BACKGROUND OF THE INVENTION

Field of the Invention

[0001] The invention disclosed herein relates generally to the field of genetic engineering. Particularly, the invention disclosed herein provides methods for biosynthetic production of phenylpropanoids and phenylpropanoid derivatives, such as chalcones and stilbenes.

Description of Related Art

[0002] Phenylpropanoids are a diverse family of phenolic compounds produced biosynthetically in plants from phenolic amino acid precursors. Phenylpropanoids and their derivatives have desirable applications, for example in the food and healthcare industries.

[0003] An exemplary phenylpropanoid derivative is naringenin, a compound that is also an intermediate in the production of downstream phenylpropanoid derivatives. Naringenin has the chemical structure:

[0004] Naringenin is produced naturally in plants, and also biosynthetically in cells genetically engineered with components of a flavonoid biosynthesis pathway (see e.g., Koopman er a/., (2012) Microbial Cell Factories 2012, 11 :155). For example, cells engineered to produce coumaroyl-CoA are further engineered with recombinant genes expressing proteins that convert coumaroyl-CoA to naringenin.

[0005] Another exemplary phenylpropanoid derivative is the stilbene resveratrol, which is also an intermediate in the production of other downstream phenylpropanoid derivatives. Resveratrol has the chemical structure:

Resveratrol is also produced using a coumaroyl-CoA precursor molecule.

[0006] Generally, stilbenes, including resveratrol, and chalcones are produced in plants and yeast through the phenyipropanoid pathway as illustrated by the reactions shown in Figure 1 and as described in U.S. 2008/0286844, which is incorporated by reference in its entirety herein.

[0007] In yeast, the starting metabolites are malonyl-CoA and either phenylalanine or tyrosine. The amino acid L-phenylalanine is converted into trans-cinnamic acid through non- oxidative deamination by L-phenylalanine ammonia lyase (PAL). Next, trans-cinnamic acid is hydroxylated at the para-position to 4-coumaric acid (4-hydroxycinnamic acid) by cinnamate-4- hydroxylase (C4H), a cytochrome P450 monooxygenase enzyme, in conjunction with NADPH:cytochrome P450 reductase (CPR). Alternatively, the amino acid L-tyrosine is directly converted into 4-coumaric acid by tyrosine ammonia lyase (TAL). The 4-coumaric acid from either pathway is subsequently activated to 4-coumaroyl-CoA by the action of 4-coumarate-CoA ligase (4CL). Within the phenyipropanoid pathway, 4-coumaroyl-CoA represents the key branching point from which phenyipropanoid derivatives, including chalcones and stilbenes, are derived. Stilbenes are synthesized via stilbene synthase (STS), also known as resveratrol synthase (RS), catalyzing condensation of a phenylpropane unit of 4-coumaroyl-CoA with malonyl-CoA, resulting in formation of resveratrol. Conversely, chalcones are synthesized via condensation of a phenylpropane unit of 4-coumaroyl-CoA with malonyl-CoA and chalcone synthase (CHS), resulting in the formation of tetrahydroxychalcone.

[0008] Current methods of producing naringenin, resveratrol, and other phenyipropanoid derivatives are limited by pathways that compete for phenylpropanoids, such as coumaroyl- CoA, as a substrate. For example, cells engineered to produce naringenin also produce phloretic acid by an unknown mechanism (see e.g., Koopman et a/., (2012) Microbial Cell Factories 2012, 1 1 :155). Phloretic acid is a side product of the phenyipropanoid pathway. It is a dihydro-phenylpropanoid, which are converted from a phenyipropanoid (e.g., p-coumaroyl-CoA) to a dihydrophenylpropanoid (e.g., p-dihydrocoumaroyl-CoA). However, the enzymes responsible for producing dihydrophenylpropanoids (and reducing naringenin production) are presently unknown.

[0009] Phenylalanine ammonia lyase (PAL), which converts L-phenylalanine to ammonia and trans-cinnamic acid, and Tyrosine ammonia lyase (TAL), which converts L-tyrosine into p- coumaric acid, are both members of the aromatic amino acid lyase family. The third member of the aromatic amino acid lyase family is histidine ammonia lyase (HAL), which converts histidine to trans-urocanic acid. Most ammonia lyases have an affinity to both phenylalanine and tyrosine, with a strong preference for phenylalanine. These enzymes are called PAL/TALs. Watts, K.T. et al. (2006), identified a single active site residue as responsible for substrate specificity, and reported that replacing the active site residue His89 with Phe in Rhodobacter sphaeroides TAL switched its substrate selectivity from tyrosine to phenylalanine (Watts, K.T. et al. (2006) Chemistry & Biology 13, 1317-1326).

[0010] Generally, PAL is a more active enzyme than TAL and, therefore, has been preferred for the production of phenylpropanoids in yeast strains such as Saccharomyces cerevisiae (see e.g. U.S. Pat. No. 8,895,287). However, finding and introducing an active, specific TAL in strains that produce phenylpropanoids and phenylpropanoid derivatives, such as S. cerevisiae, may result in a substantial increase in the carbon flux going through the phenylpropanoid pathway and, therefore, in an increased production of phenylpropanoids or phenylpropanoid derivatives, including chalcones and stilbenes.

[0011] Expression of the heterologous phenylpropanoid pathway through use of both PAL and TAL has been reported (see Koopman, F. ef al., 2012, Microbial Cell Factories, 11 :155). Koopman, F. et al. (2012) id., used TAL from Rhodobacter capsulatus (RcTAL). However, even after deregulating synthesis of aromatic amino acids, thereby increasing the available tyrosine, RcTAL shows very poor activity and, thus, cannot be used in industrial applications. Accordingly, there remains a need for expression of active, specific TALs in yeast, which produces high yields of phenylpropanoids or phenylpropanoid derivatives.

SUMMARY OF THE INVENTION

[0012] It is against the above background that the present invention provides certain advantages and advancements over the prior art.

[0013] Although this invention disclosed herein is not limited to specific advantages or functionality, the invention disclosed herein provides a recombinant host comprising a recombinant gene encoding a tyrosine ammonia lyase (TAL) polypeptide, wherein the host is capable of producing a phenylpropanoid or a phenylpropanoid derivative compound, and wherein the TAL polypeptide uses tyrosine as a preferred substrate.

[0014] The invention further provides a method of producing a phenylpropanoid or a phenylpropanoid derivative compound, comprising growing a recombinant host as described herein in a culture medium under conditions in which the recombinant genes are expressed, wherein the phenylpropanoid or the phenylpropanoid derivative compound is synthesized by the recombinant host.

[0015] In some aspects, the gene encoding the TAL polypeptide encodes a polypeptide having the amino acid sequence set forth in SEQ ID NO:31.

[0016] In some aspects, the gene encoding the TAL polypeptide encodes a polypeptide having at least 65% identity to the amino acid sequence set forth in SEQ ID NO:31.

[0017] In some aspects, the gene encoding the TAL polypeptide is overexpressed in the recombinant host.

[0018] In some aspects, the recombinant host containing the TAL gene is capable of producing an increased yield of a phenylpropanoid or a phenylpropanoid derivative compound, as compared to a recombinant host not comprising the gene encoding a TAL polypeptide.

[0019] In some aspects, the recombinant host containing the TAL gene produces an increased yield of a phenylpropanoid or a phenylpropanoid derivative compound, as compared to a recombinant host not comprising the gene encoding a TAL polypeptide.

[0020] In some aspects, the recombinant host containing the TAL gene produces an increased yield of one or more of (1 ) resveratrol and (2) coumaric acid, as compared to a recombinant host not comprising the gene encoding the TAL polypeptide.

[0021] In some aspects of the recombinant host or methods disclosed herein, the recombinant host further comprises a recombinant gene encoding:

(a) a stilbene synthase (STS) polypeptide; or

(b) a chalcone synthase (CHS) polypeptide.

[0022] In some aspects of the recombinant host or methods disclosed herein, the recombinant host further comprises one or more of:

(a) a gene encoding a L-phenylalanine ammonia lyase (PAL) polypeptide;

(b) a gene encoding a cinnamate-4-hydroxylase (C4H) polypeptide;

(c) a gene encoding a NADPHxytochrome P450 reductase polypeptide; (d) a gene encoding a 4-coumarate-CoA ligase (4CL) polypeptide; or

(e) a gene encoding a chalcone isomerase (CHI) polypeptide

wherein at least one of the genes is a recombinant gene.

[0023] In some aspects of the recombinant host or methods disclosed herein, the phenylpropanoid compound is coumaric acid.

[0024] In some aspects of the recombinant host or methods disclosed herein, the phenylpropanoid derivative compound is a stilbenoid compound or a chalcone compound.

[0025] In some aspects of the recombinant host or methods disclosed herein, the stilbene is resveratrol or a resveratrol derivative.

[0026] In some aspects of the recombinant host or methods disclosed herein, the chalcone is naringenin or a naringenin derivative.

[0027] In some aspects of the recombinant host or methods disclosed herein, the recombinant host comprises a microorganism that is a yeast cell, a plant cell, a mammalian cell, an insect cell, a fungal cell, or a bacterial cell.

[0028] In some aspects of the recombinant host or methods disclosed herein, the bacterial cell comprises Escherichia bacteria cells, Lactobacillus bacteria cells, Lactococcus bacteria cells, Cornebacterium bacteria cells, Acetobacter bacteria cells, Acinetobacter bacteria cells, or Pseudomonas bacterial cells.

[0029] In some aspects of the recombinant host or methods disclosed herein, the yeast cell is a cell from Saccharomyces cerevisiae, Schizosaccharomyces pombe, Yarrowia lipolytica, Candida glabrata, Ashbya gossypii, Cyberlindnera jadinii, Pichia pastoris, Kluyveromyces lactis, Hansenula polymorpha, Candida boidinii, Arxula adeninivorans, Xanthophyllomyces dendrorhous, or Candida albicans species.

[0030] In some aspects of the recombinant host or methods disclosed herein, the yeast cell is a Saccharomycete.

[0031] In some aspects of the recombinant host or methods disclosed herein, the yeast cell is a cell from the Saccharomyces cerevisiae species.

[0032] In some aspects, the method disclosed herein further comprises recovering the phenylpropanoid or the phenylpropanoid derivative compound from the culture media.

[0033] In some aspects, the method disclosed herein further comprises isolating the phenylpropanoid or the phenylpropanoid derivative compound from the culture medium.

[0034] These and other features and advantages will be more fully understood from the following detailed description taken together with the accompanying claims. BRIEF DESCRIPTION OF THE DRAWINGS

[0035] The following detailed description can be best understood when read in conjunction with the following drawings in which;

[0036] Figure 1 shows the phenylpropanoid pathway branching from p-coumaroyl-CoA to a variety of phenylpropanoid derivatives. Two branches of the pathway are shown, with exemplary phenylpropanoid derivatives produced by two PKS type III enzymes: chalcone synthase (CHS) and stilbene synthase (STS). Also shown is a branch producing dihydrophenylpropanoid derivatives via the action of a reductase enzyme. Other enzyme abbreviations are:

phenylalanine lyase (PAL or TAL); cinnamate-4-hydroxylase (C4H) which requires the activity of a reductase (CPR); 4-Coumaroyl-CoA ligase (4CL); and chalcone isomerase (CHI).

[0037] Figure 2 shows the PAL and TAL activity of fifteen different enzymes, via production of products of the phenylpropanoid pathway derived from either cinnamic acid as a result of PAL activity or coumaric acid as a result of TAL activity.

[0038] Figure 3 shows production of resveratrol and secondary metabolites in a resveratrol- producing yeast strain overexpressing Asa/ TAL and the direct parental resveratrol-producing yeast strain which contained all necessary genes leading to resveratrol production (control). Secondary metabolites are likely produced due to conversion of starting metabolites malonyl- CoA, phenylalanine and/or tyrosine into intermediates (e.g., coumaric acid) and side produces (e.g., phloretic acid), rather than resveratrol.

DETAILED DESCRIPTION

[0039] All publications, patents and patent applications cited herein are hereby expressly incorporated by reference for all purposes.

[0040] Because many phenylpropanoid derivatives are useful as, inter alia, pharmaceutical compounds, there is a need for efficient methods of their production. For example, the chalcone naringenin, and the stilbene resveratrol, are useful for controlling blood sugar levels, as well as other potential uses to improve human health.

[0041] Accordingly, provided herein are materials and methods useful for biosynthesis of phenylpropanoid derivatives, including chalcones and stilbenes. In certain embodiments, the disclosure provides recombinant hosts and methods for biosynthesis of naringenin and other chalcones. In alternative embodiments, the disclosure provides recombinant hosts and methods for biosynthesis of resveratrol and other stilbenes.

[0042] Before describing the disclosed methods and compositions in detail, a number of terms will be defined. As used herein, the singular forms "a", "an", and "the" include plural referents unless the context clearly dictates otherwise. For example, reference to a "nucleic acid" means one or more nucleic acids.

[0043] It is noted that terms like "preferably," "commonly," and "typically" are not utilized herein to limit the scope of the claimed invention or to imply that certain features are critical, essential, or even important to the structure or function of the claimed invention. Rather, these terms are merely intended to highlight alternative or additional features that can or cannot be utilized in a particular embodiment of this invention.

[0044] For the purposes of describing and defining the present invention it is noted that the terms "increase", "increases", "increased", "greater", 'higher", and "lower" are utilized herein to represent non-quantitative comparisons, values, measurements, or other representations to a stated reference or control.

[0045] For the purposes of describing and defining the present invention, it is noted that the terms such as "preferred substrate" and "primary substrate" are interchangeable and utilized herein to represent non-quantitative comparisons, values, measurements, or other representations regarding stated substrates.

[0046] For the purposes of describing and defining this invention it is noted that the terms "substantial" and "substantially" are utilized herein to represent the inherent degree of uncertainty that can be attributed to any quantitative comparison, value, measurement, or other representation. The terms "substantial" and "substantially" are also utilized herein to represent the degree by which a quantitative representation can vary from a stated reference without resulting in a change in the basic function of the subject matter at issue.

[0047] Methods well known to those skilled in the art can be used to construct the genetic expression constructs and recombinant cells disclosed herein. These methods include in vitro recombinant DNA techniques, synthetic techniques, in vivo recombination techniques, and polymerase chain reaction (PCR) techniques. See, for example, techniques as described in Maniatis et al., 1989, MOLECULAR CLONING: A LABORATORY MANUAL, Cold Spring Harbor Laboratory, New York; Ausubel et al„ 1989, CURRENT PROTOCOLS IN MOLECULAR BIOLOGY, Greene Publishing Associates and Wiley Interscience, New York, and PCR Protocols: A Guide to Methods and Applications (Innis et al., 1990, Academic Press, San Diego, CA).

[0048] As used herein, the terms "polynucleotide", "nucleotide", "oligonucleotide", and "nucleic acid" can be used interchangeably to refer to nucleic acid comprising DNA, RNA, derivatives thereof, or combinations thereof.

[0049] As used herein, the terms "microorganism," "microorganism host," "microorganism host cell," "recombinant host," "host cell," and "recombinant host cell" can be used interchangeably. As used herein, the term "recombinant host" is intended to refer to a host, the genome of which has been augmented by at least one DNA sequence. Such DNA sequences include but are not limited to genes or DNA sequences that are not naturally present, that are not normally transcribed into RNA, nor translated into protein ("expressed") natively in the cell, and other genes or DNA sequences one desires to introduce into a host. It will be appreciated that typically the genome of a recombinant host described herein is augmented through stable introduction of one or more recombinant genes. Generally, introduced DNA is not originally resident in the host that is the recipient of the DNA, but it is within the scope of this disclosure to isolate a DNA segment from a given host, and to subsequently introduce one or more additional copies of that DNA into the same host, e.g., to enhance production of the product of a gene or alter the expression pattern of a gene. In some instances, the introduced DNA will modify or even replace an endogenous gene or DNA sequence by, e.g., homologous recombination or site-directed mutagenesis. Suitable recombinant hosts include microorganisms.

[0050] As used herein, the term "gene" refers to a polynucleotide unit comprised of at least one of the DNA sequences disclosed herein, or any DNA sequences encoding the amino acid sequences disclosed herein, or any DNA sequence that hybridizes to the complement of the coding sequence disclosed herein. Preferably, the term includes coding and non-coding regions, and preferably all sequences necessary for normal gene expression including promoters, enhancers, and other regulatory sequences.

[0051] As used herein, the term "recombinant gene" refers to a gene or DNA sequence that is introduced into a recipient host, regardless of whether the same or a similar gene or DNA sequence may already be present in such a host. "Introduced," or "augmented" in this context, is known in the art to mean introduced or augmented by the hand of man. Thus, a recombinant gene can be a DNA sequence from another species, or can be a DNA sequence that originated from or is present in the same species, but has been incorporated into a host by recombinant methods to form a recombinant host. It will be appreciated that a recombinant gene that is introduced into a host can be identical to a DNA sequence that is normally present in the host being transformed, and is introduced to provide one or more additional copies of the DNA to thereby permit overexpression or modified expression of the gene product of that DNA. The recombinant genes are particularly encoded by cDNA.

[0052] As used herein, the term "engineered biosynthetic pathway" refers to a biosynthetic pathway that occurs in a recombinant host, as described herein, and does not naturally occur in the host. In some embodiments, the engineered biosynthetic pathway comprises enzymes naturally produced by the host, wherein in certain embodiments the extent and amount of expression of the genes encoding these enzymes are altered in the recombinant host; in some embodiments these enzymes are underexpressed, or their expression is eliminated, in the recombinant host.

[0053] As used herein, the term "endogenous" gene refers to a gene that originates from and is produced or synthesized within a particular organism, tissue, or cell.

[0054] As used herein, the terms "heterologous sequence" and "heterologous coding sequence" are used to describe a sequence derived from a species other than the recombinant host. In some embodiments, the recombinant host is an S. cerevisiae cell, and a heterologous sequence is derived from an organism other than S. cerevisiae. A heterologous coding sequence, for example, can be from a prokaryotic microorganism, a eukaryotic microorganism, a plant, an animal, an insect, or a fungus different than the recombinant host expressing the heterologous sequence. In some embodiments, a coding sequence is a sequence that is native to the host.

[0055] It will be appreciated that because of the degeneracy of the genetic code, a number of nucleic acids can encode a particular polypeptide; i.e., for many amino acids, there is more than one nucleotide triplet that serves as the codon for the amino acid. Thus, codons in the coding sequence for a given polypeptide can be modified such that optimal expression in a particular microorganism is obtained, using appropriate codon bias tables for that microorganism. Nucleic acids may also be optimized to a GC-content preferable to a particular microorganism, and/or to reduce the number of repeat sequences. As isolated nucleic acids, these modified sequences can exist as purified molecules and can be incorporated into a vector or a virus for use in constructing modules for recombinant nucleic acid constructs. In addition, heterologous nucleic acids can be modified for increased or even optimal expression in the relevant microorganism. Thus, in some embodiments of the methods and compositions disclosed herein, heterologous nucleic acids have been codon optimized for expression in the relevant microorganism. Codon optimization may be performed by routine methods known in the art (See e.g., Welch, M., et al. (2011 ), Methods in Enzymology 498:43-66).

Chalcone and Stilbene Synthesis

[0056] As used herein, the terms "chalcone" and "chalconoid" are interchangeable and refer to derivatives the compound of formula (I):

wherein formula (I) may be substituted at one or more suitable positions. Exemplary substituents include, but are not limited to, halogen, cyano, nitro, C-|-C ₆ alkyl, C C ₆ haloalkyl, C C ₆ hydroxyalkyl, hydroxy, C C ₆ alkoxy, thiol, C^Ce alkylthio, amino, CrC ₆ alkyl amino, di- C C ₆ alkyl amino, carboxyl, C C ₆ alkoxycarbonyl, amido, and glycosyl.

[0057] As used herein, the terms "stilbene" and "stilbenoid" are interchangeable and refer to compounds based on the compound of formula (II):

wherein formula (II) may be substituted at one or more suitable positions. Exemplary substituents include, but are not limited to, halogen, cyano, nitro, C C ₆ alkyl, C C ₆ haloalkyl, Ci-C ₆ hydroxyalkyl, hydroxy, C C ₆ alkoxy, thiol, C C ₆ alkylthio, amino, C-i-C ₆ alkyl amino, di- C C ₆ alkyl amino, carboxyl, C C ₆ alkoxycarbonyl, amido, and glycosyl.

[0058] As used herein, the term "phenylpropanoid" refers to compounds based on a 3- phenylprop-2-enoate backbone. Examples of such compounds include, but are not limited to, cinnamic acid, coumaric acid, caffeic acid, ferulic acid, 5-hydroxyferulic acid, sinapinic acid, cinnamoyl-CoA, p-coumaroyl-CoA, and the like.

[0059] As used herein, the term "phenylpropanoid derivative" refers to any compound derived from, synthesized from, or biosynthesized from a phenylpropanoid; i.e. a phenylpropanoid derivative includes any compound for which a phenylpropanoid compound is a precursor or intermediate. Examples of phenylpropanoid derivatives include, but are not limited to, stilbenoid compounds and chalcone compounds. Specific examples of phenylpropanoid derivatives include, but are not limited to, naringenin, resveratrol, pinosylvin, pinocembrin chalcone, and pinocembrin.

[0060] As used herein, the term "dihydrophenylpropanoid" refers to compounds based on a phenylpropanoate backbone. Examples of such compounds include, but are not limited to, dihydrocinnamic acid, phloretic acid, 3,4-dihydroxyhydrocinnamic acid, hydroferulic acid, dihydrocoumaroyl-CoA, dihydrocinnamoyl-CoA, and the like.

[0061] As used herein, the term "dihydrophenylpropanoid derivative" refers to any compound derived from, synthesized from, or biosynthesized from a dihydrophenylpropanoid; i.e. a dihydrophenylpropanoid derivative includes any compound for which a dihydrophenylpropanoid compound is a precursor or intermediate. Examples of dihydrophenylpropanoid derivatives include, but are not limited to, dihydrostilbenoid compounds and dihydrochalcone compounds. Specific examples of dihydrophenylpropanoid derivatives include, but are not limited to, phloretin, phlorizin, dihydropinosylvin, dihydropinosylvincarboxylate, 3-O-methyldihydropinosylvincarboxylate, 4-isoprenyl-3-0- methyldihydropinosylvincarboxylate (amorfrutin 1 ), 3-O-methyldihydropinosylvin, 4-isoprenyl-3- O-methyldihydropinosylvin (amorfrutin 2), 5-hydroxy-lunularic acid, and dihydroresveratrol.

[0062] As used herein, the terms "phenylpropanoid pathway," "phenylpropanoid derivative pathway," "phenylpropanoid derivative synthesis pathway," and "phenylpropanoid derivative biosynthesis pathway" are interchangeable and refer to any biosynthesis pathway in which a phenylpropanoid is a precursor or intermediate.

[0063] "Optional" or "optionally" means that the subsequently described event or circumstance may or may not occur, and that the description includes instances where said event or circumstance occurs and instances in which it does not.

[0064] As used herein, "reduced expression" refers to expression of a gene or protein at a level lower than the native expression of the gene or protein. For example, in some embodiments the activity of a reductase is reduced by decreasing the amount of protein product, or expression, of a gene encoding the reductase.

[0065] Reduction or elimination (i.e., disruption) of expression of a gene can be accomplished by any known method, including insertions, missense mutations, frame shift mutations, deletion, substitutions, or replacement of a DNA sequence, or any combinations thereof. Insertions include the insertion of the entire genes, which may be of any origin. Reduction or elimination of gene expression can, for example, comprise altering or replacing a promoter, an enhancer, or splice site of a gene, leading to inhibition of production of the normal gene product partially or completely. In some embodiments, reduction or elimination of gene expression comprises altering the total level of the protein product expressed in the cell or organism. In other embodiments, disruption of a gene comprises reducing or eliminating the activity of the protein product of the gene in a cell or organism. In some embodiments of the disclosure, the disruption is a null disruption, wherein there is no significant expression of the gene. In some embodiments the disruption of a gene in a host or organism occurs on both chromosomes, in which case it is a homozygous disruption. In other embodiments the disruption of a gene in a host or organism occurs on only one chromosome, leaving the other chromosomal copy intact, in which case it is a heterozygous gene disruption. In still other embodiments each copy of a gene in a host or organism is disrupted differently.

[0066] Reduction or elimination of gene expression may also comprise gene knock-out or knock-down. A "gene knock-out" refers to a cell or organism in which the expression of one or more genes is eliminated. A "gene knock-down" refers to a cell or organism in which the level of one or more genes is reduced, but not completely eliminated.

[0067] In some embodiments, the recombinant host further comprises one or more polypeptides of a phenylpropanoid derivative biosynthesis pathway. In some embodiments, recombinant genes are provided that catalyze formation of intermediates in the biosynthesis of chalcones, stilbenes, or other phenylpropanoid derivatives. Intermediates comprise, inter alia, cinnamic acid, cinnamoyl-CoA, p-coumaric acid, p-coumaroyl CoA, naringenin, and resveratrol.

[0068] In some embodiments, a L-phenylalanine ammonia lyase (PAL) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said PAL is a PAL (EC 4.3.1.5) from a plant belonging to the genus of Arabidopsis, Brassica, Citrus, Phaseolus, Pinus, Populus, Solanum, Prunus, Vitis, Zea, Agastache, Ananas, Asparagus, Bromheadia, Bambusa, Beta, Betula, Cucumis, Camellia, Capsicum, Cassia, Catharanthus, Cicer, Citrullus, Coffea, Cucurbita, Cynodon, Daucus, Dendrobium, Dianthus, Digitalis, Dioscorea, Eucalyptus, Gallus, Ginkgo, Glycine, Hordeum, Helianthus, Ipomoea, Lactuca, Lithospermum, Lotus, Lycopersicon, Medicago, Malus, Manihot. Medicago, Mesembryanthemum, Nicotiana, Olea, Oryza, Pisum, Persea, Petroselinum, Phalaenopsis, Phyllostachys, Physcomitrella, Picea. Pyrus. Quercus, Raphanus, Rehmannia, Rubus, Sorghum, Sphenostylis, Stellaria. Stylosanthes. Triticum, Trifolium, Triticum, Vaccinium, Vigna, or Zinnia or a microorganism belonging to the genus Agaricus, Aspergillus, Ustilago, Rhodobacter, or Rhodotorula. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the PAL is an Arabidopsis thaliana PAL, e.g., A. thaliana PAL2 (SEQ ID NO: 15).

[0069] In some embodiments, a tyrosine ammonia lyase (TAL) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said TAL is capable of using both tyrosine and phenylalanine as substrates. In some embodiments, said TAL does not use phenylalanine as its primary or preferred substrate. In some embodiments, said TAL uses tyrosine as its primary or preferred substrate. In some embodiments, said TAL has a Km for Phenylalanine which is higher than its Km for Tyrosine and/or said TAL has a Kcat for Phenylalanine which is lower than its Kcat for Tyrosine. In some embodiments, said TAL is a TAL (EC 4.3.1.5) from yeast belonging to the genus Rhodotorula or a bacterium belonging to the genus Rhodobacter. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the TAL is a Rhodobacter capsulatus TAL, e.g., R. capsulatus TAL (SEQ ID NO. ). In some embodiments, the TAL is an Aeromonas salmonicida TAL, e.g., A. salmonicida subsp. salmonicida A449 (Asal) TAL (SEQ ID NO:31 ).

[0070] In some embodiments, a cinnamate 4-hydroxylase (C4H) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said C4H is a C4H (EC 1.14.13.1 1 ) from a plant belonging to the genus of Arabidopsis, Citrus, Phaseolus, Pin us. Populus, Solanum, Vitis, Zea, Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitreila, Ruta, Saccharum, or Vigna or from a microorganism belonging to the genus Aspergillus. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the C4H is Arabidopsis thaliana C4H (SEQ ID NO:2).

[0071] In some embodiments, a 4-coumarate-CoA ligase (4CL) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said 4CL can be a 4CL (EC 6.2.1.12) from a plant belonging to the genus of Abies, Arabidopsis, Brassica, Citrus, Larix, Phaseolus, Pinus, Populus, Solanum, Vitis, Zea, e.g., Z. mays, Agastache, Amorpha, Cathaya, Cedrus, Crocus, Festuca, Glycine, Juglans, Keteleeria, Lithospermum, Lolium, Lotus, Lycopersicon, Malus, Medicago, Mesembryanthemum, Nicotiana, Nothotsuga, Oryza, Pelargonium, Petroselinum, Physcomitrella, Picea. Prunus, Pseudolarix. Pseudotsuga. Rosa, Rubus, Ryza, Saccharum, Suaeda, The/lungiella, Triticum, or Tsuga, a microorganism belonging to the genus Aspergillus, Neurospora, Yarrowia, Mycosphaerella. Mycobacterium, Neisseria, Streptomyces, or Rhodobacter, or a nematode belonging to the genus Ancylostoma. Caenorhabditis. Haemonchus, Lumbricus, Meilodogyne. Strongyloidus, or Pristionchus. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the 4CL is an Arabidopsis thaliana 4CL, e.g., A. thaliana 4CL2 (SEQ ID NO:3).

[0072] In some embodiments, the disclosure provides a recombinant host engineered to express recombinant polypeptides that catalyze the formation of stilbenoids from p-coumaroyl- CoA. Thus, in some embodiments, recombinant host further comprises one or more stiibene synthase genes.

[0073] In some embodiments, a stiibene synthase (STS) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said STS is an STS (EC 2.3.1 .95) from a plant belonging to the genus of Arachis, Rheum. Vitis, Pinus, Piceea, Lilium, Eucalyptus, Parthenocissus. Cissus, Calochortus. Polygonum, Gnetum, Artocarpus, Nothofagus. Phoenix, Festuca, Carex, Veratrum, Bauhinia, or Pterolobium. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the STS is Vitis pseudoreticulata STS (SEQ ID NO:4).

[0074] In some embodiments, an NADPH: cytochrome P450 reductase (CPR) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said CPR is a CPR (EC 1.6.2.4) from a plant belonging to genus Arabidopsis, e.g., A. thaliana, a plant belonging to genus Citrus, e.g., Citrus x sinensis, or Citrus x paradisi, a plant belonging to genus Phaseolus, e.g., P. vulgaris, a plant belonging to genus Pinus, e.g., P. taeda, a plant belonging to genus Populus, e.g., P. deltoides, R. tremuloides, or R. trichocarpa, a plant belonging to genus Solanum, e.g., S. tuberosum, a plant belonging to genus Vitis, e.g., Vitis vinifera, a plant belonging to genus Zea, e.g., Z. mays, or other plant genera, e.g., Ammi, Avicennia, Camellia, Camptotheca, Catharanthus, Glycine, Helianthus, Lotus, Mesembryanthemum, Physcomitrella, Ruta, Saccharum, or Vigna. See, e.g., WO 2006/089898, which has been incorporated by reference in its entirety. In some embodiments, the CPR is an Arabidopsis thaliana CPR, e.g., A. thaliana ATR2 (SEQ ID NO:5). [0075] In some embodiments, the disclosure provides recombinant host engineered to express recombinant polypeptides that catalyze the formation of phenylpropanoid derivatives, such as chalcones and stilbenoids.

[0076] In some embodiments, a chalcone synthase (CHS) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, said CHS is a Hordeum vulgare CHS, e.g., H. vulgare CHS2 (SEQ ID NO:7).

[0077] In some embodiments, a chalcone isomerase (CHI) polypeptide can be expressed, overexpressed, or recombinantly expressed in said microorganism. In some embodiments, the CHI is a Petunia hybrida CHI, e.g., P. hybrida CHI1 (SEQ ID NO:9) or P. hybrida CHI2 (SEQ ID NO:11 ).

[0078] In another aspect, the disclosure provides methods of producing a chalcone or a stilbene compound, comprising growing a recombinant host as disclosed herein in a culture medium under conditions in which the recombinant genes are expressed, and wherein said compound is synthesized by the recombinant host.

[0079] In some embodiments, the methods of the disclosure are used to produce a chalcone compound. In some embodiments, the chalcone compound is naringenin or a naringenin derivative. In addition to naringenin, some embodiments disclosed herein are useful for producing other chalcones, e.g., Isoliquiritigenin (liquiritigenin chalcone), Butein (Butin chalcone), Pinocembrin chalcone, Eriodictyol chalcone and Homoeriodictyol chalcone.

[0080] In some embodiments, the methods of the disclosure are used to produce a stilbenoid compound. In some embodiments the stilbene compound is resveratrol. In addition to resveratrol, some embodiments of the present disclosure are useful for producing other stilbenoids, e.g. Piceatannol, Dihydroresveratrol, Resveratrol 3-O-glucoside (Piceid, polydatin), epsilon-Viniferin, delta-Viniferin and Pallidol.

[0081] In some embodiments, the methods of producing a chalcone or a stilbene compound further comprise harvesting the said compound. In some embodiments, the methods of producing a chalcone or a stilbene compound further comprise isolating said compound.

Functional Homologs

[0082] Functional homologs of the polypeptides described above may also be suitable for use in producing phenylpropanoid derivatives in a recombinant host as provided herein. A functional homolog is a polypeptide that has sequence similarity to a reference polypeptide, and that carries out one or more of the biochemical or physiological function(s) of the reference polypeptide. A functional homolog and the reference polypeptide can be a natural occurring polypeptide, and the sequence similarity can be due to convergent or divergent evolutionary events. As such, functional homologs are sometimes designated in the literature as homologs, or orthologs, or paralogs. Variants of a naturally occurring functional homolog, such as polypeptides encoded by mutants of a wild type coding sequence, can themselves be functional homologs. Functional homologs can also be created via site-directed mutagenesis of the coding sequence for a polypeptide, or by combining domains from the coding sequences for different naturally-occurring polypeptides ("domain swapping"). Techniques for modifying genes encoding functional polypeptides described herein are known and include, inter alia, directed evolution techniques, site-directed mutagenesis techniques and random mutagenesis techniques, and can be useful to increase specific activity of a polypeptide, alter substrate specificity, alter expression levels, alter subcellular location, or modify polypeptide-polypeptide interactions in a desired manner. Such modified polypeptides are considered functional homologs. The term "functional homolog" is sometimes applied to the nucleic acid that encodes a functionally homologous polypeptide.

[0083] Functional homologs can be identified by analysis of nucleotide and polypeptide sequence alignments. For example, performing a query on a database of nucleotide or polypeptide sequences can identify homologs of phenylpropanoid or phenylpropanoid derivative biosynthesis pathway polypeptides. Sequence analysis can involve BLAST, Reciprocal BLAST, or PSI-BLAST analysis of non-redundant databases using, e.g., a phenylalanine ammonia lyase, tyrosine ammonia lyase, chalcone isomerase, or stilbene synthase amino acid sequence as the reference sequence. Amino acid sequence is, in some instances, deduced from the nucleotide sequence. Those polypeptides in the database that have greater than 40% sequence identity are candidates for further evaluation for suitability as a phenylpropanoid derivative biosynthesis pathway polypeptide. Amino acid sequence similarity allows for conservative amino acid substitutions, such as substitution of one hydrophobic residue for another or substitution of one polar residue for another. If desired, manual inspection of such candidates can be carried out in order to narrow the number of candidates to be further evaluated. Manual inspection can be performed by selecting those candidates that appear to have domains present in phenylpropanoid derivative biosynthesis pathway polypeptides, e.g., conserved functional domains. [0084] Conserved regions can be identified by locating a region within the primary amino acid sequence of a phenylpropanoid derivative biosynthesis pathway polypeptide that is a repeated sequence, forms some secondary structure (e.g., helices and beta sheets), establishes positively or negatively charged domains, or represents a protein motif or domain. See, e.g., the Pfam web site describing consensus sequences for a variety of protein motifs and domains on the World Wide Web at sanger.ac.uk/Software/Pfam/ and pfam.janelia.org/. The information included at the Pfam database is described in Sonnhammer ef a/., Nucl. Acids Res., 26:320-322 (1998); Sonnhammer et al., Proteins, 28:405-420 (1997); and Bateman ef al., Nucl. Acids Res., 27:260-262 (1999). Conserved regions also can be determined by aligning sequences of the same or related polypeptides from closely related species. Closely related species preferably are from the same family. In some embodiments, alignment of sequences from two different species is adequate to identify such homologs.

[0085] Typically, polypeptides that exhibit at least about 40% amino acid sequence identity are useful to identify conserved regions. Conserved regions of related polypeptides exhibit at least 45% amino acid sequence identity (e.g., at least 50%, at least 60%, at least 70%, at least 80%, or at least 90% amino acid sequence identity). In some embodiments, a conserved region exhibits at least 92%, 94%, 96%, 98%, or 99% amino acid sequence identity.

[0086] For example, homologs suitable for producing naringenin in a recombinant host include recombinant homologs of chalcone synthase and/or chalcone isomerase genes.

[0087] Methods to modify the substrate specificity of a given polypeptide, such as, for example, a phenylalanine ammonia lyase, tyrosine ammonia lyase, chalcone synthase, chalcone isomerase, or stilbene synthase, are known to those skilled in the art, and include without limitation site-directed/rational mutagenesis approaches, random directed evolution approaches and combinations in which random mutagenesis/saturation techniques are performed near the active site of the enzyme. For example see Osmani et al., 2009, Phytochemistry 70: 325-347.

[0088] A candidate sequence typically has a length that is from 80% to 200% of the length of the reference sequence, e.g., 82, 85, 87, 89, 90, 93, 95, 97, 99, 100, 105, 110, 1 15, 120, 130, 140, 150, 160, 170, 180, 190, or 200% of the length of the reference sequence. A functional homolog polypeptide typically has a length that is from 95% to 105% of the length of the reference sequence, e.g., 90, 93, 95, 97, 99, 100, 105, 1 10, 115, or 120% of the length of the reference sequence, or any range between. A % identity for any candidate nucleic acid or polypeptide relative to a reference nucleic acid or polypeptide can be determined as follows. A reference sequence (e.g., a nucleic acid sequence or an amino acid sequence described herein) is aligned to one or more candidate sequences using the computer program ClustalW (version 1.83, default parameters), which allows alignments of nucleic acid or polypeptide sequences to be carried out across their entire length (global alignment). Chenna et a/., 2003, Nucleic Acids Res. 31 (13):3497-500.

[0089] ClustalW calculates the best match between a reference and one or more candidate sequences, and aligns them so that identities, similarities and differences can be determined. Gaps of one or more residues can be inserted into a reference sequence, a candidate sequence, or both, to maximize sequence alignments. For fast pairwise alignment of nucleic acid sequences, the following default parameters are used: word size: 2; window size: 4; scoring method: %age; number of top diagonals: 4; and gap penalty: 5. For multiple alignment of nucleic acid sequences, the following parameters are used: gap opening penalty: 10.0; gap extension penalty: 5.0; and weight transitions: yes. For fast pairwise alignment of protein sequences, the following parameters are used: word size: 1 ; window size: 5; scoring method:%age; number of top diagonals: 5; gap penalty: 3. For multiple alignment of protein sequences, the following parameters are used: weight matrix: blosum; gap opening penalty: 10.0; gap extension penalty: 0.05; hydrophilic gaps: on; hydrophilic residues: Gly, Pro, Ser, Asn, Asp, Gin, Glu, Arg, and Lys; residue-specific gap penalties: on. The ClustalW output is a sequence alignment that reflects the relationship between sequences. ClustalW can be run, for example, at the Baylor College of Medicine Search Launcher site on the World Wide Web (searchlauncher.bcm.tmc.edu/multi-align/multi-align.html) and at the European Bioinformatics Institute site on the World Wide Web (ebi.ac.uk/clustalw).

[0090] To determine percent identity of a candidate nucleic acid or amino acid sequence to a reference sequence, the sequences are aligned using ClustalW, the number of identical matches in the alignment is divided by the length of the reference sequence, and the result is multiplied by 100. It is noted that the percent identity value can be rounded to the nearest tenth. For example, 78.11 , 78.12, 78.13, and 78.14 are rounded down to 78.1 , while 78.15, 78.16, 78.17, 78.18, and 78.19 are rounded up to 78.2.

[0091] It will be appreciated that functional homologs, e.g. of enzymes involved in phenylpropanoid derivative biosynthesis, can include additional amino acids that are not involved in the enzymatic activities carried out by the enzymes. Recombinant Nucleic Acids

[0092] A recombinant gene encoding a polypeptide described herein comprises the coding sequence for that polypeptide, operably linked in sense orientation to one or more regulatory regions suitable for expressing the polypeptide. Because many microorganisms are capable of expressing multiple gene products from a polycistronic mRNA, multiple polypeptides can be expressed under the control of a single regulatory region for those microorganisms, if desired. A coding sequence and a regulatory region are considered operably linked when the regulatory region and coding sequence are positioned so that the regulatory region is effective for regulating transcription or translation of the sequence. Typically, the translation initiation site of the translational reading frame of the coding sequence is positioned between one and about fifty nucleotides downstream of the regulatory region for a monocistronic gene.

[0093] In many cases, the coding sequence for a polypeptide described herein is identified in a species other than the recombinant host, i.e., is a heterologous nucleic acid. Thus, if the recombinant host is a microorganism, the coding sequence can be from other prokaryotic or eukaryotic microorganisms, from plants or from animals. In some case, however, the coding sequence is a sequence that is native to the host and is being reintroduced into that organism. A native sequence can often be distinguished from the naturally occurring sequence by the presence of non-natural sequences linked to the exogenous nucleic acid, e.g., non-native regulatory sequences flanking a native sequence in a recombinant nucleic acid construct. In addition, stably transformed exogenous nucleic acids typically are integrated at positions other than the position where the native sequence is found. "Regulatory region" refers to a nucleic acid having nucleotide sequences that influence transcription or translation initiation and rate, and stability and/or mobility of a transcription or translation product. Regulatory regions include, without limitation, promoter sequences, enhancer sequences, response elements, protein recognition sites, inducible elements, protein binding sequences, 5 ^' and 3 ^' untranslated regions (UTRs), transcriptional start sites, termination sequences, polyadenylation sequences, introns, and combinations thereof. A regulatory region typically comprises at least a core (basal) promoter. A regulatory region also can include at least one control element, such as an enhancer sequence, an upstream element or an upstream activation region (UAR). A regulatory region is operably linked to a coding sequence by positioning the regulatory region and the coding sequence so that the regulatory region is effective for regulating transcription or translation of the sequence. For example, to operably link a coding sequence and a promoter sequence, the translation initiation site of the translational reading frame of the coding sequence is typically positioned between one and about fifty nucleotides downstream of the promoter. A regulatory region can, however, be positioned as much as about 5,000 nucleotides upstream of the translation initiation site, or about 2,000 nucleotides upstream of the transcription start site.

[0094] The choice of regulatory regions to be included depends upon several factors, including, but not limited to, efficiency, selectability, inducibility, desired expression level, and preferential expression during certain culture stages. It is a routine matter for one of skill in the art to modulate the expression of a coding sequence by appropriately selecting and positioning regulatory regions relative to the coding sequence. It will be understood that more than one regulatory region can be present, e.g., introns, enhancers, upstream activation regions, transcription terminators, and inducible elements.

Recombinant Hosts

[0095] Recombinant hosts can be used to express polypeptides for phenylpropanoid derivative production, including mammalian, insect, plant, and algal cells. A number of prokaryotes and eukaryotes are also suitable for use in constructing the recombinant microorganisms described herein, e.g., gram-negative bacteria, yeast, and fungi. A species and strain selected for use as a phenylpropanoid derivative production strain is first analyzed to determine which production genes are endogenous to the strain and which genes are not present. Genes for which an endogenous counterpart is not present in the strain are advantageously assembled in one or more recombinant constructs, which are then transformed into the strain in order to supply the missing function(s).

[0096] The constructed and genetically engineered microorganisms provided herein can be cultivated using conventional fermentation processes, including, inter alia, chemostat, batch, fed-batch cultivations, continuous perfusion fermentation, and continuous perfusion cell culture.

[0097] Carbon sources of use in the instant method include any molecule that can be metabolized by the recombinant host to facilitate growth and/or production of the phenylpropanoid derivative. Examples of suitable carbon sources include, but are not limited to, sucrose (e.g., as found in molasses), fructose, xylose, ethanol, glycerol, glucose, cellulose, starch, cellobiose or other glucose comprising polymer. In embodiments employing yeast as a host, for example, carbons sources such as sucrose, fructose, xylose, ethanol, glycerol, and glucose are suitable. The carbon source can be provided to the host organism throughout the cultivation period or alternatively, the organism can be grown for a period of time in the presence of another energy source, e.g., protein, and then provided with a source of carbon only during the fed-batch phase.

[0098] Exemplary prokaryotic and eukaryotic species are described in more detail below. However, it will be appreciated that other species can be suitable. For example, suitable species can be in a genus such as Agaricus, Aspergillus, Bacillus, Candida, Corynebacterium, Eremothecium, Escherichia, Fusarium/Gibberella, Kluyveromyces, Laetiporus, Lentinus, Phaffia, Phanerochaete, Pichia, Physcomitrella, Rhodoturula, Saccharomyces, Schizosaccharomyces, Sphaceloma, Xanthophyllomyces or Yarrowia. Exemplary species from such genera include Lentinus tigrinus, Laetiporus sulphureus, Phanerochaete chrysosporium, Pichia pastoris, Cyberlindnera jadinii, Physcomitrella patens, Rhodoturula glutinis 32, Rhodoturula mucilaginosa, Phaffia rhodozyma UBV-AX, Xanthophyllomyces dendrorhous, Fusarium fujikuroi/Gibberella fujikuroi, Candida utilis, Candida glabrata, Candida albicans, and Yarrowia lipolytica.

[0099] In some embodiments, a microorganism can be a prokaryote such as Escherichia coli, Saccharomyces cerevisiae, Rhodobacter sphaeroides, Rhodobacter capsulatus, or Rhodotorula toruloides.

[00100] In some embodiments, a microorganism can be an Ascomycete such as Gibberella fujikuroi, Kluyveromyces lactis, Schizosaccharomyces pombe, Aspergillus niger, Yarrowia lipolytica, Ashbya gossypii, or Saccharomyces cerevisiae.

[00101] In some embodiments, a microorganism can be an algal cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis species.

[00102] In some embodiments, a microorganism can be a cyanobacterial cell such as Blakeslea trispora, Dunaliella salina, Haematococcus pluvialis, Chlorella sp., Undaria pinnatifida, Sargassum, Laminaria japonica, Scenedesmus almeriensis.

Saccharomyces spp.

[00103] Saccharomyces is a widely used chassis organism in synthetic biology, and can be used as the recombinant microorganism platform. For example, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for S. cerevisiae, allowing for rational design of various modules to enhance product yield. Methods are known for making recombinant microorganisms. Aspergillus spp.

[00104] Aspergillus species such as A. oryzae, A. niger and A. sojae are widely used microorganisms in food production and can also be used as the recombinant microorganism platform. Nucleotide sequences are available for genomes of A. nidulans, A. fumigatus, A. oryzae, A, clavatus, A. flavus, A. niger, and A. terreus, allowing rational design and modification of endogenous pathways to enhance flux and increase product yield. Metabolic models have been developed for Aspergillus. Generally, A. niger is cultured for the industrial production of a number of food ingredients such as citric acid and gluconic acid, and thus species such as A. niger are generally suitable for producing phenylpropanoid derivatives.

Escherichia coli

[00105] Escherichia coli, another widely used platform organism in synthetic biology, can also be used as the recombinant microorganism platform. Similar to Saccharomyces, there are libraries of mutants, plasmids, detailed computer models of metabolism and other information available for E. coli, allowing for rational design of various modules to enhance product yield. Methods similar to those described above for Saccharomyces can be used to make recombinant E. coli microorganisms.

Agaricus, Gibberella, and Phanerochaete spp.

[00106] Agaricus, Gibberella, and Phanerochaete spp. can be useful because they are known to produce large amounts of isoprenoids in culture. Thus, precursors for producing large amounts of phenylpropanoid derivatives are already produced by endogenous genes.

Arxula adeninivorans (Blastobotrys adeninivorans)

[00107] Arxula adeninivorans is a dimorphic yeast (it grows as a budding yeast like the baker's yeast up to a temperature of 42°C, above this threshold it grows in a filamentous form) with unusual biochemical characteristics. It can grow on a wide range of substrates and can assimilate nitrate. It has successfully been applied to the generation of strains that can produce natural plastics or the development of a biosensor for estrogens in environmental samples.

Yarrowia lipolytica.

[00108] Yarrowia lipolytica is a dimorphic yeast (see Arxula adeninivorans) and belongs to the family Hemiascomycetes. The entire genome of Yarrowia lipolytica is known. Yarrowia species is aerobic and considered to be non-pathogenic. Yarrowia is efficient in using hydrophobic substrates (e.g. alkanes, fatty acids, oils) and can grow on sugars. It has a high potential for industrial applications and is an oleaginous microorganism. Yarrowia lipolyptica can accumulate lipid content to approximately 40% of its dry cell weight and is a model organism for lipid accumulation and remobilization. See e.g. Nicaud, 2012, Yeast 29(10):409-18; Beopoulos et al., 2009, Biohimie 91 (6):692-6; Bankar et al., 2009, Appl Microbiol Biotechnol. 84(5):847-65.

Rhodotorula sp.

[00109] Rhodotorula is a unicellular, pigmented yeast. The oleaginous red yeast, Rhodotorula glutinis, has been shown to produce lipids and carotenoids from crude glycerol (Saenge ef al., 2011 , Process Biochemistry 46(1):210-8). Rhodotorula toruloides strains have been shown to be an efficient fed-batch fermentation system for improved biomass and lipid productivity (Li er a/., 2007, Enzyme and Microbial Technology 41 :312-7).

Rhodosporidium toruloides

[00110] Rhodosporidium toruloides is an oleaginous yeast and useful for engineering lipid- production pathways (See e.g. Zhu et al., 2013, Nature Commun. 3:1112; Ageitos et al., 2011 , Applied Microbiology and Biotechnology 90(4): 1219-27).

Rhodobacter spp.

[00111] Rhodobacter can be used as the recombinant microorganism platform. Similar to E. coli, there are libraries of mutants available as well as suitable plasmid vectors, allowing for rational design of various modules to enhance product yield. Isoprenoid pathways have been engineered in membraneous bacterial species of Rhodobacter for increased production of carotenoid and CoQ10. See, U.S. Patent Publication Nos. 20050003474 and 20040078846. Methods similar to those described above for E. coli can be used to make recombinant Rhodobacter microorganisms.

Candida boidinii

[00112] Candida boidinii is a methylotrophic yeast (it can grow on methanol). Like other methylotrophic species such as Hansenuia polymorpha and Pichia pastoris, it provides an excellent platform for producing heterologous proteins. Yields in a multigram range of a secreted foreign protein have been reported. A computational method, IPRO, recently predicted mutations that experimentally switched the cofactor specificity of Candida boidinii xylose reductase from NADPH to NADH.

Hansenuia polymorpha (Pichia angusta) [00113] Hansenula polymorphs is another methylotrophic yeast (see Candida boidinii). It can furthermore grow on a wide range of other substrates; it is thermo-tolerant and can assimilate nitrate (see also Kluyveromyces lactis). It has been applied to producing hepatitis B vaccines, insulin and interferon alpha-2a for the treatment of hepatitis C, furthermore to a range of technical enzymes.

Kluyveromyces lactis

[00114] Kluyveromyces lactis is yeast regularly applied to producing kefir. It can grow on several sugars, most importantly on lactose which is present in milk and whey. It has successfully been applied among others for producing chymosin (an enzyme that is usually present in the stomach of calves) for producing cheese. Production takes place in fermenters on a 40,000 L scale.

Pichia pastoris

[00115] Pichia pastoris is a methylotrophic yeast (see Candida boidinii and Hansenula polymorpha). It provides an efficient platform for producing foreign proteins. Platform elements are available as a kit and it is worldwide used in academia for producing proteins. Strains have been engineered that can produce complex human N-glycan (yeast glycans are similar but not identical to those found in humans).

Physcomitrella spp.

[00116] Physcomitrella mosses, when grown in suspension culture, have characteristics similar to yeast or other fungal cultures. This genera is becoming an important type of cell for producing plant secondary metabolites, which can be difficult to produce in other types of cells.

Methods of Producing Phenylpropanoid Derivatives

[00117] Recombinant hosts described herein can be used in methods to produce phenylpropanoid derivatives.

[00118] For example, the method can include growing the recombinant host in a culture medium under conditions in which phenylpropanoid derivative biosynthesis genes are expressed. The recombinant host can be grown in a fed batch or continuous process. Typically, the recombinant host is grown in a fermentor at a defined temperature(s) for a desired period of time. Depending on the particular host used in the method, other recombinant genes such as phenylalanine ammonia lyase (PAL), tyrosine ammonia lyase (TAL), cinnamate 4-hydroxylase (C4H), cytochrome P450 reductase (CPR), 4-coumarate-CoA ligase (4CL), stilbene synthase (STS), chalcone synthase (CHS), or chalcone isomerase (CHI) can also be present and expressed. Levels of substrates and intermediates, e.g., phenylalanine, tyrosine, cinnamic acid, coumaric acid, dihydrocinnamic acid or phloretic acid, can be determined by extracting samples from culture media for analysis according to published methods. In some embodiments, the culture medium does not contain a phenylpropanoid precursor or intermediate from an external source (i.e., phenylpropanoid precursors or intermediates are not added to the culture medium).

[00119] The genes described herein can be expressed in yeast using any of a number of known promoters. Strains that overproduce phenylpropanoids are known and can be used as acceptor molecules in the production of phenylpropanoids or phenylpropanoid derivatives.

[00120] In some embodiments, enzymes may be screened for TAL and/or PAL activity. In some embodiments, the corresponding DNA sequence for the enzymes to be screened for PAL and TAL activity are codon optimized for optimal expression in Saccharomyces cerevisiae. In some embodiments, each PAL/TAL enzyme is cloned together with all necessary genes for the production of naringenin from cinnamic acid (C4H-CPR, 4CL, CHS and CHI), to measure PAL+TAL activity, or from coumaric acid (4CL, CHS and CHI), to measure TAL activity alone. In some embodiments, the genes are then introduced in a single step into a Saccharomyces cerevisiae yeast strain which does not produce phenylpropanoids or phenylpropanoid derivatives ("non-producer yeast strain").

[00121] In some embodiments, transformants are inoculated in a 96 deep well plate and incubated overnight in 500μΙ of SC-URA medium at 30°C and 400 rpm. In some embodiments, 50μΙ of the overnight culture are inoculated to a new 96 deep well plate containing 500μΙ of DELFT medium plus 4% w/v glucose. In some embodiments, after 72 hours of growth under the same conditions, the OD600 is measured to estimate cell growth and samples taken to measure cinnamic acid, coumaric acid, naringenin and phloretic acid by HPLC as follows. In some embodiments, a sample of the culture (300μΙ) is mixed with 96% EtOH (300μΙ) on a shaking table and centrifuged. In some embodiments, supernatant (100μΙ) is used for HPLC analysis. In some embodiments, measurements are taken using pure compounds as standards.

[00122] In some embodiments, the TAL gene is encoded by the nucleotide sequence of any one of SEQ ID NOS:12, 14, 16, 18, 20, 22, 24, 26, 28, 30, 32, 34, 36, 38, 40, 42, 44, 46 or 48. In some embodiments, the TAL polypeptide is any one of SEQ ID NOS:13, 15, 17, 19, 21 , 23, 25, 27, 29, 31 , 33, 35, 37, 39, 41 , 43, 45, 47 or 49. In some embodiments, the TAL polypeptide is from Aeromonas salmonicida subsp. salmonicida A449 (Asal) (SEQ ID NO:31 ). In some embodiments, the TAL polypeptide has at least 50% identity to the amino acid sequence in SEQ ID NO:31. In some embodiments, the TAL polypeptide has at least 65% identity to the amino acid sequence in SEQ ID NO:31. In some embodiments, the TAL gene is encoded by the nucleotide sequence of SEQ ID NO:30. In some embodiments, the TAL gene has at least 60% identity to SEQ ID NO:30.

[00123] In some embodiments, the TAL activity of Asal is comparable with previously known TALs from Rhodotorula graminis (Vanneli, J. et al, (2007), Enzyme and Microbial Technology 41 , 413-422) and PAL4 from Bambusa oldhamii (Hseih, L. et al. (2010), Phytochemistry 71 ) while Asal possess much higher specificity that the two mentioned TALs (see e.g., Figure 2). In some embodiments, the Asal TAL also has higher activity than previously reported specific TALs as RcTAL from Rodobacter capsulatus (Koopman, F. et al. (2012); Kyndt, J A. ef al. (2002), FEBS Letters 512, 240-244) and Sam8 from Saccharothrix espanaensis (Berner, M. ef al. (2006), J Bacteriol. Apr;188(7):2666-73) (see e.g., Figure 2). In some embodiments, the activity of Asal TAL is more than five (5) times as active as previously reported specific TALs, such as, for example but not limited to, RcTAL from R. capsulatus and Sam8 from S. espanaensis.

[00124] After the recombinant host has been grown in culture for the desired period of time, phenylpropanoid derivatives (such as naringenin or resveratrol) can then be recovered from the culture using various techniques known in the art. In some embodiments, a permeabilizing agent can be added to aid the feedstock entering into the host, and to aid in product release from the host. For example, a crude iysate of the cultured microorganism can be centrifuged to obtain a supernatant. The resulting supernatant can then be applied to a chromatography column, e.g., a C-18 column, and washed with water to remove hydrophilic compounds, followed by elution of the compound(s) of interest with a solvent such as methanol. The compound(s) can then be further purified by preparative HPLC according to methods known in the art.

[00125] It will be appreciated that the various genes and modules discussed herein can be present in two or more recombinant hosts rather than a single host. When a plurality of recombinant host is used, they can be grown in a mixed culture to produce phenylpropanoid derivatives.

[00126] Alternatively, the two or more hosts each can be grown in a separate culture medium and the product of the first culture medium, e.g., a naringenin or resveratrol precursor, can be introduced into second culture medium to be converted into a subsequent intermediate, or into an end product such as, for example, naringenin. The product produced by the second, or final host is then recovered. It will also be appreciated that in some embodiments, a recombinant host is grown using nutrient sources other than a culture medium and utilizing a system other than a fermentor.

[00127] In some embodiments, phenylpropanoid derivatives are produced in vivo through expression of one or more enzymes involved in a phenylpropanoid derivative biosynthetic pathway in a recombinant host. For example, a naringenin-producing or resveratrol-producing recombinant host expressing recombinant genes encoding, one or more of an Arabidopsis thaliana phenylalanine ammonia lyase (PAL2) polypeptide, a gene encoding a Ammi majus cinnamate 4-hydroxylase (CH4) polypeptide, a gene encoding a Arabidopsis thaliana 4- coumarate-CoA ligase (4CL2) polypeptide, a gene encoding a Hordeum vulgare chalcone synthase 2 (CHS2) polypeptide, a gene encoding a cytochrome P450 reductase (CPR1) polypeptide can be used to produce a chalcone compound, e.g. naringenin, in vivo.

[00128] In some embodiments, phenylpropanoid derivatives are produced in vivo through expression of one or more enzymes involved in a phenylpropanoid derivative biosynthetic pathway in a recombinant host. For example, a naringenin-producing or resveratrol-producing recombinant host expressing recombinant genes encoding, one or more of an Aeromonas salmonicida tyrosine ammonia lyase (Asal TAL) polypeptide, a gene encoding a Arabidopsis thaliana 4-coumarate-CoA ligase (4CL2) polypeptide, a gene encoding a Hordeum vulgare chalcone synthase 2 (CHS2) polypeptide, a gene encoding a cytochrome P450 reductase (CPR1 ) polypeptide can be used to produce a chalcone compound, e.g. naringenin, in vivo.

[00129] As another example, a stilbenoid (such as resveratrol)-producing recombinant host wherein one or more genes encoding a Saccharomyces cerevisiae trans-2-enoyl-CoA reductase polypeptide are underexpressed or unexpressed, and expressing recombinant genes encoding one or more of an Arabidopsis thaliana phenylalanine ammonia lyase (PAL2) polypeptide, a gene encoding a Ammi majus cinnamate 4-hydroxylase (CH4) polypeptide, a gene encoding a Arabidopsis thaliana 4-coumarate-CoA ligase (4CL2) polypeptide, and a gene encoding a stilbene synthase (STS) polypeptide, can be used to produce a stilbenoid compound, e.g. resveratrol, in vivo.

[00130] As another example, a stilbenoid (such as resveratrol)-producing recombinant host wherein one or more genes encoding a Saccharomyces cerevisiae trans-2-enoyl-CoA reductase polypeptide are underexpressed or unexpressed, and expressing recombinant genes encoding one or more of an Aeromonas salmonicida tyrosine ammonia lyase (Asal_TAL) polypeptide, a gene encoding a Arabidopsis thaliana 4-coumarate-CoA ligase (4CL2) polypeptide, and a gene encoding a stilbene synthase (STS) polypeptide, can be used to produce a stilbenoid compound, e.g. resveratrol, in vivo.

[00131] In some embodiments, phenylpropanoid derivatives are produced through contact of a precursor of the desired compound with one or more enzymes involved in the phenylpropanoid derivative biosynthesis pathway in vitro. For example, contacting tyrosine with a tyrosine ammonia lyase, a 4-coumarate-CoA ligase and a chalcone synthase polypeptide can result in production of a naringenin or naringenin derivative compound in vitro. In some embodiments, a naringenin precursor is produced through contact of an upstream naringenin precursor with one or more enzymes involved in the naringenin pathway in vitro.

[00132] In some embodiments, a phenylpropanoid derivative precursor is produced by bioconversion. For bioconversion to occur, a recombinant host expressing one or more enzymes involved in the phenylpropanoid derivative biosynthesis pathway takes up and modifies a phenylpropanoid derivative precursor in the cell; following modification in vivo, the phenylpropanoid derivative remains in the cell and/or is excreted into the culture medium. For example, a recombinant host expressing a gene encoding a tyrosine ammonia lyase, a 4- coumarate-CoA ligase and a chalcone synthase polypeptide can take up tyrosine and convert it to naringenin in the cell; following conversion in vivo, a naringenin compound is excreted into the culture medium.

[00133] In some embodiments, phenylpropanoid derivatives as disclosed herein are isolated and purified to homogeneity (e.g., at least 90%, 92%, 94%, 96%, or 98% pure). In other embodiments, phenylpropanoid derivatives are isolated as an extract from a recombinant host or in vitro production method. In this respect, phenylpropanoid derivatives may be isolated, but not necessarily purified to homogeneity. Desirably, the amount of phenylpropanoid derivatives produced can be from about 1 mg/L to about 20,000 mg/L or higher. For example about 1 to about 100 mg/L, about 30 to about 100 mg/L, about 50 to about 200 mg/L, about 100 to about 500 mg/L, about 100 to about 1 ,000 mg/L, about 250 to about 5,000 mg/L, about 1 ,000 to about 15,000 mg/L, or about 2,000 to about 10,000 mg/L of phenylpropanoid derivatives can be produced. In general, longer culture times will lead to greater amounts of product. Thus, the recombinant microorganism can be cultured for from 1 day to 7 days, from 1 day to 5 days, from 3 days to 5 days, about 3 days, about 4 days, or about 5 days.

[00134] In some embodiments, a resveratrol-producing yeast strain without TAL activity is transformed with a plasmid containing the TAL gene from Aeromonas salmonicida (Asal TAL, SEQ ID NO:31 ). In some embodiments, the plasmid allows multiple integration of the TAL gene at the Ty1 regions present throughout the yeast genome. In some embodiments, the TAL gene is under the control of the strong promoter pPGK1 and results in overexpression of the gene.

[00135] In some embodiments, the resulting strain in which the TAL gene is overexpressed is compared with the direct parental strain (control) which contains all necessary genes leading to resveratrol production. In some embodiments, levels of resveratrol and pathway intermediates (coumaric and phloretic acid) are measured after growing the indicated strains for 3 days in minimal medium. In some embodiments, extraction of compounds is carried out by mixing ethanol with a fermentation sample to a final concentration of 50% by volume, and centrifugation for 5 minutes at 3222 x g.

[00136] In some embodiments, resveratrol titers are increased by at least 25% in the daughter strain in which A. salmonicida (Asal) TAL is overexpressed over the control parent strain. In some embodiments, a 25% increase in resveratrol titer is an increase in resveratrol production of approximately 375 mg/L. In some embodiments, coumaric acid accumulation is increased by 2.5 times in the daughter strain in which Asal TAL is overexpressed over the control parent strain. In some embodiments, a 2.5 times increase in coumaric acid accumulation is an increase of approximately 108 mg/L, when compared with the control parent strain. In some embodiments, the potential resveratrol flux, measured as the sum of resveratrol and major side products, is increased by 26,2 % in the new strain harboring and overexpressing Asal TAL.

EXAMPLES

[00137] The Examples that follow are illustrative of specific embodiments disclosed herein and various uses thereof. They are set forth for explanatory purposes only and are not to be taken as limiting.

Example 1: Screening of TAL enzymes

[00138] PAL/TAL enzymes were screened for PAL and TAL activity. The nucleotide and amino acid sequences are identified herein as SEQ ID NOS:12-49.

[00139] Each nucleotide sequence was codon optimized for optimal expression in Saccharomyces cerevisiae. Each PAUTAL enzyme was cloned together with all necessary genes for the production of naringenin from cinnamic acid (C4H-CPR, 4CL, CHS and CHI), to measure PAL and TAL activity, or coumaric acid (4CL, CHS and CHI), to measure TAL activity alone. The genes were then introduced in a single step into a S. cerevisiae yeast strain which does not produce phenylpropanoids or phenylpropanoid derivatives ("non-producer yeast strain").

[00140] Transformants were inoculated in a 96 deep well plate and incubated overnight in 500μΙ of SC-URA medium at 30°C and 400 rpm. Subsequently, 50μΙ of the overnight culture was inoculated to a new 96 deep well plate containing 500μΙ of DELFT medium plus 4% w/v glucose. After 72 hours of growth under the same conditions, the OD600 was measured to estimate cell growth and samples were taken to measure cinnamic acid, coumaric acid, naringenin and phloretic acid by HPLC as follows. A sample of the culture (300μΙ) was mixed with 96% EtOH (300μΙ) on a shaking table and centrifuged. Supernatant (100μΙ) was used for HPLC analysis. Measurements were taken using pure compounds as standards. Phloretin could not be measured because the peak overlaps with cinnamic acid.

[00141] Figure 2 shows that the TAL from Aeromonas salmonicida subsp. salmonicida A449 (Asal) (SEQ ID NOS:30 and 31 ) possesses much higher specificity to tyrosine as a substrate than the TAL from Rhodotorula graminls (SEQ ID NOS:36 and 37) (Vanneli, J. et at. (2007), Enzyme and Microbial Technology 41 , 413-422) and PAL4 from Bambusa oldhamii (SEQ ID NOS:38 and 39) (Hseih, L. ef a/. (20 0), Phytochemistry 71 ). Figure 2 also shows that Asal TAL has more than five times the activity of both RcTAL from Rodobacter capsulatus (SEQ ID NOS:20 and 21) (Koopman, F. et al. (2012); Kyndt, J A. et al. (2002), FEBS Letters 512, 240- 244) and Sam8 from Saccharothrix espanaensis (SEQ ID NOS:22 and 23) (Bemer, M. et al. (2006), J Bacterid. Apr;188(7):2666-73).

Example 2: Resveratrol production in a resveratrol-producing strain overexpressing Aeromonas salmonicida TAL (SEQ ID NOS:30 and 31)

[00142] A resveratrol-producing yeast strain without TAL activity was transformed with a plasmid containing the TAL gene from Aeromonas salmonicida (Asal TAL, SEQ ID NOS:30 and 31 ). The plasmid allowed multiple integration of the TAL gene at the Ty1 regions present throughout the yeast genome. The TAL gene was under the control of the strong promoter pPGK1 and results in overexpression of the gene.

[00143] The resulting strain in which the TAL gene was overexpressed was compared with the direct parental strain (control) which contained all necessary genes leading to resveratrol production. [00144] Levels of resveratrol and pathway intermediates (coumaric and phloretic acid) were measured after growing the indicated strains for 3 days in minimal medium. Extraction of compounds was carried out by mixing ethanol with a fermentation sample to a final concentration of 50% by volume, and centrifugation for 5 minutes at 3222 x g.

[00145] The results show that resveratrol titers were increased by 25% (an increase of approximately 375 mg/L) in the daughter strain in which A. salmonicida TAL was overexpressed. Additionally, coumaric acid accumulation was shown to be increased by 2.5 times (an increase of approximately 108 mg/L) when compared with the control parent strain. Overall, the potential resveratrol flux measured as the sum of resveratrol and major side products was increased by 26,2 % in the new strain harboring and overexpressing Asal TAL. (Figure 3).

[00146] Sequences

Table 1. Nucleic acid and amino acid sequences.

Protein sequence from tyrosine ammonia lyase (TAL) of Rhodobacter

SEQ ID NO:1

capsulatus

TLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCARAHARLEHAIAEQRHIY GITTGFGPLANRLIG

ADQGAELQQNLIYHLATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAV LNAGFAPEVPAQGTVGASG DLTPLAHMVLALQGRGRMIDPSGRVQEAGAVMDRLCGGPLTLAARDGLALVNGTSAMTAI AALTGVEAARAIDAAL RHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALDGAGRVCRTLTAARRLTAA DLRPEDHPAQDAYSLR VVPQLVGAVWDTLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDALN AALVTLAGLVERQIAR LTDEKLNKGLPAFLHGGQAGLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNGANQDW SMGTIAARRARAQLL PLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRPLAGHIEAV AQGLRHPSAAADPPA

Protein sequence from cinnamate 4-hydroxylase (C4H) of Arabidopsis

SEQ ID NO:2

thaliana

MDLLLLEKSLIAVFVAVILATVISKLRGKKLKLPPGPIPIPIFGNWLQVGDDLNHRNLVD YAKKFGDLFLLRMGQR NLWVSSPDLTKEVLLTQGVEFGSRTRNVVFDIFTGKGQDMVFTVYGEHWRKMRRIMTVPF FTNKWQQNREGWEF EAASVVEDVKKNPDSATKGIVLRKRLQLMMYNNMFRIMFDRRFESEDDPLFLRLKALNGE RSRLAQSFEYNYGDFI PILRPFLRGYLKICQDVKDRRIALFKKYFVDERKQIASSKPTGSEGLKCAIDHILEAEQK GEINEDNVLYIVENIN VAAIETTLWSIEWGIAELVNHPEIQSKLRNELDTVLGPGVQVTEPDLHKLPYLQAVVKET LRLRMAIPLLVPHMNL HDAKLAGYDIPAESKILVNAWWLANNPNSWKKPEEFRPERFFEEESHVEANGNDFRYVPF GVGRRSCPGIILALPI LGITIGRMVQNFELLPPPGQSKVDTSE GGQFSLHILNHSIIVMKPRNC

Protein sequence from 4-coumarate-CoA ligase 2 (4CL2) of

SEQ ID O:3

Arabidopsis thaliana

MTTQDVIVNDQNDQKQCSNDVIFRSRLPDIYIPNHLPLHDYIFENISEFAAKPCLINGPT GEVYTYADVHVTSRKL AAGLHNLGVKQHDVVMILLPNSPEVVLTFLAASFIGAITTSANPFFTPAEISKQAKASAA KLIVTQSRYVDKIKNL QNDGVLIVTTDSDAIPENCLRFSELTQSEEPRVDSIPEKISPEDVVALPFSSGTTGLPKG VMLTHKGLVTSVAQQV DGENPNLYFNRDDVILCVLPMFHIYALNSIMLCSLRVGATILIMPKFEITLLLEQIQRCK VTVAMWPPIVLAIAK SPETEKYDLSSVRMVKSGAAPLGKELEDAISAKFPNAKLGQGYGMTEAGPVLAMSLGFAK EPFPVKSGACGTVVRN AEMKILDPDTGDSLPRNKPGEICIRGNQIMKGYLNDPLATASTIDKDGWLHTGDVGFIDD DDELFIVDRLKELIKY KGFQVAPAELESLLIGHPEINDVAWAMKEEDAGEVPVAFVVRSKDSNISEDEIKQFVSKQ VVFYKRINKVFFTDS IPKAPSGKILRKDLRARLANGLMN

SEQ ID NO:4 Protein sequence from stilbene synthase (STS) Vitis pseudoreticulata

MASVEEIRNAQRAKGPATILAIGTATPDHCVYQSDYADYYFRVTKSEHMTALKKKFN RICDKSMIKKRYIHLTEEM LEEHPNIGAYMAPSLNIRQEIITAEVPKLGKEAALKALKEWGQPKSKITHLVFCTTSGVE MPGADYKLANLLGLEP SVRRVMLYHQGCYAGGTVLRTT DLAENNAGARVLWCPEITWTFRGPSEDALDSLVGQALFGDGSAAVIVGSDP DISIERPLFQLVSAAQTFIPNFAGAIAGNLREVGLTFHLWPNVPTLISENIENCLTQAFD PLGISDWNSLFWIAHP

GGPAILDAVEAKLNLDKKKLEATRHVLSEYGNVSSACVLFILDEMRKKSLKGERATT GEGLGWGVLFGFGPGLTIE TVVLHSIPMVTN

Protein sequence from NAD PH: cytochrome P450 reductase (CPR) of

SEQ ID NO:5

Arabidopsis thaliana ATR2

MSSSSSSSTSMIDLMAAIIKGEPVIVSDPANASAYESVAAELSSMLIENRQFAMIVTTSI AVLIGCIVMLVWRRSG SGNSKRVEPLKPLVIKPREEEIDDGRKKVTIFFGTQTGTAEGFAKALGEEAKARYEKTRF KIVDLDDYAADDDEYE EKLKKEDVAFFFLATYGDGEPTDNAARFYKWFTEGNDRGEWLKNLKYGVFGLGNRQYEHF NKVAKWDDILVEQGA QRLVQVGLGDDDQCIEDDFTAWREALWPELDTILREEGDTAVATPYTAAVLEYRVSIHDS EDAKFNDINMANGNGY TVFDAQHPYKANVAVKRELHTPESDRSCIHLEFDIAGSGLTYETGDHVGVLCDNLSETVD EALRLLDMSPDTYFSL HAEKEDGTPISSSLPPPFPPCNLRTALTRYACLLSSPKKSALVALAAHASDPTEAERLKH LASPAGKDEYSKWVVE SQRSLLEVMAEFPSAKPPLGVFFAGVAPRLQPRFYSISSSPKIAETRIHVTCALVYEKMP TGRIHKGVCSTWMKNA VPYEKSENCSSAPIFVRQSNFKLPSDSKVPIIMIGPGTGLAPFRGFLQERLALVESGVEL GPSVLFFGCRNRRMDF IYEEELQRFVESGALAELSVAFSREGPTKEYVQHKMMDKASDIWNMISQGAYLYVCGDAK GMARDVHRSLHTIAQE QGSMDSTKAEGFVKNLQTSGRYLRDVW

DNA sequence encoding chalcone synthase (CHS2) of Hordeum

SEQ ID O:6

vulgare, codon optimized for expression in S. cerevisiae

ATGGCTGCAGTAAGATTGAAAGAAGTTAGAATGGCACAGAGGGCTGAAGGTTTAGCTACA GTTTTAGCAATCGGTA CTGCCGTTCCAGCTAATTGTGTTTATCAAGCTACCTATCCAGATTATTATTTTAGGGTTA CTAAAAGTGAGCACTT GGCAGATTTAAAGGAGAAGTTTCAAAGAATGTGTGACAAATCAATGATTAGAAAGAGACA CATGCACTTGACCGAG GAAATATTGATCAAGAACCCAAAGATCTGTGCACACATGGAGACCTCATTGGATGCTAGA CACGCCATCGCATTAG TTGAAGTTCCCAAATTGGGCCAAGGTGCAGCTGAGAAGGCCATTAAGGAGTGGGGCCAAC CCTTGTCTAAGATTAC TCATTTGGTATTTTGCACAACATCCGGCGTTGACATGCCCGGTGCTGATTACCAATTAAC AAAGTTGTTAGGTTTG TCCCCTACAGTCAAAAGGTTAATGATGTACCAACAAGGTTGCTTTGGTGGTGCAACTGTT TTGAGATTGGCAAAAG ATATCGCTGAAAATAATAGAGGTGCCAGAGTGTTAGTCGTTTGTTCCGAGATAACTGCTA TGGCCTTCAGAGGTCC ATGCAAGAGTCATTTAGATTCCTTGGTAGGTCATGCCTTGTTCGGTGATGGTGCCGCTGC TGCAATTATAGGCGCT GACCCAGACCAATTAGACGAACAACCAGTTTTCCAGTTGGTATCAGCTTCTCAGACTATA TTACCAGAATCAGAAG GTGCCATAGATGGCCATTTAACAGAAGCTGGTTTAACTATACATTTATTAAAAGATGTTC CTGGTTTAATTTCAGA GAACATTGAACAGGCTTTGGAGGATGCCTTTGAACCTTTAGGTATTCATAACTGGAATTC AATTTTCTGGATTGCA CATCCTGGTGGCCCTGCCATTTTAGACAGAGTTGAAGATAGAGTAGGATTGGATAAGAAG AGAATGAGGGCTTCTA GGGAAGTGTTATCTGAATACGGAAATATGTCTAGTGCCTCTGTGTTGTTTGTGTTAGATG TCATGAGGAAAAGTTC TGCTAAAGACGGATTGGCAACCACAGGAGAAGGAAAAGATTGGGGAGTGTTGTTTGGATT CGGACCAGGCTTGACT GTAGAAACCTTAGTGTTGCATAGTGTCCCAGTCCCTGTCCCTACTGCAGCTTCTGCATGA

SEQ ID NO:7 Protein sequence of CHS2 from Hordeum vulgare

MAAVRLKEVRMAQRAEGLATVLAIGTAVPANCVYQATYPDYYFRVTKSEHLADLKEKFQR MCDKSMIRKRHMHLTE EILIKNPKICAHMETSLDARHAIALVEVPKLGQGAAEKAIKEWGQPLSKITHLVFCTTSG VDMPGADYQLTKLLGL SPTVKRLMMYQQGCFGGATVLRLAKDIAENNRGARVLVVCSEITAMAFRGPCKSHLDSLV GHALFGDGAAAAIIGA DPDQLDEQPVFQLVSASQTILPESEGAIDGHLTEAGLTIHLLKDVPGLISENIEQALEDA FEPLGIHNWNSIFWIA HPGGPAILDRVEDRVGLDKKRMRASREVLSEYGNMSSASVLFVLDVMRKSSAKDGLATTG EGKDWGVLFGFGPGLT VETLVLHSVPVPVPTAASA

DNA sequence encoding chalcone isomerase A (CHI-A) of Petunia

SEQ ID NO:8

hybrida

ATGTCTCCACCAGTTTCTGTTACAAAAATGCAAGTCGAAAATTATGCTTTTGCACCAACA GTGAACCCTGCCGGTT CCACCAATACTTTGTTCTTAGCTGGAGCAGGCCATAGAGGTCTAGAGATTGAAGGAAAGT TTGTGAAATTCACAGC CATAGGCGTATACCTTGAGGAAAGTGCTATCCCATTTTTGGCAGAAAAGTGGAAAGGTAA GACCCCTCAGGAGTTA ACTGATAGCGTCGAGTTCTTTAGGGACGTGGTTACAGGTCCATTCGAAAAGTTTACCAGA GTAACTATGATTCTAC CTCTTACAGGAAAGCAATATTCTGAGAAAGTCGCCGAAAACTGTGTTGCTCACTGGAAGG GCATAGGTACCTACAC TGATGACGAAGGAAGGGCAATCGAGAAATTCTTGGATGTGTTTAGATCAGAAACATTCCC ACCTGGTGCTTCCATT ATGTTTACTCAGAGTCCATTAGGCTTGTTAACCATCAGCTTTGCCAAGGACGATTCAGTT ACCGGTACTGCAAATG CTGTAATCGAGAACAAACAACTATCAGAAGCCGTCCTTGAATCCATTATTGGAAAGCATG GTGTGAGTCCTGCAGC CAAATGCTCTGTTGCCGAGAGAGTAGCAGAATTGTTAAAAAAGAGCTATGCTGAAGAGGC CTCAGTGTTCGGCAAA CCAGAAACCGAAAAGTCCACAATACCTGTTATCGGTGTGTAG

SEQ ID NO:9 Protein sequence of CHI-A (CHI1) from Petunia hybrida

MSPPVSVTKMQVENYAFAPTVNPAGSTNTLFLAGAGHRGLEIEGKFVKFTAIGVYLEESA IPFLAEKWKGKTPQEL TDSVEFFRDVVTGPFEKFTRVTMILPLTGKQYSEKVAENCVAHWKGIGTYTDDEGRAIEK FLDVFRSETFPPGASI MFTQSPLGLLTISFAKDDSVTGTANAVIENKQLSEAVLESIIGKHGVSPAAKCSVAERVA ELLKKSYAEEASVFGK PETEKSTIPVIGV

DNA sequence encoding chalcone isomerase B (CHI-B) of Petunia

SEQ ID NO:10

hybrida

ATGTCTCCATCTGTTTCTGTTACTAAAGTCCAAGTGGAAAATTATGTCTTTCCTCCAACA GTGAAGCCTCCAGCTA GTACCAAAACTTTGTTCTTAGGTGGAGCAGGCCATAGAGGTCTAGATGTTGAGGGAAAGT TTGTGAAATTCACAGT TATTGGCGTATACCTTGAAGAGAGCGCCGTCCAGTTTTTGGCTCCTAAGTGGAAAGGTAA GTCTGCAGAAGAATTA ATACACTCAGTTGACTTCTTTAGGGATATCGTGACCGGTCCATTCGAGAAGTTTACTAGA GTTAGGTTCATTCTAC CTCTTACAGGAAAGCAATTTTCCGAAAAAGTAGCCGAAAACTGTGTCGCTCATTGGAAGG CAACCGGCACTTATAG TGACGCCGGTAGCAGAGCTATAGAGAAATTCTTGAATGTGGTTAAGTCTGAAACATTTTT ACCAGGAGCATCAATC TTGTTTACCCAGTCCCCTTTAGGTAGTCTAACTATTTCTTTCACAAAAGATGACAGCATA TCCGAAGCTGGCAACG CCGTAATCGAGAACAAACAGTTTAGTGAGGCCGTCCTTGAGACTATTATTGGTGAACACG GAGTTAGTCCAGCTGC CAAGTGCTCTATAGCAGCTAGAATGTCAGAATTGTTCAAAAACAGCTTATTTTGA

ATGGAGATTAACGGGGCACACAAGAGCAACGGAGGAGGAGTGGACGCTATGTTATGCGGC GGAGACATCAAGACAA AGAACATGGTGATCAACGCGGAGGATCCTCTCAACTGGGGAGCTGCAGCGGAGCAAATGA AAGGTAGCCATTTGGA TGAAGTGAAGAGAATGGTTGCTGAGTTTAGGAAGCCAGTTGTGAATCTTGGTGGTGAGAC TCTGACCATTGGACAA GTGGCTGCGATCTCAACTATTGGTAACAGTGTGAAGGTGGAGCTATCGGAGACAGCTAGA GCCGGTGTGAATGCTA GTAGTGATTGGGTTATGGAGAGTATGAACAAAGGCACTGATAGTTATGGTGTTACTACTG GTTTTGGTGCTACTTC TCATCGGAGAACCAAAAACGGTGTCGCACTTCAGAAGGAACTTATTAGATTCCTTAACGC CGGAATATTCGGAAGC ACGAAAGAAACAAGCCACACATTGCCACACTCCGCCACAAGAGCCGCCATGCTTGTACGA ATCAACACTCTCCTCC AAGGATTTTCCGGTATCCGATTTGAGATTCTCGAAGCAATTACCAGTTTCCTCAACAACA ACATCACTCCATCTCT CCCCCTCCGTGGTACAATCACCGCCTCCGGAGATCTCGTTCCTCTCTCCTACATCGCCGG ACTTCTCACCGGTCGT CCCAATTCCAAAGCTACTGGTCCCAACGGTGAAGCTTTAACAGCAGAGGAAGCTTTCAAA TTAGCAGGAATCAGCT CCGGATTCTTTGATCTCCAGCCTAAGGAAGGTCTCGCGCTAGTCAATGGCACGGCGGTTG GATCTGGAATGGCGTC AATGGTGTTATTCGAAACGAATGTTCTCTCTGTTTTGGCTGAGATTTTGTCGGCGGTTTT CGCAGAGGTGATGAGT GGTAAGCCTGAGTTCACCGATCATCTCACTCACAGACTTAAACATCATCCCGGTCAAATC GAAGCGGCGGCGATAA TGGAGCATATCCTCGACGGAAGCTCGTACATGAAATTAGCTCAGAAGCTTCACGAGATGG ATCCGTTACAGAAACC TAAACAAGATCGTTACGCTCTTCGTACTTCTCCTCAATGGTTAGGTCCTCAAATCGAAGT GATCCGTTACGCAACG AAATCGATCGAGCGTGAGATTAACTCCGTCAACGATAATCCGTTGATCGATGTTTCGAGG AACAAGGCGATTCACG GTGGTAACTTCCAAGGAACACCAATCGGAGTTTCAATGGATAACACGAGATTGGCGATAG CAGCGATTGGTAAACT CATGTTTGCTCAATTCTCAGAGCTTGTGAATGATTTCTACAACAATGGTTTACCCTCGAA TCTAACCGCTTCGAGG AATCCAAGTTTGGATTATGGATTCAAGGGAGCTGAGATTGCAATGGCTTCTTATTGTTCA GAGCTTCAATACTTAG CTAATCCTGTGACTAGCCATGTTCAATCAGCAGAGCAACATAACCAAGATGTCAACTCTT TGGGACTAATCTCGTC TCGCAAAACTTCTGAAGCTGTTGATATTCTCAAGCTTATGTCAACAACGTTCCTCGTTGC GATTTGTCAAGCTGTG GATTTGAGACATTTGGAGGAGAATTTGAGACAGACTGTGAAGAACACTGTCTCTCAAGTG GCGAAGAAAGTTCTTA CTACTGGAGTCAATGGTGAGCTTCATCCTTCTCGCTTCTGCGAAAAGGATTTACTCAAAG TTGTAGACCGTGAACA AGTCTACACATACGCGGATGATCCTTGTAGCGCAACGTACCCGTTGATTCAGAAGCTGAG ACAAGTTATTGTTGAC CATGCTTTGATCAATGGTGAGAGTGAGAAGAATGCAGTGACTTCAATCTTCCATAAGATT GGAGCTTTCGAGGAGG AGCTTAAGGCAGTGCTACCGAAAGAAGTGGAAGCAGCAAGAGCAGCCTACGATAACGGAA CATCGGCTATCCCGAA CAGGATCAAGGAATGTAGGTCGTATCCATTGTATAGATTCGTGAGGGAAGAGCTTGGAAC AGAGCTTTTGACCGGA GAGAAAGTGACGTCGCCTGGAGAAGAGTTCGACAAGGTTTTCACGGCGATTTGTGAAGGT AAAATCATTGATCCGA TGATGGAATGTCTCAACGAGTGGAACGGAGCTCCCATTCCAATATGTTAA

SEQ ID NO:13 Protein sequence of PAL1 from Arabidopsis thaliana

MEINGAHKSNGGGVDAMLCGGDIKTKNMVINAEDPLNWGAAAEQMKGSHLDEVKRMVAEF RKPVVNLGGETLTIGQ VAAISTIGNSVKVELSETARAGVNASSDWVMESMNKGTDSYGVTTGFGATSHRRTKNGVA LQKELIRFLNAGIFGS TKETSHTLPHSATRAAMLVRINTLLQGFSGIRFEILEAITSFLNNNITPSLPLRGTITAS GDLVPLSYIAGLLTGR PNSKATGPNGEALTAEEAFKLAGISSGFFDLQPKEGLALVNGTAVGSGMASMVLFETNVL SVLAEILSAVFAEVMS GKPEFTDHLTHRLKHHPGQIEAAAIMEHILDGSSYM LAQKLHEMDPLQKPKQDRYALRTSPQWLGPQIEVIRYAT KSIEREINSVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELV NDFYNNGLPSNLTASR NPSLDYGFKGAEIAMASYCSELQYLANPVTSHVQSAEQHNQDVNSLGLISSR TSEAVDILKLMSTTFLVAICQAV DLRHLEENLRQTVKNTVSQVAKKVLTTGVNGELHPSRFCEKDLLKVVDREQVYTYADDPC SATYPLIQKLRQVIVD HALINGESEKNAVTSIFHKIGAFEEELKAVLPKEVEAARAAYDNGTSAIPNRIKECRSYP LYRFVREELGTELLTG EKVTSPGEEFDKVFTAICEGKIIDPMMECLNE NGAPIPIC

DNA sequence encoding phenylalanine ammonia lyase 2 (PAL2) from

SEQ ID O:14

Arabidopsis thaliana codon optimized for expression in S. cerevisiae

ATGGACCAAATTGAAGCAATGCTATGCGGTGGTGGTGAAAAGACCAAGGTGGCCGTA ACGACAAAAACTCTTGCAG ATCCTTTGAATTGGGGTCTGGCAGCTGACCAGATGAAAGGTAGCCATCTGGATGAAGTTA AGAAGATGGTTGAGGA ATACAGAAGACCAGTCGTAAATCTAGGCGGCGAGACATTGACGATAGGACAGGTAGCTGC TATTTCGACCGTTGGC GGTTCAGTGAAGGTAGAACTTGCAGAAACAAGTAGAGCCGGAGTTAAGGCTTCATCAGAT TGGGTCATGGAAAGTA TGAACAAGGGCACAGATTCCTATGGCGTTACCACAGGCTTTGGTGCTACCTCTCATAGAA GAACTAAAAATGGCAC TGCTTTGCAAACAGAACTGATCAGATTCCTTAACGCCGGTATTTTCGGTAATACAAAGGA AACTTGCCATACATTA CCCCAATCGGCAACAAGAGCTGCTATGCTTGTTAGGGTGAACACTTTGTTGCAAGGTTAC TCTGGAATAAGGTTTG AAATTCTTGAGGCCATCACTTCACTATTGAACCACAACATTTCTCCTTCGTTGCCCTTAA GAGGAACAATAACTGC CAGCGGTGATTTGGTTCCCCTTTCATATATCGCAGGCTTATTAACGGGAAGACCTAATTC AAAGGCCACTGGTCCA GACGGAGAATCCTTAACCGCTAAGGAAGCATTTGAGAAAGCTGGTATTTCAACTGGTTTC TTTGATTTgCAACCCA AGGAAGGTTTAGCCCTGGTGAATGGCACCGCTGTCGGCAGCGGTATGGCATCCATGGTGT TGTTTGAAGCTAACGT ACAAGCAGTTTTGGCCGAAGTTTTGTCCGCAATTTTTGCCGAAGTCATGAGTGGAAAACC TGAGTTTACTGATCAC TTGACCCACAGGTTAAAACATCACCCAGGACAAATTGAAGCAGCAGCTATCATGGAGCAC ATTTTGGACGGCTCTA GCTACATGAAGTTAGCCCAGAAGGTTCATGAAATGGACCCTTTGCAAAAACCCAAACAAG ATAGATATGCTTTAAG GACATCCCCACAATGGCTTGGCCCTCAAATTGAAGTAATTAGACAAGCTACAAAGTCTAT AGAAAGAGAGATCAAC TCTGTTAACGATAATCCACTTATTGATGTGTCGAGGAATAAGGCAATACATGGAGGCAAT TTCCAGGGTACACCCA TAGGAGTCAGTATGGATAATACCAGGCTTGCCATAGCCGCAATTGGCAAATTAATGTTTG CCCAATTTTCTGAATT GGTCAATGACTTCTACAATAACGGTTTGCCTTCGAATCTGACCGCATCTTCTAACCCTAG TCTTGATTATGGTTTC AAAGGTGCTGAGATAGCAATGGCAAGCTATTGTTCAGAGCTGCAATATCTAGCCAACCCA GTAACCTCTCATGTAC AATCAGCCGAACAACACAATCAGGATGTTAATTCTTTGGGCCTGATTTCATCAAGAAAAA CAAGCGAGGCCGTTGA TATCCTTAAATTAATGTCCACAACATTTTTAGTGGGTATATGCCAGGCCGTAGATTTgAG ACACTTGGAAGAGAAT TTGAGACAGACAGTGAAAAATACCGTATCACAGGTTGCAAAAAAGGTTCTAACTACAGGT ATCAATGGTGAATTGC ACCCATCAAGATTCTGTGAAAAAGATTTATTAAAAGTTGTAGATAGAGAACAAGTATTTA CTTACGTTGACGATCC ATGTAGCGCTACTTATCCATTGATGCAGAGATTGAGACAAGTTATTGTAGATCACGCTTT ATCCAATGGTGAAACT GAGAAAAATGCCGTTACTTCAATATTCCAAAAGATAGGTGCCTTTGAAGAAGAACTGAAG GCAGTTTTACCAAAGG AAGTCGAAGCTGCTAGAGCCGCATACGGAAATGGTACTGCCCCTATACCAAATAGAATCA AAGAGTGTAGGTCGTA CCCTTTGTACAGATTCGTTAGAGAAGAGTTGGGAACCAAATTACTAACTGGTGAAAAAGT CGTTAGCCCAGGTGAA GAATTTGACAAGGTATTCACAGCTATGTGCGAGGGAAAGTTGATAGATCCACTTATGGAT TGCTTGAAAGAGTGGA ATGGTGCACCTATTCCAATCTGCTAA

SEQ ID NO:15 Protein sequence of PAL2 from Arabidopsis thaliana

MDQIEAMLCGGGEKTKVAVTTKTLADPLNWGLAADQMKGSHLDEVKKMVEEYRRPVVNLG GETLTIGQVAAISTVG GSVKVELAETSRAGVKASSDWVMESMNKGTDSYGVTTGFGATSHRRTKNGTALQTELIRF LNAGIFGNTKETCHTL PQSATRAAMLVRVNTLLQGYSGIRFEILEAITSLLNHNISPSLPLRGTITASGDLVPLSY IAGLLTGRPNSKATGP DGESLTAKEAFEKAGISTGFFDLQPKEGLALVNGTAVGSGMASMVLFEANVQAVLAEVLS AIFAEVMSGKPEFTDH LTHRLKHHPGQIEAAAIMEHILDGSSYMKLAQKVHEMDPLQKPKQDRYALRTSPQWLGPQ IEVIRQATKSIEREIN SVNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGL PSNLTASSNPSLDYGF KGAEIAMASYCSELQYLANPVTSHVQSAEQHNQDVNSLGLISSRKTSEAVDILKLMSTTF LVGICQAVDLRHLEEN LRQTVKNTVSQVAKKVLTTGINGELHPSRFCEKDLLKVVDREQVFTYVDDPCSATYPLMQ RLRQVIVDHALSNGET EKNAVTSIFQKIGAFEEELKAVLPKEVEAARAAYGNGTAPIPNRIKECRSYPLYRFVREE LGTKLLTGEKVVSPGE EFDKVFTAMCEGKLIDPLMDCLKEWNGAPIPIC

SEQ ID NO: 16 DNA sequence encoding phenylalanine ammonia lyase/tyrosine ammonia lyase (PAL/TAL) from Rhodosporidium toruloides codon optimized for expression in S. cerevisiae

ATGGCTCCATCATTGGATTCTATTTCTCATTCTTTTGCAAACGGTGTTGCATCTGCAAAA CAAGCTGTTAATGGTG CATCTACTAATTTGGCAGTTGCTGGTTCTCATTTACCAACTACCCAAGTTACACAAGTTG ATATTGTTGAAAAGAT GTTAGCAGCACCTACTGATTCTACCTTGGAATTGGATGGTTACTCTTTAAATTTAGGTGA TGTTGTTTCTGCAGCT AGAAAGGGTAGACCAGTTAGAGTTAAAGATTCTGATGAAATTAGATCTAAAATTGATAAA TCTGTTGAATTTTTGA GATCTCAATTATCAATGTCAGTTTATGGTGTTACAACTGGTTTCGGTGGTTCAGCTGATA CTAGAACTGAAGATGC AATTTCTTTACAAAAGGCATTGTTGGAACATCAATTATGTGGTGTTTTGCCTTCATCATT CGATTCTTTTAGATTA GGTAGAGGTTTAGAAAACTCTTTGCCATTAGAAGTTGTTAGAGGTGCAATGACAATTAGA GTTAATTCTTTAACAA GAGGTCATTCTGCTGTTAGATTGGTTGTTTTAGAAGCTTTGACTAACTTTTTGAACCATG GTATTACTCCAATTGT TCCATTAAGAGGTACAATTTCTGCATCTGGTGATTTGTCTCCTTTGTCTTATATTGCAGC TGCTATTTCAGGTCAT CCAGATTCAAAGGTTCATGTTGTTCATGAAGGTAAGGAAAAGATTTTATATGCAAGAGAA GCTATGGCTTTATTTA ATTTAGAACCAGTTGTTTTAGGTCCTAAGGAAGGTTTAGGTTTAGTTAACGGTACAGCTG TTTCAGCATCTATGGC TACCTTAGCTTTGCATGATGCTCATATGTTATCTTTGTTATCTCAATCATTAACAGCTAT GACTGTTGAAGCTATG GTTGGTCATGCTGGTTCTTTTCATCCATTCTTGCATGATGTTACCAGACCTCATCCAACA CAAATTGAAGTTGCTG GTAATATTAGAAAGTTGTTAGAAGGTTCTAGATTCGCAGTTCATCATGAAGAAGAAGTTA AAGTTAAGGATGATGA AGGTATTTTGAGACAAGATAGATACCCATTGAGAACTTCACCACAATGGTTGGGTCCATT GGTTTCTGATTTGATT CATGCTCATGCAGTTTTGACCATTGAAGCAGGTCAATCTACAACAGATAATCCATTGATT GATGTTGAAAACAAAA CATCACATCATGGTGGTAATTTTCAAGCAGCTGCTGTTGCTAATACAATGGAAAAGACAA GATTAGGTTTGGCACA AATTGGTAAGTTAAATTTCACACAATTAACTGAAATGTTGAATGCAGGTATGAATAGAGG TTTGCCATCTTGTTTG GCAGCTGAAGATCCTTCATTATCTTATCATTGTAAAGGTTTGGATATTGCAGCAGCAGCT TATACTTCAGAATTAG GTCATTTAGCAAATCCAGTTACTACACATGTTCAACCAGCTGAAATGGCTAATCAAGCTG TTAATTCTTTAGCATT GATTTCAGCTAGAAGAACCACTGAATCAAACGATGTTTTGTCATTATTATTAGCTACTCA TTTATATTGTGTTTTA CAAGCTATTGATTTGAGAGCAATTGAATTTGAATTTAAAAAGCAATTTGGTCCAGCTATT GTTTCATTAATTGATC AACATTTTGGTTCTGCAATGACTGGTTCAAATTTGAGAGATGAATTAGTTGAAAAGGTTA ACAAGACCTTGGCTAA AAGATTAGAACAAACTAACTCTTACGATTTGGTTCCAAGATGGCATGATGCTTTTTCTTT TGCTGCAGGTACAGTT GTTGAAGTTTTGTCATCTACCTCATTGTCTTTGGCAGCTGTTAACGCTTGGAAAGTTGCT GCTGCTGAATCAGCTA TTTCATTAACTAGACAAGTTAGAGAAACTTTTTGGTCTGCTGCTTCAACTTCTTCACCTG CTTTGTCTTACTTGTC TCCAAGAACTCAAATTTTGTACGCTTTCGTTAGAGAAGAATTGGGTGTTAAAGCTAGAAG AGGTGATGTTTTCTTA GGTAAGCAAGAAGTTACTATTGGTTCTAATGTTTCTAAAATTTACGAAGCTATTAAATCA GGTAGAATTAATAACG TTTTGTTGAAGATGTTAGCATAA

SEQ ID NO:17 Protein sequence of PAL/TAL from Rhodosporidium toruloides

MAPSLDSISHSFANGVASAKQAVNGASTNLAVAGSHLPTTQVTQVDIVEKMLAAPTDSTL ELDGYSLNLGDWSAA RKGRPVRVKDSDEIRSKIDKSVEFLRSQLSMSVYGVTTGFGGSADTRTEDAISLQKALLE HQLCGVLPSSFDSFRL GRGLENSLPLEVVRGAMTIRVNSLTRGHSAVRLVVLEALTNFLNHGITPIVPLRGTISAS GDLSPLSYIAAAISGH PDSKVHVVHEGKEKILYAREAMALFNLEPWLGPKEGLGLVNGTAVSASMATLALHDAHML SLLSQSLTAMTVEAM VGHAGSFHPFLHDVTRPHPTQIEVAGNIRKLLEGSRFAVHHEEEVKVKDDEGILRQDRYP LRTSPQWLGPLVSDLI HAHAVLTIEAGQSTTDNPLIDVENKTSHHGGNFQAAAVANTMEKTRLGLAQIGKLNFTQL TEMLNAGMNRGLPSCL AAEDPSLSYHCKGLDIAAAAYTSELGHLANPVTTHVQPAEMANQAVNSLALISARRTTES NDVLSLLLATHLYCVL QAIDLRAIEFEFKKQFGPAIVSLIDQHFGSAMTGSNLRDELVEKVNKTLAKRLEQTNSYD LVPRWHDAFSFAAGTV VEVLSSTSLSLAAVNAWKVAAAESAISLTRQVRETFWSAASTSSPALSYLSPRTQILYAF VREELGVKARRGDVFL GKQEVTIGSNVSKIYEAIKSGRINNVLLKMLA

DNA sequence encoding tyrosine ammonia lyase (TAL) from

SEQ ID NO:18 Rhodobacter capsulatus codon optimized for expression in S.

cerevisiae (Fluxome)

ATGACCCTGCAATCTCAAACAGCTAAAGATTGTTTGGCTTTGGATGGTGCCTTGACATTA GTTCAATGCGAAGCGA TAGCAACCCATAGAAGTAGAATCTCTGTAACACCAGCCCTACGTGAGAGATGTGCTAGAG CACATGCTAGGTTAGA ACATGCAATAGCCGAACAGCGACACATATATGGGATAACGACAGGCTTCGGGCCACTTGC TAACAGGCTGATCGGA GCAGACCAGGGTGCTGAATTACAACAGAACCTIATCTACCATTTGGCAACCGGAGTTGGC CCCAAATTATCATGGG CCGAAGCCAGAGCTTTAATGCTCGCTCGTTTGAATAGTATACTACAAGGTGCTTCTGGTG CTAGCCCTGAAACAAT TGATAGGATCGTTGCAGTCTTAAATGCCGGATTTGCCCCGGAAGTCCCAGCCCAAGGAAC CGTTGGTGCTTCGGGT GACTTAACTCCGTTAGCACACATGGTATTAGCATTGCAAGGCAGAGGTCGTATGATTGAT CCTTCAGGGAGAGTTC AAGAAGCCGGCGCTGTCATGGATAGGTTGTGTGGAGGCCCTTTAACATTGGCTGCCAGAG ATGGCCTCGCCTTAGT AAATGGTACATCTGCCATGACAGCTATTGCCGCATTGACCGGTGTGGAGGCTGCAAGAGC GATTGATGCAGCGCTT AGACATTCCGCAGTCTTGATGGAGGTCCTGTCAGGGCATGCTGAGGCTTGGCACCCTGCC TTTGCGGAATTGCGTC CGCATCCAGGACAATTACGCGCCACTGAGAGGTTAGCTCAAGCATTGGACGGCGCAGGTA GAGTCTGCCGGACTCT TACAGCCGCTAGGCGTCTAACTGCAGCTGATCTGAGACCAGAAGATCATCCAGCTCAAGA TGCATATTCACTTCGA GTAGTTCCTCAGCTGGTTGGTGCCGTATGGGATACGTTGGATTGGCACGACAGGGTTGTG ACTTGCGAACTTAACT CCGTGACCGACAATCCAATTTTCCCCGAGGGTTGTGCGGTTCCAGCACTACACGGTGGAA ACTTTATGGGCGTACA TGTGGCACTAGCTTCTGACGCTTTAAATGCAGCGTTGGTTACATTAGCTGGTCTAGTTGA AAGGCAGATTGCAAGA CTTACTGATGAGAAGTTGAATAAGGGTTTGCCTGCTTTTTTGCATGGAGGCCAAGCAGGT TTACAATCAGGTTTCA TGGGAGCTCAGGTTACTGCTACTGCTTTGCTAGCGGAAATGAGAGCTAACGCGACTCCCG TGTCCGTTCAAAGCCT CAGCACCAATGGTGCAAATCAAGACGTGGTAAGTATGGGTACGATTGCCGCGAGACGAGC AAGAGCTCAACTTTTA CCTCTGICICAAATCCAAGCGATTTTGGCACTGGCTCTTGCACAAGCCATGGATCTCCTA GACGATCCTGAAGGAC AAGCCGGTTGGTCCTTAACGGCAAGAGATTTAAGAGACCGTATACGGGCTGTCAGTCCAG GGTTGCGCGCAGATAG ACCACTAGCGGGTCATATTGAAGCTGTGGCTCAAGGTCTAAGACACCCCTCGGCAGCTGC CGATCCACCTGCTTAA

SEQ ID N0:19 Protein sequence of TAL from Rhodobacter capsulatus (Fluxome)

MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCARAHARLEHAIAEQ RHIYGITTGFGPLANRLIG ADQGAELQQNLIYHLATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAVLNA GFAPEVPAQGTVGASG DLTPLAHMVLALQGRGRMIDPSGRVQEAGAVMDRLCGGPLTLAARDGLALVNGTSAMTAI AALTGVEAARAIDAAL RHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALDGAGRVCRTLTAARRLTAA DLRPEDHPAQDAYSLR VVPQLVGAVWDTLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDALN AALVTLAGLVERQIAR LTDEKLNKGLPAFLHGGQAGLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNGANQDV VSMGTIAARRARAQLL PLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRPLAGHIEAV AQGLRHPSAAADPPA

DNA sequence encoding tyrosine ammonia lyase (TAL) from

SEQ ID NO:20 Rhodobacter capsulatus codon optimized for expression in S.

cerevisiae (see Koopman, F. et al. (2012))

ATGACCTTACAATCCCAAACTGCCAAAGACTGCTTAGCCTTAGACGGTGCCTTGACCTTG GTTCAATGTGAAGCAA TTGCCACACATAGATCCAGAATAAGTGTCACCCCAGCTTTGAGAGAAAGATGCGCTAGAG CACATGCCAGATTAGA ACACGCTATTGCAGAACAAAGACACATCTATGGTATAACTACAGGTTTTGGTCCTTTGGC TAATAGATTAATAGGT GCCGATCAAGGTGCTGAATTGCAACAAAACTTAATCTACCATTTGGCTACTGGTGTTGGT CCAAAATTGTCTTGGG CCGAAGCTAGAGCATTGATGTTGGCAAGATTGAACTCAATCTTGCAAGGTGCATCTGGTG CCTCACCTGAAACAAT CGACAGAATTGTTGCTGTCTTAAACGCTGGTTTCGCACCAGAAGTCCCTGCCCAAGGTAC TGTAGGTGCTTCCGGT GACTTGACACCATTGGCACATATGGTTTTGGCCTTACAAGGTAGAGGTAGAATGATTGAT CCTAGTGGTAGAGTTC AAGAAGCCGGTGCTGTCATGGACAGATTATGTGGTGGTCCATTGACTTTAGCTGCAAGAG ATGGTTTGGCTTTAGT TAATGGTACTTCTGCCATGACAGCTATCGCCGCTTTGACAGGTGTTGAAGCAGCCAGAGC TATTGATGCTGCATTA AGACATTCCGCAGTATTAATGGAAGTTTTGAGTGGTCATGCAGAAGCCTGGCACCCAGCT TTTGCAGAATTGAGAC CACACCCTGGTCAATTAAGAGCTACCGAAAGATTAGCCCAAGCTTTGGATGGTGCAGGTA GAGTTTGCAGAACCTT GACTGCCGCTAGAAGATTGACAGCAGCCGACTTAAGACCAGAAGATCATCCTGCACAAGA CGCCTATTCTTTGAGA GTTGTCCCACAATTAGTTGGTGCTGTCTGGGATACTTTGGACTGGCACGATAGAGTAGTT ACCTGTGAATTGAACT CAGTCACTGATAACCCAATATTTCCTGAAGGTTGCGCTGTACCTGCATTACATGGTGGTA ATTTCATGGGTGTACA CGTTGCATTGGCCTCCGACGCTTTAAACGCTGCATTAGTAACATTGGCTGGTTTAGTTGA AAGACAAATCGCAAGA TTGACCGATGAAAAGTTGAATAAGGGTTTGCCAGCATTTTTGCATGGTGGTCAAGCAGGT TTACAATCAGGTTTCA TGGGTGCTCAAGTTACAGCTACCGCATTGTTAGCAGAAATGAGAGCCAACGCTACCCCTG TCTCTGTACAATCTTT GTCAACTAATGGTGCTAACCAAGATGTCGTATCAATGGGTACTATCGCCGCTAGAAGAGC AAGAGCCCAATTGTTG CCATTGTCTCAAATCCAAGCAATCTTGGCTTTAGCATTGGCCCAAGCTATGGACTTGTTA GATGACCCTGAAGGTC AAGCAGGTIGGTCCTTGACAGCCAGAGACTTAAGAGATAGAAITAGAGCTGTTAGTCCAG GTTTGAGAGCTGATAG ACCTTTAGCAGGTCATATAGAAGCAGTCGCACAAGGTTTGAGACATCCATCCGCCGCAGC AGACCCTCCAGCCTAA

Protein sequence of TAL from Rhodobacter capsulatus (see

SEQ ID NO:21

Koopman, F. et al. (2012))

MTLQSQTAKDCLALDGALTLVQCEAIATHRSRISVTPALRERCARAHARLEHAIAEQRHI YGITTGFGPLANRLIG ADQGAELQQNLIYHLATGVGPKLSWAEARALMLARLNSILQGASGASPETIDRIVAVLNA GFAPEVPAQGTVGASG DLTPLAHMVLALQGRGRMIDPSGRVQEAGAVMDRLCGGPLTLAARDGLALVNGTSAMTAI AALTGVEAARAIDAAL RHSAVLMEVLSGHAEAWHPAFAELRPHPGQLRATERLAQALDGAGRVCRTLTAARRLTAA DLRPEDHPAQDAYSLR VVPQLVGAVWDTLDWHDRVVTCELNSVTDNPIFPEGCAVPALHGGNFMGVHVALASDALN AALVTLAGLVERQIAR LTDEKLNKGLPAFLHGGQAGLQSGFMGAQVTATALLAEMRANATPVSVQSLSTNGANQDV VSMGTIAARRARAQLL PLSQIQAILALALAQAMDLLDDPEGQAGWSLTARDLRDRIRAVSPGLRADRPLAGHIEAV AQGLRHPSAAADPPA

DNA sequence encoding tyrosine ammonia lyase (TAL) (SAM8) from

SEQ ID NO:22 Saccharothrix espanaensis codon optimized for expression in S.

cerevisiae

ATGACACAGGTAGTTGAAAGGCAGGCAGATAGGCTTAGTTCCAGGGAATATCTTGCCAGG GTCGTCAGGTCCGCTG GTTGGGATGCTGGTTTGACTTCCTGTACTGATGAGGAAATCGTGAGAATGGGTGCTAGTG CCAGAACAATTGAAGA GTACTTGAAGTCCGATAAACCTATATACGGCTTAACACAAGGATTTGGTCCACTTGTTCT ATTTGATGCCGATAGT GAATTAGAGCAAGGAGGTTCTTTAATCTCTCATCTAGGTACAGGCCAAGGTGCTCCTTTG GCCCCAGAAGTGTCAA GACTAATCTTATGGTTGAGAATACAGAATATGAGAAAAGGTTATTCCGCAGTGTCACCTG TATTCTGGCAGAAGTT AGCCGATCTATGGAATAAGGGTTTCACACCAGCTATTCCAAGGCACGGTACTGTCTCCGC ATCTGGCGATTTGCAG CCACTTGCTCATGCTGCTTTAGCATTCACTGGCGTTGGAGAAGCATGGACAAGAGATGCT GACGGCAGATGGAGCA CTGTTCCTGCAGTAGACGCTTTGGCTGCTTTGGGTGCAGAACCATTTGATTGGCCAGTTA GAGAGGCATTAGCTTT TGTTAATGGTACTGGCGCCTCATTGGCAGTAGCCGTGCTAAACCATAGGAGTGCTTTAAG ATTAGTGAGAGCCTGT GCCGTGTTGTCCGCAAGGTTAGCCACATTGCTTGGTGCCAATCCTGAGCATTATGATGTA GGTCATGGCGTTGCAA GAGGCCAAGTTGGTCAATTGACTGCAGCAGAATGGATCAGGCAAGGTTTACCTAGAGGTA TGGTCAGAGACGGAAG TAGGCCATTGCAAGAACCATACTCCTTAAGATGTGCTCCTCAAGTTTTAGGTGCCGTTTT GGACCAGTTAGATGGA GCTGGTGACGTATTAGCTAGGGAAGTCGACGGTTGTCAGGACAATCCTATAACTTACGAA GGAGAGTTGTTGCATG GTGGTAATTTCCATGCAATGCCAGTTGGTTTCGCATCTGATCAAATAGGTTTAGCAATGC ATATGGCCGCTTACTT GGCAGAAAGGCAGCTTGGTTTATTAGTTAGCCCTGTTACAAACGGTGACCTTCCACCAAT GTTAACCCCTAGGGCT GGTAGAGGCGCAGGACTAGCAGGTGTGCAGATATCCGCTACCAGTTTTGTTAGTAGAATT AGGCAGTTGGTGTTTC CTGCAAGCTTGACAACTTTGCCTACCAACGGATGGAATCAAGATCACGTCCCAATGGCAT TGAATGGCGCAAATTC AGTATTCGAAGCCTTAGAGTTGGGATGGTTAACTGTTGGTAGCTTGGCAGTAGGTGTTGC CCAATTAGCCGCCATG ACAGGTCACGCTGCTGAGGGTGTTTGGGCAGAACTTGCTGGTATTTGCCCTCCACTTGAT GCTGATAGACCTTTGG GAGCAGAAGTGAGGGCTGCTAGGGATCTTTTGTCTGCCCACGCTGATCAATTGTTAGTCG ATGAAGCTGATGGAAA AGACTTCGGATAATGA

SEQ ID NO:23 Protein sequence of TAL (SAM8) from Saccharothrix espanaensis

MTQVVERQADRLSSREYLARWRSAGWDAGLTSCTDEEIVRMGASARTIEEYLKSDKP IYGLTQGFGPLVLFDADS ELEQGGSLISHLGTGQGAPLAPEVSRLILWLRIQNMRKGYSAVSPVFWQKLADLWNKGFT PAIPRHGTVSASGDLQ PLAHAALAFTGVGEAWTRDADGRWSTVPAVDALAALGAEPFDWPVREALAFVNGTGASLA VAVLNHRSALRLVRAC AVLSARLATLLGANPEHYDVGHGVARGQVGQLTAAEWIRQGLPRGMVRDGSRPLQEPYSL RCAPQVLGAVLDQLDG AGDVLAREVDGCQDNPITYEGELLHGGNFHAMPVGFASDQIGLAMHMAAYLAERQLGLLV SPVTNGDLPPMLTPRA GRGAGLAGVQISATSFVSRIRQLVFPASLTTLPTNGWNQDHVPMALNGANSVFEALELGW LTVGSLAVGVAQLAAM TGHAAEGVWAELAGICPPLDADRPLGAEVRAARDLLSAHADQLLVDEADGKDFG

DNA sequence encoding phenylalanine ammonia lyase (PAL) from

SEQ ID NO:24 Petroselinum crispum codon optimized for expression in S.

cerevisiae

ATGGAAAATGGTAATGGTGCTACTACAAATGGCCATGTTAACGGTAATGGAATGGATTTT TGTATGAAAACCGAGG ACCCATTGTATTGGGGCATTGCAGCCGAAGCTATGACTGGTTCTCACTTAGATGAGGTAA AGAAAATGGTCGCAGA ATACAGAAAGCCTGTGGTTAAACTAGGTGGAGAAACACTTACCATATCACAAGTAGCCGC TATCTCAGCAAGGGAC GGAAGTGGTGTCACTGTGGAGTTGTCCGAAGCTGCTAGAGCAGGAGTTAAGGCTTCTTCA GATTGGGTAATGGACT CCATGAACAAAGGTACAGATAGTTATGGCGTCACCACTGGTTTCGGAGCCACAAGCCATA GGAGAACCAAGCAGGG CGGCGCATTACAAAAAGAACTAATTAGATTCTTGAATGCTGGTATATTCGGAAACGGTTC TGATAATACTTTGCCA CACTCAGCTACCAGGGCAGCTATGTTAGTTAGAATCAACACTTTGTTACAGGGCTACTCC GGAATTAGATTTGAAA TCCTTGAAGCCATCACCAAGTTTCTAAACCAAAATATTACACCTTGCTTGCCATTAAGGG GTACTATTACCGCAAG TGGCGACCTAGTGCCTTTGTCTTACATAGCTGGTTTACTTACTGGTAGACCAAACAGCAA AGCCGTTGGTCCTACT GGCGTAATCTTGTCACCAGAAGAGGCCTTTAAGTTAGCTGGTGTCGAAGGAGGTTTCTTT GAATTGCAACCTAAAG AAGGCCTTGCCTTGGTGAATGGAACAGCAGTTGGTTCCGGTATGGCCAGTATGGTATTAT TCGAAGCTAACATTCT AGCCGTCTTGGCAGAGGTTATGTCTGCTATATTTGCCGAAGTGATGCAGGGCAAGCCAGA GTTCACCGATCACTTA ACTCACAAACTTAAACATCATCCTGGACAAATCGAGGCAGCTGCCATTATGGAACACATA TTGGATGGCTCCGCAT ACGTTAAGGCTGCACAAAAGTTGCATGAAATGGACCCACTACAGAAGCCTAAACAAGATA GGTATGCTTTGAGAAC CTCACCTCAGTGGTTAGGTCCACAAATCGAGGTAATTAGAAGCTCCACTAAGATGATTGA AAGGGAGATCAATAGT GTCAACGACAATCCTCTTATCGATGTGTCAAGAAACAAAGCCATTCACGGCGGAAATTTT CAAGGTACCCCAATAG GCGTTTCTATGGACAACACAAGACTAGCAATCGCTGCCATTGGAAAGTTGATGTTTGCAC AGTTCAGCGAGTTAGT GAATGATTTTTACAATAACGGCCTTCCTTCCAACCTATCTGGCGGCAGGAACCCATCATT AGATTATGGATTCAAA GGTGCTGAAATAGCCATGGCATCCTACTGTAGCGAGCTACAGTTTTTGGCTAATCCTGTC ACTAACCATGTTCAAT CCGCCGAACAGCACAATCAAGACGTGAACAGTTTAGGTCTTATTTCATCTAGAAAGACCA GTGAGGCCGTTGAGAT ATTGAAACTAATGTCCACAACTTTCTTAGTAGGCTTGTGCCAGGCTATTGATCTTAGACA CTTAGAAGAAAATCTA AAGTCAACCGTCAAAAACACAGTTTCTAGTGTGGCTAAAAGGGTATTGACTATGGGAGTC AATGGTGAGTTACATC CAAGCAGATTTTGTGAAAAGGACCTTTTGAGGGTTGTGGATAGAGAATACATATTCGCCT ACATCGATGACCCTTG CAGTGCAACATATCCACTAATGCAGAAACTAAGACAAACATTGGTTGAGCACGCTCTTAA GAATGGCGATAACGAA AGGAATTTGAGTACTTCTATTTTTCAGAAAATAGCAACCTTCGAGGACGAACTAAAGGCA TTGTTACCTAAAGAAG TAGAGAGTGCTAGGGCCGCACTAGAAAGTGGAAACCCAGCTATCCCTAATAGAATTGAAG AGTGTAGGTCCTACCC ACTTTATAAGTTTGTCAGAAAGGAGTTGGGTACAGAATACTTAACCGGCGAGAAGGTTAC TAGTCCAGGCGAAGAA TTTGAGAAAGTGTTCATAGCCATGAGTAAGGGAGAAATTATCGATCCATTGTTAGAGTGT TTGGAGTCCTGGAACG GTGCACCACTACCTATTTGCTAA

SEQ ID NO:25 Protein sequence of PAL from Petroselinum crispum

MENGNGATTNGHVNGNGMDFCMKTEDPLYWGIAAEAMTGSHLDEVKKMVAEYRKPVVKLG GETLTISQVAAISARD GSGVTVELSEAARAGVKASSDWVMDSMNKGTDSYGVTTGFGATSHRRTKQGGALQKELIR FLNAGIFGNGSDNTLP HSATRAAMLVRINTLLQGYSGIRFEILEAITKFLNQNITPCLPLRGTITASGDLVPLSYI AGLLTGRPNSKAVGPT GVILSPEEAFKLAGVEGGFFELQPKEGLALVNGTAVGSGMASMVLFEANILAVLAEVMSA IFAEVMQGKPEFTDHL THKLKHHPGQIEAAAIMEHILDGSAYVKAAQKLHEMDPLQKPKQDRYALRTSPQWLGPQI EVIRSSTKMIEREINS VNDNPLIDVSRNKAIHGGNFQGTPIGVSMDNTRLAIAAIGKLMFAQFSELVNDFYNNGLP SNLSGGRNPSLDYGFK GAEIAMASYCSELQFLANPVTNHVQSAEQHNQDVNSLGLISSRKTSEAVEILKLMSTTFL VGLCQAIDLRHLEENL KSTVKNTVSSVAKRVLTMGVNGELHPSRFCEKDLLRVVDREYIFAYIDDPCSATYPLMQK LRQTLVEHALKNGDNE RNLSTSIFQKIATFEDELKALLPKEVESARAALESGNPAIPNRIEECRSYPLYKFVRKEL GTEYLTGEKVTSPGEE FEKVFIAMSKGEIIDPLLECLESWNGAPLPIC

DNA sequence encoding phenylalanine ammonia lyase (PAL) from

SEQ ID NO:26

Aspergillus niger codon optimized for expression in S. cerevisiae

ATGTTGGACAAGCACATCCCAGACGGTCACTTAGAAACCACTAGCGCCCACTGGAGG GATTTAAACCAAGTTGTTC AAAACGGTGAATTATCTATTGACGGTTACTCCTTGTCCTTGGCCGATGTTGTTGCTGTCG CTAAGTATGGTTGCCA ACCAAGATTGACTGACAAGCCAGAGACTATTGATGCTATTAACGGTTCTGTCATCGCCTT GGCTGAATGTTTAAGG GATGGTCATCACATTTACGGTGTTAACACTGGTTTTGGTGGTTCTGCCGATTCCAGAACC AACCAGACCACTACTT TGCAAAGCTCCTTGTTGCAATTGTTGCAATCCGGTATCTTAACTGCTTCTGACACTACCA ATGAAGGTTTGCAGTT GAACTTGCAAGGTCAAAGCAGCCATTCTATGCCATCTGAGTGGGTTAAAGCTACCATGTT GGTTCGTTCTAACTCT GTCGCTAGAGGCCATTCTGCTGTCAGCTTGCCAGCTATTTCCGCCATTTTGAGATTGATC AGAGAAGATATCGTCC CAGTTATTCCATTGAGAGGTACTATCTCCGCTTCCGGTGACTTGATGCCATTGGCTTACG TTGTCGGTGCCATTGA AGGTTCTCCAGGTATTTACGTTAGAGTCAAGGATGGTTCTGAACATCAAGTCGTTACCGC TCAAAAGGCCCTACAA ACTATCGGTGCTAAGGGTGTTACTTTGGGCCCTAAAGAGGGTTTAGGTTTGGTCAATGGT ACTGCTGCTTCTGGTG CCTTAGCTGGTTTGGTTTTGTATGAGGCTCATCAATTGGCCGTCTTGGCTCAAGCTGTCA CCGCCTTAACTGTCGA AGCTATTCAAGGTTCTACCGAATCCTTTCACCCTTTTATCGCTCAAGTCCGTCCACATGA AGGTCAGATCGAGGCT GCTGAAAACATCCTATCTCTATTAAAAGGTAGCTTGTTGGCCAGAGGTAGCTCTACTACC CAAACCAGAACCGGTC TAGTCCAAGACAGATACTCCTTGAGAACTGCTTCTCAATGGATCGGTCCTCAATTGGAAG ATTTATTGTTGGCCGA CAGACAGGTCCAAGTCGAACTAAATTCTACCAGCGACAACCCATTAATCGATACTGGTTC TAAAACTTTCTACACT GGTGGTAACTTCCAAGCTACCAGCATTACCTCCGCTATGGAAAAGACTAGGTTGGCTTTG CAAATGTTCGGTAAGA TGTTATTCGTCCAATGTAATGAAATGATCGACCCAAACTTGAACAACGGTCTACCTACCA ACTTGGTTGCTGATGA CCCATCCTTGTCCTTCACCATGAAAGGCGTCGATATCAACATGGCTGCTTATATGTCTGA ATTGGCTTACTTGGCT AATCCAGTCTCCTCCCACGTTCAAACTGCTGAAATGCAAAACCAAGCCTTGAACTCCTTG GCTTTCGTTAGCGCTA GGTATACTATGAAAGCTGTTGATATCGTCTCTATGATGGGTGCTTGTGCTTTGTATGTCG CTTGTCAAGCCTTAGA CTTGAGGGTCTTGCAATTGCGTTTCTTCCAAAGAGTCCAAGGTGTCGCTAAAGAAATCGC TCACGGTGCCTTTGGT AAGGCCTTGGAACCTTTCGAAATCGACCAGGTTGCTGATCACTTGTCTGAAGCTATTCAA AACTCCTGGCCATCTA CCTCTAGGTTGGACTTGAGAGACAGATGCAAAAGGGTTGCTGAAATGTTTATCCCAGTCT TGTTCGGTGCTTTGTT GCAAATTATCCCACAGAACAGACAAACCTCTGATTTATTCACCGCCATCTCTGCTTGTAA GATGATTTCCGTTTTT AAGTTGGAAGGCGTTTACAGAGAAGTTTTCGCTGAATTTTGCACTTCCCAACCTACCGCT GACTTTTTGGGTACCG GTACTAAGGAAATCTACACCTTCATCAGACACGACTTGAGAGTCCCATTCCACCAGGGTT TCGTCGAACATCCATC CGCCTCTCAAACCGACTTACCAGAAACTATCAACGGTAGAGTTAAAAAGACCGTCGGTGG TTGGATTTCTGTCGTT TACGAAGCCTTGAGAAATGGTACCTTAAGCGGTACTATTTTGAACTCCTTCCAACAATAA

SEQ ID NO:27 Protein sequence of PAL from Aspergillus niger

MLDKHIPDGHLETTSAHWRDLNQVVQNGELSIDGYSLSLADWAVAKYGCQPRLTDKPETI DAINGSVIALAECLR DGHHIYGVNTGFGGSADSRTNQTTTLQSSLLQLLQSGILTASDTTNEGLQLNLQGQSSHS MPSEWVKATMLVRSNS VARGHSAVSLPAISAILRLIREDIVPVIPLRGTISASGDLMPLAYVVGAIEGSPGIYVRV KDGSEHQVVTAQKALQ TIGAKGVTLGPKEGLGLVNGTAASGALAGLVLYEAHQLAVLAQAVTALTVEAIQGSTESF HPFIAQVRPHEGQIEA AENILSLLKGSLLARGSSTTQTRTGLVQDRYSLRTASQWIGPQLEDLLLADRQVQVELNS TSDNPLIDTGSKTFYT GGNFQATSITSAMEKTRLALQMFGKMLFVQCNEMIDPNLNNGLPTNLVADDPSLSFTMKG VDINMAAYMSELAYLA NPVSSHVQTAEMQNQALNSLAFVSARYTMKAVDIVSMMGACALYVACQALDLRVLQLRFF QRVQGVAKEIAHGAFG KALEPFEIDQVADHLSEAIQNSWPSTSRLDLRDRCKRVAEMFIPVLFGALLQIIPQNRQT SDLFTAISACKMISVF KLEGVYREVFAEFCTSQPTADFLGTGTKEIYTFIRHDLRVPFHQGFVEHPSASQTDLPET INGRVKKTVGGWISVV YEALRNGTLSGTILNSFQQ

DNA sequence encoding phenylalanine ammonia lyase (PAL) from

SEQ ID NO:28 Puccinia graminis f. sp. tritici codon optimized for expression in S.

cerevisiae

ATGGCTCATGCTGATTTGTGCTCCGCTATTTCTAGAGAATTGGAAGAACACATCTCCAAC AAGAGAGAAATTATCT TGGACGGTCATGGTTTGACTTCTACTGGTGTTGTTGGTGCTGCTAGATACAATGTTCAAG CCAAGATTTCTAACGA TCCAGCCTTGATTACTGTCGTTGAAGAATCTGTTGAATTCTTGGCCTCTAAGTTGGATAC TGCTATCTATGGTGTT ACCACCTTCTCTGAAAGATTAGATCCAAGACCAACTAACGCTTGCAACTTTTACTTTCAA GGTTCTGCTGATACCA GATCTGATTCTACTGCTGACTTGCAAATGGCTTTCTTGGAACATCAAGTTTCTGGTGTTT TGCCATTGTCCTCTAG ATCTACTTCTGCTGGTTTGTATTTGTCCGATCCAATGAACAATGTTATGCCTGAAGCTAT TACCAGAGGTGCCATT TTGATTAGAATCAACTCTTTGGTTAGAGGTCACTCCGCTTTGAGATTTCAAGTTTTGGAA ACTTTGATCACCTTGT TGAACAAGAACTTGACTCCAATGGTTCCATTGAGAGGTTCTATTTCTGCTTCTGGTGATT TGATGCCTTTGTCTTA TGTTGCTGCTGCAATTTGTGGTCATCCAGCCATTAGAATTATCGATAGATCTTCAGCCGA TGGTCACATCGAAATT ATGCCAGCTTCTGATGCTTTGACTAAGCACGGTATTACTCCAATAGTTTTGGGTCCAAAA GAAGGTTTGGCCATCT CTAATGGTACTGCTTTTTCTGCATCTGCTGCTTGTTTGGTTGCTCATGATTCTCATATGT TGTTGATGTTGGCTCA AGGTTTGACATCTATGACTGTTGAAGCTATGATGGGTCAAGCTCAATCTTTCGATCCTTT CATTCACGAAACTTGT AGACCACATCCAGGTCAAGTTGAAGTTGCTAAGACTATCAGATCTATGTTCGAAGGTTCC AGATTGGTTATCCACA TGGATGAAGAAAGATCCGTTGATCAAGAAAAGGACCAAGGTATCTTGAGACAAGATAGAT ATGCTTTGAGAACTGC TCCACAATGGTTGGGTCCACAATTAGAAGAATTGGTTACTGTCAACAAGACCTTGTGCAG AGAAATCAATGCTACT ACTGATAACCCATTGATCGACATCAAGAACAAAAAGATCTTGAACGGTGGTAACTTCCAA GCCATGTCTATTACCA ATTCTATGGAAAAGACCAGATCCTCCTTGGAATCCATTGGTAAATTGTCTTTCGCTCAAG CCATCGAATTGATGAA CTGTACTATGTCTAAAGGTTTGCCTTCTTGTTTGGCTGGTGATGAACCATCTACTAATTA CCATACAAAGGGTTTG GATATTAACATGGCTGCTTACACTGCTGAATTGGGTTTTTTGGCTTCTCCAGTTTCTACC CATGTTCAATCTGCTG AACAACACAATCAATCCGTTAACTCATTGGCTTTGGTTTCTGCCAGATACACAATTCAAG CTGTCGAAGTTTTGTC CATGTTGTTGTCATCTCACTTGTACGTTGTCTGCATGGCTATTGATTTGAGAGTTATCGA CCAAATGTTTCAAAAA GAATTGAAGGGTTTGTTGCCAGTTTTGTTGGATTCCCATTTTAAGTCTAGACCAACTCAA GCTGCTGATCCATTGA TTGGTGCTTTAGCTTCTAGATTGGAAGCTACTGCTTCTTTGGATTCTGAAGCTAGATTTT TGTCCGCTTTCAAGCA AACCTTGCATGTTATTTTAGCCTTCCCAGTTGATTTGGAAGAAGCTAGAAGTTGGCCATC ATTTGCTGCTTCTCAA TCTACTTTGTTGTACAAGAGAACCAGAGATCAATACTTCGAAAACTCCGAATCTTTCTTG GCTGAAAAGTGGTTGG GTAAAAAGAACAAGCACTTGTACCACTTCGTCAGAAAAGAATTAGGTATCGGTCCTAGAA GAGGTGATGTTAGATT GGGTAGACATGAAGGTTCCGTTTCCATTGATGTTTCTAAGATCTACGAATCCGTCAGATC CGGTGAATTATACAAG TTTATGAACAGAATGTTTTAA

SEQ ID NO;29 Protein sequence of PAL from Puccinia graminis f. sp. tritici

MAHADLCSAISRELEEHISNKREIILDGHGLTSTGVVGAARYNVQAKISNDPALI VVEESVEFLASKLDTAIYGV TTFSERLDPRPTNACNFYFQGSADTRSDSTADLQMAFLEHQVSGVLPLSSRSTSAGLYLS DPMNNVMPEAITRGAI LIRINSLVRGHSALRFQVLETLITLLNKNLTPMVPLRGSISASGDLMPLSYVAAAICGHP AIRIIDRSSADGHIEI MPASDALTKHGITPIVLGPKEGLAISNGTAFSASAACLVAHDSHMLLMLAQGLTSMTVEA MMGQAQSFDPFTHETC RPHPGQVEVAKTIRSMFEGSRLVIHMDEERSVDQEKDQGILRQDRYALRTAPQWLGPQLE ELVTVNKTLCREINAT TDNPLIDIKNKKILNGGNFQAMSITNSMEKTRSSLESIGKLSFAQAIELMNCTMSKGLPS CLAGDEPSTNYHTKGL DINMAAYTAELGFLASPVSTHVQSAEQHNQSVNSLALVSARYTIQAVEVLSMLLSSHLYW CMAIDLRVIDQMFQK ELKGLLPVLLDSHFKSRPTQAADPLIGALASRLEATASLDSEARFLSAFKQTLHVILAFP VDLEEARSWPSFAASQ STLLYKRTRDQYFENSESFLAEKWLGKKNKHLYHFVRKELGIGPRRGDVRLGRHEGSVSI DVSKIYESVRSGELYK FMNRMF

DNA sequence encoding tyrosine ammonia lyase (TAL) from

SEQ ID NO:30 Aeromonas salmonicida subsp. salmonicida A449 (Asal) codon

optimized for expression in S. cerevisiae

ATGAACAAGTCCGAAATGAAGTACGTTACCTTTGGTGCTGAACCATTGACCATTGAAGAT GTTGTTGCTTTGGCTG AAAGAAGAGCTAGACCAGCTTTGAATAGAGATCCAGCTTTTATGGCCAGAATTCAAAGAG GTGCTGATTTCTTGGA TAGATTATTGGCCGAAGAAGGTGTTATCTACGGTGTTACTACTGGTTATGGTGATTCTGT TACTAGACCAGTTCCA GCTGAATTGGTTCCAGAATTGCCATTGCATTTGACTAGATTTCATGGTTGTGGTTTGGGT GAAGATTTGGAATTGG ATGCTGGTAGAGCTGTTTTGGCTACTAGATTGTGTTCTTTGGCTCAAGGTGTTTCTGGTG TTTCACCAGGTTTGTT GGAAAGATTATGTTGGTTGTTGGAACAAGACTTGATCCCAAGAATTCCTGAAGAAGGTTC TGTTGGTGCTTCTGGT GATTTGACTCCATTGTCTTATGTTGCTGCTGTATTGGTTGGTGAAAGAGAATTGCATCAC GACGGTGCTTTAAGAC CAGCTGCTGAAGTTTATCAAGAATTGGGTATTACCCCATTGACCTTAAGACCAAAAGAAG GTTTGGCTTTGATGAA CGGTACTTCTGTTATGACTGCTTTAGCTTGTTTGGCTTATGCTAGAGCTGATTACTTAAT GCAATTGGCCACTAGA ATTACCGCCTTGGTTTCTGTTGCTATGGGTGGTAATGCTTTTCATTTCGACGAAAGATTA TTCGCCGTTAAGCCAC ATCCAGGTATGCAAGGTATTGCTGCTTGGTTGAGATCTGATTTGGTTGCTGGTGAATTGC CAAGACATTCTGATAG ATTGCAAGACAGATACTCTTTGAGATGTGCCCCACATGTTATTGGTGTTGTTGCAGATTC TTTGCCATGGTGGAGA CAATTGATTGAAAACGAATTGAACTCCGCCAACGATAACCCATTGATTGATGGTGAAGGT GAACATGTTATGCATG GTGGTCATTTTTACGGTGGTCATATTGCTATGGCTATGGATTCTATGAAGACCGCTATTG CTAATTTGGCCGATTT GTTGGACAGACAATTGGCTCAATTGGTTGATACCAAGTTTAATGGTGGTTTGCCATCTAA TTTGTCTGGTGCTCCA GCTGGTAGACAAATGATCAATCATGGTTTTAAGGCCGTTCAAATTGGTGTTAGTGCTTGG ACTGCTGAAGCTTTGA AACAAACTATGCCAGCTTCTGTTTTCTCCAGATCTACCGAATGTCACAATCAAGACAAAG TCTCCATGGGTACAAT TGCTGCTAGAGATGCTTTGAGAGTTTTGACTTTGACTGAACAAGTTGGTGCTGCTTGTTT GTTGGCTGCTGTTCAA GGTGTAGAATTGAGATTAGCTCAACCTACTCCATTCACTAGACCATTATCTCCAGCTTTA GCTCACATGGTTCAAC AAGTTAGAGCTGAATTTGCCCCATTATTGGAAGATAGAGCCTTAGAACAAGAATTGAGAG CTTTGATTGCCAGAAT CAGATTGAGACACTACCCTTTGTACCAAGAATCCTCTTTGTGA CTTCGAAGGTATTGCCGAACAAAACATTCCAATCTACGGTGTTACTACTGGTTACGGTGA AATGATCTATATGCAA GTCGACAAGTCCAAAGAAGTTGAATTGCAAACTAACTTGGTCAGATCTCATTCTGCTGGT GTTGGTCCATTATTCG CTGAAGATGAAGCTAGAGCTATAGTTGCTGCTAGATTGAATACTTTGGCTAAAGGTCATT CAGCTGTCAGACCAAT TATCTTGGAAAGATTGGCTCAATACTTGAACGAAGGTATCACTCCAGCTATTCCAGAAAT TGGTTCTTTGGGTGCT TCTGGTGATTTGGCTCCATTGTCTCATGTTGCTTCTACTTTGATTGGTGAAGGTTACGTT TTGAGAGATGGTAGAC CAGTTGAAACTGCTCAAGTTTTGGCTGAAAGAGGTATTGAACCATTGGAATTGAGATTCA AAGAAGGTTTGGCCTT GATTAACGGTACTTCTGGTATGACTGGTTTGGGTTCTTTAGTTGTTGGTAGAGCTTTGGA ACAAGCTCAACAAGCT GAAATAGTTACCGCCTTGTTGATAGAAGCTGTTAGAGGTTCTACTTCTCCATTCTTAGCT GAAGGTCATGATATTG CTAGACCACATGAAGGTCAAATTGATACTGCTGCTAATATGAGAGCTTTGATGAGAGGTT CTGGTTTGACAGTTGA ACATGCTGATTTGAGAAGAGAATTACAAAAGGACAAAGAAGCCGGTAAGGACGTTCAAAG ATCTGAAATCTACTTG CAAAAGGCCTACTCCTTGAGAGCTATTCCTCAAGTTGTAGGTGCAGTTAGAGATACCTTG TATCATGCTAGACACA AGTTGAGAATCGAATTGAATTCCGCTAACGACAACCCTTTGTTCTTTGAAGGTAAAGAAA TTTTCCACGGTGCCAA CTTTCATGGTCAACCTATTGCTTTTGCTATGGACTTCGTTACCATTGCTTTGACTCAATT GGGTGTTTTAGCCGAA AGACAAATCAACAGAGTTTTGAACAGACACTTGTCTTACGGTTTGCCAGAATTTTTGGTT TCAGGTGATCCAGGTT TACATTCTGGTTTTGCTGGTGCTCAATATCCAGCTACTGCTTTGGTTGCTGAAAACAGAA CTATTGGTCCAGCTTC TACACAATCTGTTCCATCTAATGGTGATAATCAAGACGTTGTCTCCATGGGTTTGATTTC TGCTAGAAATGCAAGA AGAGTCTTGTCCAACAACAACAAGATTTTGGCAGTCGAATATTTGGCTGCTGCTCAAGCT GTTGATATTTCTGGTA GATTCGATGGTTTGTCTCCAGCTGCTAAAGCAACTTATGAAGCTGTAAGAAGATTGGTTC CAACCTTGGGTGTTGA CAGATATATGGCTGATGATATTGAATTGGTTGCCGATGCTTTGTCTAGAGGTGAATTTTT GAGAGCCATTGCTAGA GAAACCGACATCCAATTGAGATAA

SEQ ID NO:35 Protein sequence of TAL from Streptomyces globisporus

MALTQVETEIVPVSVDGETLTVEAVRRVAEERATVDVPAESIAKAQKSREIFEGIAEQNI PIYGVTTGYGEMIYMQ VDKSKEVELQTNLVRSHSAGVGPLFAEDEARAIVAARLNTLAKGHSAVRPIILERLAQYL NEGITPAIPEIGSLGA SGDLAPLSHVASTLIGEGYVLRDGRPVETAQVLAERGIEPLELRFKEGLALINGTSGMTG LGSLVVGRALEQAQQA EIVTALLIEAVRGSTSPFLAEGHDIARPHEGQIDTAANMRALMRGSGLTVEHADLRRELQ KDKEAGKDVQRSEIYL QKAYSLRAIPQVVGAVRDTLYHARHKLRIELNSANDNPLFFEGKEIFHGANFHGQPIAFA MDFVTIALTQLGVLAE RQINRVLNRHLSYGLPEFLVSGDPGLHSGFAGAQYPATALVAENRTIGPASTQSVPSNGD NQDVVSMGLISARNAR RVLSNNNKILAVEYLAAAQAVDISGRFDGLSPAAKATYEAVRRLVPTLGVDRYMADDIEL VADALSRGEFLRAIAR ETDIQLR

DNA sequence encoding phenylalanine ammonia lyase/tyrosine

SEQ ID NO:36 ammonia lyase (PAL/TAL) from Rhodotorula graminis codon

optimized for expression in S. cerevisiae

ATGGCCCCATCTTTGGATTCTTTGGCTACTACTTTGGCTAACGGTTTCACTAATGGTTCT CATGCTGCTCCAACAA AATCTGCTGCTGGTCCAACTTCTGCTTTGAGAAGAACTCCAGGTTTGGATGGTCATGCTG CACATCAATCTCAATT GGAAATCGTTCAAGAATTATTGTCCGATCCAACCGATGATGTTGTTGAATTGTCTGGTTA CTCTTTGACCGTTAGA GATGTTGTAGGTGCTGCTAGAAAAGGTAGAAGAGTTAGAGTTCAAAACGACGACGAAATT AGAGCCAGAGTTGATA AGTCTGTTGATTTCTTGAAGGCCCAATTGCAAAACTCTGTTTACGGTGTTACTACTGGTT TTGGTGGTTCTGCTGA TACAAGAACTGAAGATGCTGTTTCCTTGCAAAAGGCCTTGATTGAACATCAATTGTGTGG TGTTACTCCAACCTCT GTTTCTTCATTTTCTGTTGGTAGAGGTTTGGAAAACACCTTGCCATTGGAAGTTGTTAGA GGTGCTATGGTTATTA GAGTCAACTCATTGACTAGAGGTCATTCCGCTGTTAGATTGGTTGTTTTGGAAGCTTTGA CCAACTTCTTGAACCA TAGAATTACTCCAATCGTCCCATTGAGAGGTTCTATTTCTGCTTCTGGTGATTTGTCTCC ATTGTCTTATATTGCT GGTGCTATTACTGGTCACCCAGATGTTAAGGTTCATGTTTTACATGAAGGTACTGAAAAG ATCATGTTCGCCAGAG AAGCTATTTCTTTGTTTGGTTTGGAAGCAGTTGTCTTGGGTCCAAAAGAAGGTTTGGGTT TGGTTAATGGTACTGC TGTTTCAGCTTCTATGGCTACTTTGTCATTGCATGATTCCCACATGTTGTCCTTGTTGTC TCAAGCTTTAACTGCC TTGACTGTTGAAGCTATGGTTGGTCAACAAGGTTCTTTTGCTCCATTCATTCATGATGTC TGTAGACCACATCCAG GTCAAGTTGAAGTTGCTAGAAACATCAGAACTTTGTTGTCCGGTTCATCTTTCGCTGTTG AACATGAAGAAGAAGT TAAGGTTAAGGACGACGAAGGTATTTTGAGACAAGATAGATATCCATTGAGAACTTCCCC ACAATTTTTGGGTCCA TTGGTTGAAGATATGATGCATGCTTACTCTACCTTGTCCTTAGAAAACAACACTACTACC GATAACCCTTTGTTGG ATGTCGAAAACAAACAAACTGCTCATGGTGGTAATTTTCAAGCTTCTGCTGTCTCTATCT CTATGGAAAAAACTAG ATTGGCTTTGGCCTTGATCGGTAAGTTGAATTTCACTCAATGCACCGAATTATTGAACGC TGCTATGAATAGAGGT TTACCATCTTGTTTGGCTGCTGAAGATCCATCTTTGAACTATCATGGTAAGGGTTTGGAT ATTCATATTGCTGCTT ACGCTTCAGAATTGGGTCATTTGGCTAATCCAGTTACTACTTTTGTTCAACCAGCCGAAA TGGGTAATCAAGCCGT TAATTCTTTAGCCTTGATTTCCGCTAGAAGAACTGCTGAAGCTAACGATGTTTTGTCTTT GTTGTTGGCTTCTCAC TTGTACTGTACATTGCAAGCAGTTGATTTGAGAGCCATGGAATTGGATTTCAAGAAGCAA TTCGATCCTTTGTTGC CTACCTTGTTACAACAACATTTGGGTACTGGTTTGGACGTTAATGCTTTGGCTTTAGAAG TCAAGAAGGCTTTGAA CAAGAGATTGGAACAAACTACCACCTACGATTTGGAACCTAGATGGCATGATGCTTTTTC TTATGCTACTGGTACT GTCGTTGAATTATTGAGTTCTTCCCCATCTGCTAACGTTACTTTGACTGCTGTTAACGCT TGGAAAGTTGCATCTG CTGAAAAGGCTATTTCCTTGACAAGAGAAGTCAGAAACAGATTCTGGCAAACTCCATCTT CTCAAGCTCCAGCTCA TGCTTATTTGTCACCAAGAACTAGAGTCTTGTACTCCTTCGTTAGAGAAGAATTAGGTGT CCAAGCTAGAAGAGGT GATGTTTTTGTTGGTGTTCAACAAGAAACCATCGGTTCCAATGTTTCAAGAATCTACGAA GCCATTAAGGACGGTA GAATCAATCACGTTTTGGTTAAGATGTTGGCTTAA

SEQ ID NO:37 Protein sequence of PAL/TAL from Rhodotorula graminis

MAPSLDSLATTLANGFTNGSHAAPTKSAAGPTSALRRTPGLDGHAAHQSQLEIVQELLSD PTDDVVELSGYSLTVR DVVGAARKGRRVRVQNDDEIRARVDKSVDFL AQLQNSVYGVTTGFGGSADTRTEDAVSLQKALIEHQLCGVTPTS VSSFSVGRGLENTLPLEWRGAMVIRVNSLTRGHSAVRLVVLEALTNFLNHRITPIVPLRG SISASGDLSPLSYIA GAITGHPDVKVHVLHEGTEKIMFAREAISLFGLEAVVLGPKEGLGLVNGTAVSASMATLS LHDSHMLSLLSQALTA LTVEAMVGQQGSFAPFIHDVCRPHPGQVEVARNIRTLLSGSSFAVEHEEEVKVKDDEGIL RQDRYPLRTSPQFLGP LVEDMMHAYSTLSLENNTTTDNPLLDVENKQTAHGGNFQASAVSISMEKTRLALALIGKL NFTQCTELLNAAMNRG LPSCLAAEDPSLNYHGKGLDIHIAAYASELGHLANPVTTFVQPAEMGNQAVNSLALISAR RTAEANDVLSLLLASH LYCTLQAVDLRAMELDFKKQFDPLLPTLLQQHLGTGLDVNALALEVKKALNKRLEQTTTY DLEPRWHDAFSYATGT VVELLSSSPSANVTLTAVNAWKVASAEKAISLTREVRNRFWQTPSSQAPAHAYLSPRTRV LYSFVREELGVQARRG DVFVGVQQETIGSNVSRIYEAIKDGRINHVLVKMLA

DNA sequence encoding phenylalanine ammonia lyase (PAL) from

SEQ ID NO:38

Bambusa oldhamii codon optimized for expression in S. cerevisiae

ATGGCTGGTAATGGTCCAATCGTTAAGGATGATCCATTGAATTGGGGTGCTGCTGCT GCAGAATTGACTGGTTCTC ATTTTGATGAAGTCAAGAGAATGGTTGCCCAATTCAGAGAACCTGTTATTAAGATTGAAG GTGCCTCTTTGAGAGT TGGTCAAGTTGCTGCTGTTGCTCAAGCTAAAGATGTTTCTGGTGTTGCTGTTGAATTGGA TGAAGAAGCTAGACCA AGAGTTAAGGCTTCTTCTGAATGGATTTTGAACTGTTTGGCTCATGGTGGTGATATCTAT GGTGTTACTACTGGTT TTGGTGGTACTTCCCATAGAAGAACAAAAGATGGTCCAGCATTGCAAGTTGAATTATTGA GACATTTGAACGCCGG TATTTTCGGTACTGGTACTGATGGTCATACTTTGCCATCTGAAGTTACTAGAGCTGCTAT GTTGGTTAGAATCAAC ACTTTGTTGCAAGGTTACTCCGGTATCAGATTCGAAATTTTGGAAGCCATTACCAAGTTG ATCAATACTGGTGTTA CACCATGTTTGCCATTGAGAGGTACTATTACTGCTTCTGGTGATTTGGTTCCATTGTCTT ATATTGCCGGTTTGAT TACTGGTAGACCAAATGCTCAAGCAGTTGCTCCAGATGGTAGAAAAGTTGATGCTGCTGA AGCTTTTAAGATTGCT GGTATAGAAGGTGGTTTCTTCAAGTTGAACCCAAAAGAAGGTTTGGCTATCGTTAACGGT ACTTCTGTTGGTTCTG CTTTGGCTGCTACTGTCTTGTATGATTGCAATGTTTTGGCCGTTTTGTCCGAAGTTTTGT CTGCTGTTTTTTGCGA AGTTATGAACGGTAAGCCAGAATACACTGATCATTTGACCCATAAGTTGAAACATCACCC AGGTTCTATTGAAGCT GCTGCTATTATGGAACATATTTTGGCTGGTTCCTCTTTCATGTCCCATGCTAAAAAAGTT AACGAAATGGACCCTT TGTTGAAGCCTAAGCAAGATAGATATGCTTTGAGAACTTCTCCACAATGGTTGGGTCCAC AAATTGAAGTTATAAG AGCAGCCACCAAGTCCATCGAAAGAGAAGTTAATTCCGTTAATGACAACCCAGTTATCGA TGTTCATAGAGGTAAG GCTTTACATGGTGGTAATTTTCAAGGTACTCCAATCGGTGTTTCTATGGACAACACTAGA TTGGCTATTGCTAACA TCGGTAAGTTGATGTTCGCTCAATTTTCCGAATTGGTCAACGAATTCTACAACAACGGTT TGACTTCTAATTTGGC CGGTTCTAGAAATCCATCTTTGGATTACGGTTTCAAGGGTACTGAAATTGCTATGGCTTC TTACTGCTCCGAATTG CAATATTTGGCTAACCCAATCACCAACCATGTTCAATCTGCTGAACAACACAATCAAGAC GTTAACTCTTTGGGTT TGGTTTCTGCTAGAAAAACAGCTGAAGCCGTTGATATCTTGAAGTTGATGAGTTCTACTT ACATGGTTGCTTTGTG CCAAGCTGTTGATTTGAGACACTTGGAAGAAAACATCAAGTCCTCTGTTAAGAACTGCGT TACCCAAGTTGCTAAA AAGGTTTTGACTATGAACCCAACCGGTGATTTGTCATCTGCTAGATTTTCTGAAAAGAAC TTGTTGACCGCCATTG ATAGAGAAGCTGTTTTCACTTATGCTGATGATCCTTGTTCTGCTAACTACCCATTGATGC AAAAATTGAGAGCCGT TTTGGTTGATCATGCTTTGACATCTGGTGATGCTGAAAGAGAACCTTCTGTTTTCTCTAA GATCACCAAGTTCGAA GAAGAATTGAGATCTGCTTTGCCAAGAGAAATTGAAGCAGCTAGAGTTGCAGTTGCTGAT GGTACAGCTCCAATTG CTAATAGAATCAAAGAATCCAGATCCTTCCCAGTCTACAGATTCGTTAGAGAAGAATTAG GTTGCGTTTACTTGAC CGGTGAAAAATTGAAATCTCCAGGTGAAGAATGCAACAAGGTTTTCATTGGTATCTCCCA AGGTAAATTGATCGAC CCAATGTTGGAATGCTTGAAAGAATGGAATGGTGAACCATTGCCAATCAACTGA

SEQ ID NO:39 Protein sequence of PAL from Bambusa oldhamii

MAGNGPIVKDDPLNWGAAAAELTGSHFDEVKRMVAQFREPVIKIEGASLRVGQVAAVAQA KDVSGVAVELDEEARP RVKASSEWILNCLAHGGDIYGVTTGFGGTSHRRTKDGPALQVELLRHLNAGIFGTGTDGH TLPSEVTRAAMLVRIN TLLQGYSGIRFEILEAITKLINTGVTPCLPLRGTITASGDLVPLSYIAGLITGRPNAQAV APDGRKVDAAEAFKIA GIEGGFF LNPKEGLAIVNGTSVGSALAATVLYDCNVLAVLSEVLSAVFCEVMNGKPEYTDHLTHKLK HHPGSIEA AAIMEHILAGSSFMSHAKKVNEMDPLLKPKQDRYALRTSPQWLGPQIEVIRAATKSIERE VNSVNDNPVIDVHRGK ALHGGNFQGTPIGVSMDNTRLAIANIGKLMFAQFSELVNEFYNNGLTSNLAGSRNPSLDY GFKGTEIAMASYCSEL QYLANPITNHVQSAEQHNQDVNSLGLVSARKTAEAVDILKLMSSTYMVALCQAVDLRHLE ENIKSSVKNCVTQVAK KVLTMNPTGDLSSARFSEKNLLTAIDREAVFTYADDPCSANYPLMQKLRAVLVDHALTSG DAEREPSVFSKITKFE EELRSALPREIEAARVAVADGTAPIANRIKESRSFPVYRFVREELGCVYLTGEKLKSPGE ECNKVFIGISQGKLID PMLECLKEWNGEPLPIN

DNA sequence encoding histidine ammonia lyase/phenylalanine

SEQ ID NO:40 ammonia lyase (HAL/PAL) from Neosartorya fischeri codon optimized for expression in S. cerevisiae

ATGCCAGCTTCTTGGGTTAGAGGTACTATGTTGGTTAGATGTAACTCTAACGCTAGAGGT CATTCTGCTGTTTCTT TGCCAGCTATTGAATCCTTGTTGAGATTGATCGAAAACCACATTACTCCAGTTGTTCCAT TGAGAGGTTCTATTTC TGCTTCTGGTGATTTGATGCCATTGTCTTATATTGCTGGTGCTATTGAAGGTTCCCCAGA TGTTTATGTTCAAGTT CAAGATTCCGACAAGACCAGAATCATGAATTCTAGAGATGCTTTGTTGTCCACTGGTTTG GAAGCTCAAACTTTGG GTCCAAAAGAAGGTTTGGGTTTGGTTAATGGTACTTCTGCTTCAGCTGCTTTGGCTTCTT TGGCTATGTATGAAGC TCATCAATTGGCCGTTTTGGTTCAAGCTTTGTCTGCTTTGACTGTTGAAGCTTTGATGGG TAATGCTGAATCTTTC CATCCATTCATTTCCGCCATTAGACCACATGATGGTCAAATTGAATGTGCCAGAAACGTC ATGTCTTTCTTGCAAG GTTCTCAATTGGCTCAAAACTTGGAAAGAAACTTGAAGGACAGAAACAGACCAGGTTTGA TCCAAGATAGATACGC TTTGAGAACTGTTCCACAATGGATTGGTCCACAATTGGAAGATTTGTTGTTGGCCCATAG ACAAGTTACCGTTGAA TTGAACTCTTCTTGCGATAACCCATTGGTTGATGCTCAATCCGATGATATTTTCTACGGT GGTAATTTCCAAGCCG TTTCTATTACATCTGCTATGGAAAAGACTAGAACCTGCTTGCAAATGTTCGGTAGATTGT TGTTTGCTCAAGCCAC CGAATTGATTGATCCATCTTTGAACAATGGTTTGCCTACCAATTTGGTTGCTGATGATCC ATCCTTGTCTTTCACT ATGAAGGGTGTTGATATTTCCATGGCTTCTTACATGGCTGAATTGGCTTACTTGGCTAAT CCAGTTTCTTCTCATG TTCAAACCGCCGAAATGCACAATCAATCTGTTAATTCTATGGCCTTCGTTTCCTCTAGAT ACACTATGCAAGCTGT CGAAATCGTTTCTTTGATGTGTGCTTGCTCTGTTTACATTGGTTGCCAAGCTTTGGATTT GAGAGTCTTGCATTTG ACATTCTTGCAAAGATCTACCCCACAATTGCATACTTTGACCTCTCATTTGTTCTCCGAA CACTTGTTTGAACCAG ATTTGGCTACTTTGAATGAAGCCTTGTCTACCCATATTCAAAAGTCTTGGCCAACTACTA CCAGATTGAACATTAC CGATAGAGTTGAAGAAGTTGTTACCTCCGCTATTCCAATTTTGTGTAGAACTTTCGCTTC TTCCACTGGTACATCT ACTTCTCAAGCTCCAACTTTCTCTGATTTGGAAACCTGGAAATCTAGAGCTTCAGCTTTG TTGAACGAAATCTACC AAGATACTGCTCATGCCTTCTTCTCTTACCAACATACTGAAGAAATGTTGGGTACTTCCT CTAAGATCTTGTACCA AACCGTTAGAAGACAATTGGGTGTTCCATTTCATCAAGGTTTCATTGAACATCCAACCGC TCAATCTGATACTTTG GGTGGTAGACCAAAAAAGACTGTTGGTTCTTGGATCTCCATTATCTACGAAGCTATTAGA GAAGGTAGATTGATGG ATCCTTTGATGGCATCTTTACAAGCTGGTGTTGCTGGTGAATCAGATACTGAAGCTGTTG ATACTTTAAAGGACGG TTCTTCTGGTAAGTGTTCTTCTTCAGGTTTGGACTGA

SEQ ID NO:41 Protein sequence of HAL/PAL from Neosartorya fischeri

MPASWVRGTMLVRCNSNARGHSAVSLPAIESLLRLIENHITPVVPLRGSISASGDLMPLS YIAGAIEGSPDVYVQV QDSDKTRIMNSRDALLSTGLEAQTLGPKEGLGLVNGTSASAALASLAMYEAHQLAVLVQA LSALTVEALMGNAESF HPFISAIRPHDGQIECARNVMSFLQGSQLAQNLERNLKDRNRPGLIQDRYALRTVPQWIG PQLEDLLLAHRQVTVE LNSSCDNPLVDAQSDDIFYGGNFQAVSITSAMEKTRTCLQMFGRLLFAQATELIDPSLNN GLPTNLVADDPSLSFT MKGVDISMASYMAELAYLANPVSSHVQTAEMHNQSVNSMAFVSSRYTMQAVEIVSLMCAC SVYIGCQALDLRVLHL TFLQRSTPQLHTLTSHLFSEHLFEPDLATLNEALSTHIQKSWPTTTRLNITDRVEEVVTS AIPILCRTFASSTGTS TSQAPTFSDLETWKSRASALLNEIYQDTAHAFFSYQHTEEMLGTSSKILYQTVRRQLGVP FHQGFIEHPTAQSDTL GGRPKKTVGSWISIIYEAIREGRLMDPLMASLQAGVAGESDTEAVDTLKDGSSGKCSSSG LD

DNA sequence encoding partial tyrosine ammonia lyase (TAL) from

SEQ ID NO:42

Rhodotorula glutinis codon optimized for expression in S. cerevisiae

ATGTCCTTGAGAAGAGATCACGGTGTTAGAAGATTGGGTAGACATCCAGATGGTGGT AGAGATTTGGCTGCTGAAG GTGTTAGATTGCATATTGCTTTGTCTCCATTCTCCTCAAGAAGAGCTTCTACTGATTCTA GAGGTCCATTCGCTTT CGAAAACAGTTTGTTGGAACATCAATTGTGCGGTGTTTTGCCAACTTCTATGGATGGTTT TGCTTTGGGTTCTGGT TTGGAAAATTCTTTGCCATTGGAAGTTGTTAGAGGTGCTATGACTTTGAGAGTCAACTCT TTGACAAGAGGTCATT CTGCTGTTAGAATCGTTGTTTTGGAAGCTTTGACTAACTTCTTGAACCATGGTATTACTC CAATCGTTCCATTGAG AGGTACTATTTCTGCTTCTGGTGATTTGTCACCATTGTCTTATATTGCTGCTTCCATTAC TGGTCACCCAGATTCT AAAGTTCATGTTGATGGTCAAATCATGTCCGCTCAAGAAGCTATTGCATTGAAAGGTTTA CAACCAGTTGTCTTGG GTCCAAAAGAAGGTTTGGGTTTAGTTAATGGTACTGCTGTTTCAGCTTCTATGGCTACTT TGGCTTTGACTGATGC TCATGTTTTGTCTTTGTTGGCTCAAGCTAATACTGCTTTGACAGTTGAAGCTATGGTTGG TCATGCTGGTTCTTTT CATCCATTCTTGCATGATGTTACTAGACCAGCTCCAGATCCAGACAGAGGTAGAGCTGCT ACTTTAGGTTTGTTGT TGGAAGGTTCTAAGTACTGCTTGTCTACTATGAGACCAAGATCCAGATCTAGAACTACTA GAGCTTCTTCTGGTAG AACTGATACAAGATCTGCTGCTAGACCTAGATTTGTCGTTTTCAGAATTCCATCCTTGTC TGCCCATAGATCTGAT CCTTTGTTGTTATGTTCTGGTGCTCAATGGTTGGGTCCATTGGTTTCTGATATGATTCAT GCCCATTCTGTCTTGT CTTTAGAAGCTGGTCAATCTACTACTGACAACCCATTGATTGACTTGGAAAACAAGATGA CTCATCATGGTGGTGC TTTTATGGCTTCTTCAGTTGGTAACACTATGGAAAAGAGATTGGTTTCCCCATCTCATTT GTGGGCTAGATTGGCT TCTTTATCTTCTCCAAGATGTTCTACTCCAGCTTGTACTGCTAGATTTCCACCAGCTTCA CCACCAAGAACTAGAT TGTGTCCAACTACAGCTAGAGTTTCTACTTCTCCACCATTGCATACTTTGAGATCCTCTG TTACTTCTAGAACCCA ATCCAGACCAACTTTCTCTAGACAAAGATGGGCTATTAGAAGATCTACCAGATCTCCATC TTCTAGACCAGTTGCT CCTCCTAGAAGAACAACTTCTTCTAGATCATCATCTCCTCCAACTTCAACTGCTTCTTGT AGAAGATCCACTTGTG CTAGATGGTCATCCTCTACTAGAAAGTCTTTATCAAGATGGTCCCCAACCTGTTCTTCAT CTACTTTAGCTAGATC AAGACAACCTACCTCCAGAACAAGATCAGCTAATAGATCTACTTCTGGTTGCTCCAGAAC TACAAGAACTACTTCA TCTTCTGGTGGTACAACCAGATCAAGAAGTAGACCAGCACCATCATCTAAACCATCTCCA GGTACTAGATGTAGAT CTAGAGCATCTACACCAGGTAGAAGTAGAGCTTTGAGAAGACCTTCTCCATCTCCTGCTC CATGTGCTACTAGATC AGGTCCTCGTCGTAGAAGAAGAAGACCAAGAAGTTCAACTAGTAGAAGAGGTTTGGCTTC CTGTACTAGATCTTCA GGTAAAACATCTGCTTCAAGACCAGCTGCTGCTACTAGTACTTCAGCTTCAAGAAGATCA AGATCCGGTCCAACTT CTGCTGCATCAACAAGAAGATCCAGAACTGCTGCTTTGTTGAGATCATCTTCAAGATGTT GGCACAAGAGAACTTT GGTTCAAGCTTCATTGGCTAGAGATTCTAAGTTGCCATTTTTGCCAGGTAGATTGAGACA AAGAAGATTCCCACCA GATTGTCATTTTCCACATGCTCCATATCCATTGGGTTTCAGATCTCATTCTGGTCCAGTT GAAACCCATATTTCTT TCGGTAGAAGACCATATTAA

SEQ ID NO:43 Protein sequence of PAL/TAL from Rhodotorula glutinis

MSLRRDHGVRRLGRHPDGGRDLAAEGVRLHIALSPFSSRRASTDSRGPFAFENSLLEHQL CGVLPTSMDGFALGSG LENSLPLEWRGAMTLRVNSLTRGHSAVRIVVLEALTNFLNHGITPIVPLRGTISASGDLS PLSYIAASITGHPDS KVHVDGQIMSAQEAIALKGLQPVVLGPKEGLGLVNGTAVSASMATLALTDAHVLSLLAQA NTALTVEAMVGHAGSF HPFLHDVTRPAPDPDRGRAATLGLLLEGSKYCLSTMRPRSRSRTTRASSGRTDTRSAARP RFVVFRIPSLSAHRSD PLLLCSGAQWLGPLVSDMIHAHSVLSLEAGQSTTDNPLIDLENKMTHHGGAFMASSVGNT MEKRLVSPSHLWARLA SLSSPRCSTPACTARFPPASPPRTRLCPTTARVSTSPPLHTLRSSVTSRTQSRPTFSRQR WAIRRSTRSPSSRPVA PPRRTTSSRSSSPPTSTASCRRSTCARWSSSTRKSLSRWSPTCSSSTLARSRQPTSRTRS ANRSTSGCSRTTRTTS SSGGTTRSRSRPAPSSKPSPGTRCRSRASTPGRSRALRRPSPSPAPCATRSGPRRRRRRP RSSTSRRGLASCTRSS GKTSASRPAAATSTSASRRSRSGPTSAASTRRSRTAALLRSSSRCWHKRTLVQASLARDS KLPFLPGRLRQRRFPP DCHFPHAPYPLGFRSHSGPVETHISFGRRPY

DNA sequence encoding phenylalanine ammonia lyase/tyrosine ammonia

SEQ ID NO:44

lyase (PAL/TAL) from Trichosporon cutaneum codon optimized for expression in S. cerevisiae

ATGTTCATCGAAACTAACGTTGCTAAGCCAGCTTCTACTAAGGCTATGAATGCTGGTTCT GCTAAAGCTGCTCCAG TTGAACCATTTGCTACTTATGCTCATTCTCAAGCTACTAAGACCGTTTCTATTGATGGTC ATACAATGAAGGTTGG TGATGTTGTTGCTGTTGCTAGACATGGTGCTAAAGTTGAATTGGCTGCTTCTGTTGCTGG TCCAGTTAGAGCTTCA GTTGATTTCAAAGAATCCAAAAAGCACACCTCCATCTACGGTGTTACTACTGGTTTTGGT GGTTCAGCTGATACAA GAACTTCTGATACTGAAGCCTTGCAAATCTCCTTGTTGGAACATCAATTGTGTGGTTTCT TGCCAACTGATGCTAC TTACGAAGGTATGTTGTTGGCTGCTATGCCAATTCCAATAGTTAGAGGTGCTATGGCTGT TAGAGTTAATTCTTGT GTTAGAGGTCACTCCGGTGTTAGATTGGAAGTTTTACAATCTTTCGCCGACTTCATCAAC AGAGGTTTGGTTCCAT GTGTTCCATTGAGAGGTACTATTTCTGCTTCTGGTGATTTGTCTCCATTGTCTTATATTG CTGGTGCTATTTGTGG TCACCCAGATGTTAAGGTTTTTGATACTGCTGCTTCTCCACCAACTGTTTTGACTTCTCC TGAAGCTATTGCTAAG TACGGTTTGAAAACTGTTAAGTTGGCCTCCAAAGAAGGTTTGGGTTTGGTTAATGGTACT GCTGTTTCTGCTGCTG CTGGTGCATTGGCATTATATGATGCTGAATGTTTGGCCATCATGTCCCAAACTAACACAG TTTTGACTGTTGAAGC TTTGGATGGTCATGTTGGTTCTTTTGCTCCATTCATCCAAGAAATTAGACCACATGCAGG TCAAATTGAAGCTGCC AGAAATATCAGACATATGTTGGGTGGTTCTAAGTTGGCTGTTCATGAAGAATCTGAATTA TTGGCTGATCAAGACG CCGGTATTTTGAGACAAGATAGATATGCTTTGAGAACCTCCGCTCAATGGATTGGTCCAC AATTGGAAGCTTTAGG TTTGGCCAGACAACAAATTGAAACCGAATTGAACTCTACCACCGATAACCCATTGATTGA TGTTGAAGGTGGTATG TTTCATCACGGTGGTAATTTTCAAGCTATGGCAGTTACTTCTGCTATGGATTCTGCTAGA ATTGTCTTGCAAAACT TGGGTAAATTGTCCTTCGCTCAAGTCACTGAATTGATCAACTGTGAAATGAATCACGGTT TGCCATCTAATTTGGC AGGTTCTGAACCATCTACTAATTACCATTGCAAGGGTTTGGATATTCATTGTGGTGCTTA TTGTGCTGAATTGGGT TTTTTGGCTAACCCAATGTCTAACCATGTTCAATCTACCGAAATGCACAATCAATCCGTT AACTCTATGGCTTTTG CTTCCGCTAGAAGAACTATGGAAGCTAACGAAGTTTTGTCCTTGTTGTTGGGTTCACAAA TGTACTGTGCTACCCA AGCCTTGGATTTGAGAGTTATGGAAGTTAAGTTCAAGATGGCCATTGTCAAGTTGTTGAA CGAAACTTTGACCAAG CACTTTGCTGCTTTTTTGACTCCAGAACAATTGGCTAAGTTGAACACTCATGCTGCTATC ACCTTGTACAAAAGAT TGAATCAAACCCCATCTTGGGATTCCGCTCCAAGATTTGAAGATGCTGCTAAACATTTGG TTGGTGTTATTATGGA TGCCTTGATGGTTAACGATGATATCACTGACTTGACTAACTTGCCAAAGTGGAAGAAAGA ATTCGCTAAAGAAGCT GGTAACTTGTACAGATCCATTTTGGTTGCTACTACTGCTGATGGTAGAAACGATTTGGAA CCAGCTGAATATTTGG GTCAAACTAGAGCTGTTTACGAAGCCGTTAGATCAGAATTGGGTGTCAAAGTTAGAAGAG GTGATGTAGCTGAAGG TAAGAGTGGTAAATCTATCGGTTCTTCCGTTGCCAAAATCGTTGAAGCTATGAGAGATGG TAGATTGATGGGTGCT GTTGGTAAGATGTTCTGA

SEQ ID NO:45 Protein sequence of PAL/TAL from Trichosporon cutaneum

MFIETNVAKPASTKAMNAGSAKAAPVEPFATYAHSQATKTVSIDGHTMKVGDVVAVARHG AKVELAASVAGPVRAS VDFKESKKHTSIYGVTTGFGGSADTRTSDTEALQISLLEHQLCGFLPTDATYEGMLLAAM PIPIVRGAMAVRVNSC VRGHSGVRLEVLQSFADFINRGLVPCVPLRGTISASGDLSPLSYIAGAICGHPDVKVFDT AASPPTVLTSPEAIAK YGLKTVKLASKEGLGLVNGTAVSAAAGALALYDAECLAIMSQTNTVLTVEALDGHVGSFA PFIQEIRPHAGQIEAA RNIRHMLGGSKLAVHEESELLADQDAGILRQDRYALRTSAQWIGPQLEALGLARQQIETE LNSTTDNPLIDVEGGM FHHGGNFQAMAVTSAMDSARIVLQNLGKLSFAQVTELINCEMNHGLPSNLAGSEPSTNYH CKGLDIHCGAYCAELG FLANPMSNHVQSTEMHNQSVNSMAFASARRTMEANEVLSLLLGSQMYCATQALDLRVMEV KFKMAIVKLLNETLTK HFAAFLTPEQLAKLNTHAAITLYKRLNQTPSWDSAPRFEDAAKHLVGVIMDALMVNDDIT DLTNLPKWKKEFAKEA GNLYRSILVATTADGRNDLEPAEYLGQTRAVYEAVRSELGVKVRRGDVAEGKSGKSIGSS VAKIVEAMRDGRLMGA VGKMF

DNA sequence encoding phenylalanine ammonia lyase/tyrosine

SEQ ID NO:46 ammonia lyase (PAL/TAL) from Phanerochaete chrysosporium codon optimized for expression in S. cerevisiae

ATGCCATCCAGAATCGACTACTACACTTCTTCTGGTAATGGTTACGCCCAATCCAGAAAA TCTTCTGCTATCTATC CAGCTTCTGCTTCTACTGGTCATGCTGCTCCATCTACTGAAAGAAAACCAGAATTATTGG ACAAGTTCGTTGAAGC CTACGACGAATTGCAATCTTACAGAGAAGGTAAGCCAGTTATCGTTGATGGTCATAACTT GTCTATTCCAGCTGTT GCTGCTACAGCTAGATTTGGTGCTGCTGTTGTTTTGGACGAAAATCCTGAAACTCACGAA AGAGTCTTGCAATCTA GAAGAGTTATCGTCGATAAGGTCAGTACCCAAAGATCTGTTTATGGTGTTTCTACAGGTT TTGGTGGTTCTGCTGA TACAAGAACTTCTGATCCATTGCAATTGGGTCATGCCTTGTTACAACATCAACACGTTGG TGTTTTGCCAACTCAA ACTGAATCTCCATTGCCAGCTTTGCCATTGGGTGATCCATTAGCTACTACTTCTATGCCT GAAGCTTGGGTTAGAG GTGCTATTTTGATTAGAATGAACTCCTTGATCAGAGGTCACTCTGGTGTTAGATGGGAAT TGATTGAAAAGATGGG TGAATTATTGAGAGAAAACATCACCCCATTGGTTCCATTGAGAGGTTCTATTTCTGCTTC AGGTGATTTGTCTCCA TTGTCTTATATTGCCGGTACTTTGATTGGTTCCCCAGCTATTAGAGTTTTTGATGGTCCA GCTTCATATGGTGCCA GAAGAATTTTGCCATCCAATATTGCTTTGGCCAATCATGGTGTTGCTCCAATTCCATTGT CATCCAAAGAACATTT GGGTATCTTGAACGGTACTGCTTTTTCAGCTTCTGTTGGTGCTTTGGCTTTGAATGAAGC TGTTCATTTGTCTTTG TTGGCTCAAGTATGTACTGCTATGGGTACTGAAGCTATGATTGGTGCAGTTGGTTCTTTC GATGCTTTCATTCATG ATACTGCTAGACCACATCCAGGTCAAGTTGAAGTTGCTAGAAATGTTAGAACCTTGTTGG AAGATTCTCAAATGGC TGTTAAGGCCGAAGATGAAGTTCATATTGCTGAAGATGAAGGTGAATTGAGACAAGACAG ATACCCATTGAGAACT GCTGCTCAATTTTTGGGTCCACAAATCGAAGATATTTTGTCTGCTCACGAAACCGTTACC TTGGAATGTAATTCTA CTACCGATAACCCATTGATCGATGGTGAAACTGGTACTGTTCATCATGGTGGTAATTTTC AAGCTATGGCCGTTAC TAATGCTATGGAAAAAACCAGATTGGCTATCCATCATATCGGTAAGTTGTTGTTTGCTCA AGCTACCGAATTGATC AACCCAATGATGAATAGAGGTTTGCCACCTAATTTGGCTGCTACTGATCCATCTCATAAT TACTTTGCTAAGGGTG TTGATATTCATTTGGCAGCTTACGTTGGTGAATTGGGTTTTTTGGCTTCTCCAGTTTCCT CCCATATTCAATCTGC TGAAATGCATAATCAAGCCGTTAATTCCTTGGCTTTGGTTTCTGCTAGATATACCATTTC CGCTTTGGATGTCTTA TCTTTGTTGACTGCTGCTTACTTGTACGTTTTGTGTCAAGCTTTGGATTTGAGAGCTATG CATAACGACTTGCAAT CATCTTTGTCAGCCATCGTTAGAGAATTATTACCAAAGCACTTTCCATCCGCTGCTAAAA GAGCTGACGCTTTGTT GCCAATTTTGGAAAGAACTATTTTCAGAGCCTTGAACTCCTCTTCTTCTGCTGACTGTAA AGCTAGAATGGTTTCA GTTGCTGCTTCAACTACTACTCCATTGGTTGATTTTTTGTCAGCTGATGCAGCTTTGGCA TCTGAATTGGCTAATA TTACTGCTTTCAGAACCGAATTAGCTACCAGAGCTGCTGATGCTTTGACTACTTTGAGAA CTCAATATTTGGAAGG TGCTAGAGGTGCAGCTCCAGCATCTAAATACTTGGGTAAAACTAGACCAGTCTACGAATT TGTTAGAGTCACTTTG AACGTTCCAATGCACGGTAGAGAAAACTTGCATAACTTTGAAATGGGTCCAGGTGTTGAA GATGGTATTATTGGTA ACAACATCTCCACCATCTACGAAGCAATTAGAGATGGTAAGATGCAAAACGTCGTAATGC AATTGGTCAAGTCCAT TAAGGCTTAA

SEQ ID NO:47 Protein sequence of PAL/TAL from Phanerochaete chrysosporium

MPSRIDYYTSSGNGYAQSRKSSAIYPASASTGHAAPSTERKPELLDKFVEAYDELQS YREGKPVIVDGHNLSIPAV AATARFGAAVVLDENPETHERVLQSRRVIVDKVSTQRSVYGVSTGFGGSADTRTSDPLQL GHALLQHQHVGVLPTQ TESPLPALPLGDPLATTSMPEAWVRGAILIRMNSLIRGHSGVRWELIEKMGELLRENITP LVPLRGSISASGDLSP LSYIAGTLIGSPAIRVFDGPASYGARRILPSNIALANHGVAPIPLSSKEHLGILNGTAFS ASVGALALNEAVHLSL LAQVCTAMGTEAMIGAVGSFDAFIHDTARPHPGQVEVARNVRTLLEDSQMAVKAEDEVHI AEDEGELRQDRYPLRT AAQFLGPQIEDILSAHETVTLECNSTTDNPLIDGETGTVHHGGNFQAMAVTNAMEKTRLA IHHIGKLLFAQATELI NPMMNRGLPPNLAATDPSHNYFAKGVDIHLAAYVGELGFLASPVSSHIQSAEMHNQAVNS LALVSARYTISALDVL SLLTAAYLYVLCQALDLRAMHNDLQSSLSAIVRELLPKHFPSAAKRADALLPILERTIFR ALNSSSSADCKARMVS VAASTTTPLVDFLSADAALASELANITAFRTELATRAADALTTLRTQYLEGARGAAPASK YLGKTRPVYEFVRVTL NVPMHGRENLHNFEMGPGVEDGIIGNNISTIYEAIRDGKMQNVVMQLVKSIKA

DNA sequence encoding tyrosine ammonia lyase 2 (TAL2) from

SEQ ID NO:48

Rhodotorula glutinis codon optimized for expression in S. cerevisiae

ATGGCCCCATCCGTTGATTCTATTGCTACTTCTGTTGCTAACTCCTTGTCCAATGGT TTACATGCTGCTGCTGCAG CTAATGGTGGTGATGTTCATAAGAAAACTGCTGGTGCTGGTTCTTTGTTGCCAACTACTG AAACTACTCAATTGGA CATCGTCGAAAGAATTTTGGCTGATGCTGGTGCAACTGATCAAATCAAATTGGATGGTTA CACTTTGACCTTGGGT GATGTTGTTGGTGCTGCTAGAAGAGGTAGATCTGTTAAGGTTGCTGATTCCCCACATATC AGAGAAAAGATTGATG CCTCTGTCGAATTCTTGAGAACCCAATTGGATAACTCTGTTTACGGTGTTACTACTGGTT TTGGTGGTTCTGCTGA TACAAGAACTGAAGATGCTATCTCCTTGCAAAAGGCTTTGTTGGAACATCAATTGTGTGG TGTTTTGCCAACTTCT ATGGATGGTTTTGCTTTGGGTAGAGGTTTGGAAAATTCATTGCCATTGGAAGTTGTTAGA GGTGCCATGACTATCA GAGTTAATTCTTTGACTAGAGGTCACTCCGCTGTTAGAATAGTTGTTTTGGAAGCTTTGA CTAACTTCTTGAACCA TGGTATTACTCCAATCGTTCCATTGAGAGGTACTATTTCTGCTTCTGGTGATTTGTCTCC ATTGTCTTATATTGCT GCTTCCATTACTGGTCACCCAGATTCTAAAGTTCATGTTGATGGTAAGATCATGTCCGCT CAAGAAGCTATTGCTT TGAAAGGTTTACAACCAGTTGTCTTGGGTCCAAAAGAAGGTTTGGGTTTGGTTAATGGTA CTGCTGTTTCAGCTTC TATGGCTACATTGGCTTTGACTGATGCTCATGTTTTGTCTTTGTTGGCTCAAGCTTTAAC TGCTTTGACAGTTGAA GCTATGGTTGGTCATGCTGGTTCATTTCATCCATTCTTGCATGATGTTACTAGACCACAT CCAACCCAAATTGAAG TTGCCAGAAACATTAGAACCTTGTTGGAAGGTTCCAAGTATGCTGTTCATCACGAAACTG AAGTTAAGGTTAAGGA TGACGAAGGTATCTTGAGACAAGATAGATACCCATTGAGATGTTCTCCACAATGGTTGGG TCCATTGGTTTCTGAT ATGATTCATGCTCATGCCGTCTTGTCTTTAGAAGCTGGTCAATCTACTACTGACAACCCA TTGATTGACTTGGAAA ACAAGATGACTCATCATGGTGGTGCTTTTATGGCTTCTTCTGTAGGTAACACTATGGAAA AGACTAGATTGGCTGT TGCTTTGATGGGTAAGGTTTCTTTCACTCAATTGACCGAAATGTTGAACGCTGGTATGAA TAGAGCTTTGCCATCA TGTTTGGCTGCTGAAGATCCATCTTTATCTTACCACTGTAAGGGTTTGGATATTGCAGCT GCTGCTTATACTTCTG AATTGGGTCATTTGGCTAACCCAGTTTCTACTCATGTTCAACCAGCTGAAATGGGTAATC AAGCTATCAATTCTTT GGCCTTGATTTCCGCTAGAAGAACAGCTGAAGCTAATGATGTTTTGAGTTTGTTGTTGGC TACCCACTTGTATTGC GTTTTACAAGCTGTTGATTTGAGAGCCATGGAATTCGAACATACCAAAGCTTTCGAACCT ATGGTCACCGAATTAT TGAAGCAACATTTTGGTGCTTTGGCTACCGCTGAAGTTGAAGATAAGGTAAGAAAGTCCA TCTACAAGAGATTGCA ACAAAACAATTCCTACGATTTGGAACAAAGATGGCACGATACTTTTTCAGTTGCTACTGG TGCTGTTGTTGAAGCT TTGGCAGGTCAAGAAGTATCTTTGGCTTCTTTGAATGCTTGGAAAGTTGCTTGTGCTGAA AAGGCTATTGCATTGA CTAGATCCGTTAGAGATTCTTTTTGGGCTGCTCCATCTTCTTCATCTCCAGCTTTGAAAT ACTTGTCTCCAAGAAC TAGAGTCTTGTACTCCTTCGTTAGAGAAGAAGTTGGTGTTAAGGCAAGAAGAGGTGACGT TTATTTGGGTAAACAA GAAGTCACCATCGGTACAAACGTTTCCAGAATCTATGAAGCCATTAAGTCCGGTAGAATT GCTCCAGTTTTGGTTA AGATGATGGCCTGA

SEQ ID NO:49 Protein sequence of PAL/TAL2 from Rhodotorula glutinis

MAPSVDSIATSVANSLSNGLHAAAAANGGDVHKKTAGAGSLLPTTETTQLDIVERILADA GATDQIKLDGYTLTLG DVVGAARRGRSVKVADSPHIREKIDASVEFLRTQLDNSVYGVTTGFGGSADTRTEDAISL QKALLEHQLCGVLPTS MDGFALGRGLENSLPLEWRGAMTIRVNSLTRGHSAVRIVVLEALTNFLNHGITPIVPLRG TISASGDLSPLSYIA ASITGHPDSKVHVDGKIMSAQEAIALKGLQPVVLGPKEGLGLVNGTAVSASMATLALTDA HVLSLLAQALTALTVE AMVGHAGSFHPFLHDVTRPHPTQIEVARNIRTLLEGSKYAVHHETEVKVKDDEGILRQDR YPLRCSPQWLGPLVSD MIHAHAVLSLEAGQSTTDNPLIDLENKMTHHGGAFMASSVGNTMEKTRLAVALMGKVSFT QLTEMLNAGMNRALPS CLAAEDPSLSYHCKGLDIAAAAYTSELGHLANPVSTHVQPAEMGNQAINSLALISARRTA EANDVLSLLLATHLYC VLQAVDLRAMEFEHTKAFEPMVTELLKQHFGALATAEVEDKVRKSIYKRLQQNNSYDLEQ RWHDTFSVATGAVVEA LAGQEVSLASLNAWKVACAEKAIALTRSVRDSFWAAPSSSSPALKYLSPRTRVLYSFVRE EVGVKARRGDVYLGKQ EVTIGTNVSRIYEAIKSGRIAPVLVKMMA

[00147] Having described the invention in detail and by reference to specific embodiments thereof, it will be apparent that modifications and variations are possible without departing from the scope of the invention defined in the appended claims. More specifically, although some aspects of the present invention are identified herein as particularly advantageous, it is contemplated that the present invention is not necessarily limited to these particular aspects of the invention.