAL); dnnamate-4-h droxylase (C4H); 4-coumarate-CoA ligase (4CL); chalcone synthase (CHS); chalcone isomerase (CHI); flavanone 3-h droxylase (F3H); dihydroflavonol 4- reductase (DFR); flavonoid 3’-h droxylase (F3Ή); flavonoid 3’, 5' droxylase (F3'5Ή); a C. sativa prenyltransferase (CsPT); or a C sativa O-methyltransferase (CsOMT).
34. The polynucleotide of claim 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts glucose into phenylalanine.
35. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts phenylalanine into cinnamic acid.
36. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts cinnamic add into p-coumaric acid, caffeic acid, or ferulic acid.
37. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts: p-coumaric add into p-coumaroyl-CoA; caffeic add into caffeoyl-CoA; or ferulic add into feruloyl-CoA.
38. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts cinnamic add into dnnamoyl-CoA.
39. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that catalyzes the addition of three molecules of malonyl-CoA to one molecule of either dnnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA to yield, respectively, either pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone.
40. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that converts: pinocembrin chalcone into pinocembrin or a functional isomer thereof; naringen chalcone (tetrahydroxychalcone) into naringenin or a functional isomer thereof; eriodictyl chalcone into eriodictyl or a functional isomer thereof; or homoeriodictyl chalcone into homoeriodictyl or a functional isomer thereof.
41. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that adds a prenyl moiety to a flavonoid precursor.
42. The polynudeotide of daim 41, wherein the encoded enzyme is a phenyl transferase obtained or derived from C. sativa.
43. The polynudeotide of any one of claims 32 or 33, wherein the encoded enzyme is involved in the metabolic pathway that adds a methyl group to a flavonoid precursor.
44. The polynudeotide of claim 43, wherein the encoded enzyme is a methyl transferase obtained or derived from C. sativa.
45. A vector comprising a nudeic add encoding at least one enzyme involved in a metabolic pathway that converts sugar to a flavonoid or flavonoid derivative.
46. The vector of claim 45, wherein the encoded enzyme is phenylalanine/ tyrosine ammonia- lyase (PAL); cinnamate-4-h droxylase (C4H); 4-coumarate-CoA ligase (4CL); chalcone synthase (CHS); chalcone isomerase (CHI); 3- flavanone 3-h droxylase (F3H); dihydroflavonol 4-reductase (DFR); flavonoid 3’-h droxylase (R3Ή); flavonoid 3’, 5' droxylase (R3'5Ή); a C. sativa prenyltransferase (CsPT); or a C. sativa O-methyltransferase (CsOMT).
47. The vector of claim 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts glucose into phenylalanine.
48. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts phenylalanine into cinnamic acid.
49. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts cinnamic acid into p-coumaric acid, caffeic add, or ferulic acid.
50. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts: p-coumaric add into p-coumaroyl-CoA; caffeic add into caffeoyl- CoA; or ferulic add into feruloyl-CoA.
51. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts cinnamic acid into dnnamoyl-CoA.
52. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that catalyzes the addition of three molecules of malonyl-CoA to one molecule of either cinnamoyl-CoA, p-coumaroyl-CoA, caffeoyl-CoA, or feruloyl-CoA to yield, respectively, either pinocembrin chalcone, naringen chalcone (tetrahydroxychalcone), eriodictyl chalcone, or homoeriodictyl chalcone.
53. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that converts: pinocembrin chalcone into pinocembrin or a functional isomer thereof; naringen chalcone (tetrahydroxychalcone) into naringenin or a functional isomer thereof; eriodictyl chalcone into eriodictyl or a functional isomer thereof; or homoeriodictyl chalcone into homoeriodictyl or a functional isomer thereof.
54. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that adds a prenyl moiety to a flavonoid precursor.
55. The vector of claim 54, wherein the encoded enzyme is a phenyl transferase obtained or derived from C. saliva.
56. The vector of any one of claims 45 or 46, wherein the encoded enzyme is involved in the metabolic pathway that adds a methyl group to a flavonoid precursor.
57. The vector of claim 56, wherein the encoded enzyme is a methyl transferase obtained or derived from C. sativa.
58. A composition, or a pharmaceutically-acceptable derivative or prodrug thereof, comprising a flavonoid or flavonoid derivative produced by a method of claim 23 or 24.
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