Engineered phenylalanine ammonia lyase polypeptides and methods of making and using the same

Abstract

The present disclosure provides polypeptides (e.g., PAL polypeptides), polynucleotides and expression vectors comprising the same, methods of producing polypeptides (e.g., PAL polypeptides), and methods of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into L-phenylalanines (e.g., 3-cyano-L-phenylalanine).

Description

ENGINEERED PHENYLALANINE AMMONIA LYASE POLYPEPTIDES ANDMETHODS OF MAKING AND USING THE SAMECROSS-REFERENCE TO RELATED APPLICATIONS

[0001] This application claims the benefit of U.S. Provisional Patent Application Serial No. 63 / 639,340 filed April 26, 2024, the entire contents of which are incorporated by reference herein.REFERENCE TO SEQUENCE LISTING SUBMITTED ELECTRONICALLY

[0002] The application contains a Sequence Listing which has been submitted electronically in XML format and is hereby incorporated by reference in its entirety. The XML file, created on July 29, 2024, is named 25844-WO-PCT_SL.XML and is 63,716 bytes in size.FIELD

[0003] This disclosure relates generally to engineered phenylalanine ammonia lyase (PAL) polypeptides and related polynucleotides, expression vectors, host cells, methods of production, and methods of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into L- phenylalanines (e.g.. 3-cyano-L-phenylalanine).BACKGROUND

[0004] Enzy mes are protein molecules that serve to accelerate the chemical reactions of living cells (often by several orders of magnitude). Without enzymes, most biochemical reactions would be too slow to carry out life processes. Enzymes display great specificity and are not permanently modified by their participation in reactions. Because they are not changed during the reactions, enzy mes may be cost effectively used as catalysts for a desired chemical transformation.

[0005] Phenylalanine ammonia lyases (PALs) are a family of enzymes that catalyze the non- oxidative deamination of L-phenylalanine to generate cinnamic acid. At high ammonia concentrations, PALs can also catalyze hydroamination of cinnamic acids to generate L- phenylalanines. Different PAL enzymes have been shown to have activity' towards arylacrylic acid analogs with different ring substitutions. Therefore, PALs hold great promise to fulfill the increasing needs for synthesizing different non-canonical amino acids in the chemical manufacturing or pharmaceutical industries. Nonetheless, use of PALs for the large-scalesynthesis of non-canonical phenylalanine analogs has been limited, at least in part due to low activity of wild-type PALs toward non-natural substrates.

[0006] Therefore, there is a need for improved PAL polypeptides, e.g., PAL polypeptides with improved activity. soluble expression, and enantioselectivity.SUMMARY

[0007] The present disclosure provides, inter alia, polypeptides (e.g., PAL polypeptides), polynucleotides encoding the same, expression vectors, host cells, methods of producing polypeptides, and methods of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into L-phenylalanmes (e.g., 3-cyano-L-phenylalanine).

[0008] In one aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises at least one substitution at one or more amino acid positions selected from 57. 70, 75, 94. 102, 227. 288, 294, 296, 306, 310. 357, 360. 485, 510, and 534. wherein the ammo acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0009] In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more amino acid positions selected from 57, 70, 75, 94, 102, 227, 288. 294, 296, 306, 310, 357. 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0010] In some embodiments, the polypeptide comprises: a) a substitution set at positions 57, 75, 94, 102, 227, 288, 360, 485, 510, and 534; b) a substitution at position 102; c) a substitution at position 306; d) a substitution set at positions 94, 102, 288. and 485; e) a substitution set at positions 57. 94. 102, 288. and 485; I) a substitution set at positions 94. 102. 288, and 485; g) a substitution set at positions 57, 75, 94, 102, 288, 360, 485, and 510; h) a substitution set at positions 57, 94, 102, 288, 310, 357, and 485; or i) a substitution set at positions 57, 70, 75, 94, 102, 227. 288, 294, 296, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0011] In some embodiments, the polypeptide comprises one or more substitutions set forth in any one of Tables 5, 6, 7, 8, or 9.

[0012] In some embodiments, the polypeptide comprises one of the following substitutions or substitution sets: a) R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R. E510G, and M534V; b) L102V; c) L306N; d) H94M, L102A, Y288V, and A485R; e) R57K, H94M, L102A, Y288P, and A485R; f) H94M, L102A, Y288P, and A485L; g) R57K, I75V, H94M, L102A, Y288V, M360T, A485R, and E510G; h) R57K, H94M, L102A, Y288V, A310P, Y357S, andA485R; or i) R57K, R70D, 175V, H94M, L102A. S227A. Y288V, K294T. N296D, M360T. A485R, E510G, and M534T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0013] In some embodiments, the amino acid sequence comprises SEQ ID NO: 18.

[0014] In some embodiments, the amino acid sequence comprises SEQ ID NO: 4.

[0015] In some embodiments, the amino acid sequence comprises SEQ ID NO: 6.

[0016] In some embodiments, the amino acid sequence comprises SEQ ID NO: 8.

[0017] In some embodiments, the amino acid sequence comprises SEQ ID NO: 10.

[0018] In some embodiments, the amino acid sequence comprises SEQ ID NO: 12.

[0019] In some embodiments, the amino acid sequence comprises SEQ ID NO: 14.

[0020] In some embodiments, the amino acid sequence comprises SEQ ID NO: 16.

[0021] In some embodiments, the amino acid sequence comprises SEQ ID NO: 20.

[0022] In another aspect, provided herein is an engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21, wherein the polypeptide comprises at least one substitution at one or more amino acid positions selected from 45, 58, 63, 82, 90, 215, 276, 282, 284, 294, 298, 345, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0023] In some embodiments, the polypeptide comprises 2, 3. 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more amino acid positions selected from 45, 58, 63, 82, 90, 215, 276, 282, 284, 294, 298, 345, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0024] In some embodiments, the polypeptide comprises: a) a substitution set at positions 45, 63. 82. 90. 215, 276. 348, 473. 498, and 522; b) a substitution at position 90; c) a substitution at position 294; d) a substitution set at positions 82, 90, 276, and 473; e) a substitution set at positions 45, 82, 90, 276, and 473; f) a substitution set at positions 82, 90, 276, and 473; g) a substitution set at positions 45, 63, 82. 90, 276, 348, 473, and 498; h) a substitution set at positions 45, 82, 90, 276, 298. 345, and 473; or i) a substitution set at positions 45. 58. 63, 82, 90, 215, 276, 282, 284, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0025] In some embodiments, the polypeptide comprises one or more substitutions set forth in Table 9.

[0026] In some embodiments, the polypeptide comprises one of the following substitutions or substitution sets: a) R45K, I63V, H82M, L90A, S215A, Y276V, M348T, A473R, E498G, and M522V; b) L90V; c) L294N; d) H82M, L90A, Y276V, and A473R; e) R45K, H82M, L90A,Y276P. and A473R; f) H82M. L90A, Y276P. and A473L; g) R45K, I63V. H82M. L90A, Y276V, M348T, A473R, and E498G; h) R45K, H82M, L90A, Y276V, A298P, Y345S, and A473R; or i) R45K, R58D, I63V, H82M, L90A, S215A, Y276V, K282T, N284D, M348T, A473R, E498G, and M522T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0027] In some embodiments, the amino acid sequence comprises any one of SEQ ID NO: 30, 23, 24, 25, 26, 27, 28, 29, or 31.

[0028] In some embodiments, the amino acid sequence comprises SEQ ID NO: 30.

[0029] In some embodiments, the polypeptide comprises an epitope tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the tag comprises the amino acid sequence of GSHHHHHHHHSG (SEQ ID NO: 22). In some embodiments, the polypeptide comprises an N- terminal methionine residue, and the epitope tag is inserted immediately following the N- terminal methionine residue.

[0030] In some embodiments of any of the preceding aspects, the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0031] In another aspect, provided herein is an engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 18, 4, 6, 8, 10, 12, 14, 16, or 20, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0032] In another aspect, provided herein is a polypeptide comprising at least 99% sequence identity to any one of SEQ ID NO: 18, 4, 6, 8, 10, 12, 14, 16, or 20.

[0033] In some embodiments of any of the preceding aspects, the polypeptide is isolated.

[0034] In some embodiments of any of the preceding aspects, the polypeptide is a phenylalanine ammonia lyase.

[0035] In some embodiments of any of the preceding aspects, the polypeptide has one or more of the following properties relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2: a) increased activity in converting 3-cyanocinnamic acid into 3- cyano-L-phenylalanine, optionally wherein the polypeptide has at least about 1. 1 -fold, about 5- fold, about 10-fold, or about 100-fold increased activity; b) increased enantioselectivity; c) increased thermostability'; and / or d) increased soluble expression.

[0036] In another aspect, provided herein is a polynucleotide encoding any one of the polypeptides disclosed herein.

[0037] In some embodiments, the polynucleotide is codon-optimized.

[0038] In some embodiments, the polynucleotide comprises SEQ ID NO: 17, 3, 5, 7, 9, 11, 13,15, or 19.

[0039] In another aspect, provided herein is a polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO: 17, 3, 5, 7, 9, 11, 13, 15, or 19.

[0040] In another aspect, provided herein is an expression vector comprising at least one of the polynucleotides disclosed herein.

[0041] In some embodiments, the polynucleotide sequence is operably linked to a control sequence.

[0042] In some embodiments, the control sequence is a promoter.

[0043] In some embodiments, the promoter is a heterologous promoter.

[0044] In another aspect, provided herein is a host cell comprising any one of the polynucleotides disclosed herein or any one of the expression vectors disclosed herein.

[0045] In some embodiments, the host cell is prokaryotic or eukaryotic.

[0046] In another aspect, provided herein is a method of producing a polypeptide, the method comprising culturing any one of the host cells disclosed herein under conditions that the polypeptide encoded by the polynucleotide is produced.

[0047] In some embodiments, the method further comprises the step of recovering the polypeptide.

[0048] In some embodiments, the method further comprises the step of purity ing the polypeptide.

[0049] In another aspect, provided herein is a method of converting 3 -cyanocinnamic acid into 3-cyano-L-phenylalanine, the method comprising contacting 3-cyanocinnamic acid with an ammonia source in the presence of any one of the polypeptides disclosed herein.

[0050] In some embodiments, the ammonia source is ammonium carbonate, ammonium carbamate, ammonium sulfate, ammonium acetate, or ammonium formate.

[0051] The summary of the technology described above is non-limiting and other features and advantages of the technology will be apparent from the following detailed description, and from the claims.BRIEF DESCRIPTION OF THE DRAWINGS

[0052] FIGS. 1A and IB show synthesis of phenylalanine (Phe)-amino acids using phenylalanine ammonia lyases (PALs). (A) Syntheses of Phe-amino acids and this work. (B) Challenges in using PAL for industrial applications.

[0053] FIG. 2A shows evaluation of PAL enzymes at different pH and lysate loading with (A) Planctomyces brasiliensis PAL (PbPAL) (SEQ ID NO: 2) and (B) Streptomyces rimosus PAL(SrPAL) (SEQ ID NO: 34). Lysate loadings are highlighted in light gray (90 v / v%), dark gray (180 v / v%) and black (270 v / v%), respectively.

[0054] FIG. 2B shows expression of PbPAL (SEQ ID NO: 2) and SrPAL (SEQ ID NO: 34) under different induction conditions. Both PbPAL and SrPAL are induced under 0.1 mM isopropyl -D-1 -thiogalactopyranoside (IPTG) with different temperatures. The highest expression in both cell lysates matches to the size of protein of interest (PbPAL: 61KDa, SrPAL: 57KDa). T: total cell lysate, S: supernatant fraction, P: precipitates post centrifugation.

[0055] FIGS. 3A-3C show directed evolution of PbPAL (SEQ ID NO: 2; labeled “WT”). (FIG. 3A) Mutations introduced and fold improvement achieved over the course of evolution. The mutations and fold improvements indicated were in companson to the previous evolution backbones. (FIG. 3B) Mutations mapped on the tetramer PbPAL model structure illustrated by spheres: surface, active site and core mutations. (FIG. 3C) The PAL variants were characterized at the same screening condition as the round 4 evolution (Table 1). The total fold improvement of activity was calculated based on the conversions of the product and the weight % of lyophilized crude extracts used in the reactions.

[0056] FIG. 3D shows protein solubility of the wild-type PbPAL (SEQ ID NO: 2; labeled “WT”) and the evolutionary variants from PALR2BB to PALRSBB. Enzy me powders from each variant were hydrolyzed and loaded into the sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) gels to assess soluble expression. The band of interest is highlighted with a light gray rectangle.

[0057] FIG. 4A shows an overlay of modeled structures between PbPAL and PbPALRSBB in the active sites. The most differential loop regions were highlighted in with arrows, wild-type (WT) (solid arrow); PbPALRSBB (dashed arrow).

[0058] FIG. 4B shows molecular dynamic simulation on the wild-type PbPAL (SEQ ID NO: 21) and PALRSBB (SEQ ID NO: 30). The amino acid positions are in reference to SEQ ID NO: 2. The root-mean-square fluctuation (RMSF) measures the average fluctuation of specific residues from reference positions over time. Dark gray: WT PbPAL. Light gray: PALRSBB.

[0059] FIG. 4C shows simulated molecular interactions between the substrate and the wildtype PbPAL (SEQ ID NO: 21) and PbPALRSBB (SEQ ID NO: 30). The top bar graphs represent different types of molecular interactions between the active site residues and the substrate based on the simulation. The bottom graphs illustrate detailed substrate atom interactions. The amino acid positions are in reference to SEQ ID NO: 2. Hydrophobic residues are shown by dark gray spheres, whereas the hydrophilic amino acids are shown in light gray. The H-bonds and water bridges are shown by light gray arrows. Pi-interactions are shown by dark gray arrow s (Phe82).The percentages shown on the arrows are the percent frequencies of the interactions over the course of the simulation. Only the residues that have >0.1% frequencies of the interactions were shown here.

[0060] FIGS. 5A and 5B show optimization of PbPAL reaction. (FIG. 5A) Evaluation of different ammonia sources. (FIG. 5B) Evaluation of PAL reaction parameters. All reactions were performed with 0.58 mmol 4 using 5 w / w% PbPALRSBB (SEQ ID NO: 18) in buffered ammonia unless noted. Conversions were determined by ultra-high performance liquid chromatography (UHPLC).DETAILED DESCRIPTION

[0061] The present disclosure relates to engineered PAL enzymes. As is described in the Examples, the present disclosure provides engineered PAL polypeptides from Planctomyces brasiliensis (PbPAL) with improved enzyme properties relative to wild-type PbPAL. including improved enzyme activity for the synthesis of L-3-cyanophenylalanine that were discovered through iterative rounds of directed evolution as described herein.

[0062] For example, the Examples describe the discovery7of a PAL variant referred to as PbPALR5BB (SEQ ID NO: 18) with >100-fold of activity7improvement and enhanced soluble protein expression relative to wild-ty pe PbPAL. The exemplary PbPALRSBB variant described herein provided high conversion (e.g., up to 97%) to 3-cyano-L-phenylalanine with low enzyme loading (<5%) in >99% enantioselectivity C'ee”), which was also demonstrated on up to 100- gram scale. In addition, the exemplary PbPALRSBB variant described herein exhibits a broad substrate scope against a variety of heterocyclic cinnamic acids with high enantioselectivity, demonstrating its synthetic utility. Other PbPAL variants disclosed herein (e.g., PbPALR2BB. PbPALRSBB, PbPALR4BB, and other PAL variants described in Tables 5, 6, 7, 8, 9, and 10) also have improved enzyme properties, including improved enzyme activity7, compared to wild-type PbPAL. Therefore, the present disclosure provides engineered PAL polypeptides that enable the biocatalytic synthesis of nonstandard phenylalanine derivatives at scale.

[0063] The present disclosure also relates to polynucleotides and expression vectors encoding the PAL polypeptides of the present disclosure, host cells comprising the polynucleotides or expression vectors, methods of producing the PAL polypeptides, and methods of converting 3- cyanocinnamic acid into 3-cyano-L-phenylalanine using the PAL polypeptides of the present disclosure.Definitions

[0064] Listed below are definitions of various terms used herein. These definitions apply to the terms as they are used throughout this specification and claims, unless otherwise limited in specific instances, either individually or as part of a larger group.

[0065] Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Generally, the nomenclature used herein and the laboratory procedures in cell culture, molecular genetics, organic chemistry, and peptide chemistry are those well-known and commonly employed in the art.

[0066] As used herein, the articles “a” and "an" refer to one or to more than one (i. e.. to at least one) of the grammatical object of the article. By way of example, “an element” means one element or more than one element. Furthermore, use of the term “including” as well as other forms, such as “include,” “includes,” and “included,” is not limiting.

[0067] As used herein, the term “about” in quantitative terms refers to plus or minus 10% of the value it modifies (rounded up to the nearest whole number if the value is not sub-dividable, such as a number of molecules or nucleotides).

[0068] As used herein, the terms “at least one” item or “one or more” item each include a single item selected from a list as well as combinations of two or more items selected from a list (e g., a list of mutations). Similarly, the terms “at least two” items and “two or more” items each include combinations of tw o items selected from a list as well as combinations of three or more items selected from a list.

[0069] All ranges disclosed herein are inclusive of the recited endpoint and independently combinable (for example, the range of “from 50 mg to 500 mg” is inclusive of the endpoints, 50 mg and 500 mg, and all the intermediate values). The endpoints of the ranges and any values disclosed herein are not limited to the precise range or value; they are sufficiently imprecise to include values approximating these ranges and / or values.

[0070] As used herein, the term “comprising” may include the embodiments “consisting of’ and “consisting essentially of.” The terms “comprise(s),” “include(s),” “having,” “has,” “may,” “contain(s),” and variants thereof, as used herein, are intended to be open-ended transitional phrases, terms, or words that require the presence of the named ingredients / steps and permit the presence of other ingredients / steps. How ever, such description should be construed as also describing compositions or processes as “consisting of’ and “consisting essentially of’ the enumerated components, which allows the presence of only the named components or compounds, along with any acceptable carriers or fluids, and excludes other components or compounds.

[0071] “Derived from” as used herein in the context of enzymes, identifies the originating enzyme, and / or the gene encoding such enzyme, upon which the enzyme was based. For example, the PbPALR5BB enzyme of SEQ ID NO: 18 was obtained by artificially evolving over multiple generations the gene encoding the wild-type PbPAL enzyme of SEQ ID NO: 2. Thus, the evolved PbPALR5BB enzyme is “derived from” the PbPAL of SEQ ID NO: 2.

[0072] As used herein, “polynucleotide” and “nucleic acid’ refer interchangeably to two or more nucleotides that are covalently linked together. The polynucleotide may be wholly comprised of ribonucleotides (i.e., RNA), wholly comprised of 2' deoxyribonucleotides (i.e., DNA), or comprised of mixtures of ribo- and 2' deoxyribonucleotides. While the nucleosides will typically be linked together via standard phosphodiester linkages, the polynucleotides may include one or more non-standard linkages. The polynucleotide may be single-stranded or double-stranded, or the polynucleotide may include both single-stranded regions and doublestranded regions. Moreover, while a polynucleotide will typically be composed of the naturally occurring encoding nucleobases (i.e., adenine, guanine, uracil, thymine, and cytosine), it may include one or more modified and / or synthetic nucleobases, such as, for example, inosine, xanthine, hypoxanthine, etc. In some embodiments, such modified or synthetic nucleobases are nucleobases encoding amino acid sequences.

[0073] The abbreviations used for the genetically encoding nucleosides are conventional and are as follows: adenosine (A); guanosine (G); cytidine (C); thymidine (T); and uridine (U). Unless specifically delineated, the abbreviated nucleosides may be either ribonucleosides or 2'- deoxyribonucleosides. The nucleosides may be specified as being either ribonucleosides or 2'- deoxyribonucleosides on an individual basis or on an aggregate basis. When nucleic acid sequences are presented as a string of one-letter abbreviations, the sequences are presented in the 5' to 3' direction in accordance with common convention, and the phosphates are not indicated.

[0074] As used herein, the terms “protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post-translational modification (e.g., glycosylation, phosphorylation, lipidation, myristoylation, ubiquitination, and the like). Included within this definition are D- and L-amino acids, and mixtures of D- and L-amino acids, as w ell as polymers comprising D- and L-amino acids, and mixtures of D- and L-amino acids. Proteins, polypeptides, and peptides may include a tag (e.g.. an epitope tag), such as a histidine tag.

[0075] As used herein, the terms “amino acid” or “residue” as used in context of the polypeptides disclosed herein refers to the specific monomer at a sequence position. Amino acids are referred to herein by either their commonly known three-letter symbols or by the one-lettersymbols recommended by International Union of Pure and Applied Chemistry (1UPAC) - International Union of Biochemistry (IUB) Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single letter codes.

[0076] The abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), try ptophan (Trp or W), ty rosine (Tyr or Y), and valine (Vai or V).

[0077] “Hydrophilic amino acid’’ or “hydrophilic residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of less than zero according to the normalized consensus hydrophobicity7scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophilic amino acids include L-Thr (T), L-Ser (S), L-His (H), L-Glu (E), L-Asn (N), L-Gln (Q). L-Asp (D). L-Lys (K). and L-Arg (R).

[0078] “Acidic amino acid” or “acidic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pK value of less than about 6 when the amino acid is included in a peptide or polypeptide. Acidic amino acids typically have negatively charged side chains at physiological pH due to loss of a hydrogen ion. Genetically encoded acidic amino acids include L-Glu (E) and L-Asp (D).

[0079] “Basic amino acid” or “basic residue” refers to a hydrophilic amino acid or residue having a side chain exhibiting a pKa value of greater than about 6 when the amino acid is included in a peptide or polypeptide. Basic amino acids typically have positively charged side chains at physiological pH due to association with hydronium ion. Genetically encoded basic amino acids include L-Arg (R) and L-Lys (K).

[0080] “Polar amino acid” or “polar residue” refers to a hydrophilic amino acid or residue having a side chain that is uncharged at physiological pH. but which has at least one bond in which the pair of electrons shared in common by two atoms is held more closely by one of the atoms. Genetically encoded polar amino acids include L-Asn (N), L-Gln (Q), L-Ser (S), and L- Thr (T).

[0081] “Hydrophobic amino acid” or “hydrophobic residue” refers to an amino acid or residue having a side chain exhibiting a hydrophobicity of greater than zero according to the normalized consensus hydrophobicity scale of Eisenberg et al., 1984, J. Mol. Biol. 179: 125-142. Genetically encoded hydrophobic amino acids include L-Pro (P), L-Ile (I), L-Phe (F), L-Val (V), L-Leu (L), L-Trp (W), L-Met (M), L-Ala (A), and L-Tyr (Y).

[0082] “Aromatic amino acid” or “aromatic residue” refers to a hydrophilic or hydrophobic amino acid or residue having a side chain that includes at least one aromatic or heteroaromatic ring. Genetically encoded aromatic amino acids include L-Phe (F), L-Tyr (Y), L-His (H), and L- Trp (W). L-His (H) histidine is also classified herein as a hydrophilic residue or as a constrained residue.

[0083] As used herein, “constrained amino acid” or “constrained residue” refers to an amino acid or residue that has a constrained geometry. Herein, constrained residues include L-Pro (P) and L-His (H). Histidine has a constrained geometry because it has a relatively small imidazole ring. Proline has a constrained geometry because it also has a five-membered ring.

[0084] “Non-polar amino acid” or “non-polar residue” refers to a hydrophobic amino acid or residue that has a side chain that is uncharged at physiological pH and that has bonds in which the pair of electrons shared in common by two atoms is generally held equally by each of the two atoms (i.e., the side chain is not polar). Genetically encoded non-polar amino acids include L-Gly (G), L-Leu (L), L-Val (V). L-Ile (I). L-Met (M), and L-Ala (A).

[0085] As used herein, “aliphatic amino acid” or “aliphatic residue” refers to a hydrophobic amino acid or residue having an aliphatic hydrocarbon side chain. Genetically encoded aliphatic amino acids include L-Ala (A), L-Val (V), L-Leu (L). and L-Ile (I).

[0086] The ability of L-Cys (C) (and other amino acids with SH-containing side chains) to exist in a peptide in either the reduced free SH or oxidized disulfide-bridged form affects whether L- Cys (C) contributes net hydrophobic or hydrophilic character to a peptide. While L-Cys (C) exhibits a hydrophobicity of 0.29 according to the normalized consensus scale of Eisenberg (Eisenberg et al.. 1984, supra), it is to be understood that for purposes of the present disclosure, L-Cys (C) is categorized into its own unique group. It is noted that L-Cys (C) is unusual in that it can form disulfide bridges with other L-Cys (C) amino acids or other sulfanyl- or sulfhydryl- containing amino acids. The “cysteine-like residues” include cysteine and other amino acids that contain sulfhydryl moieties that are available for formation of disulfide bridges.

[0087] As used herein, “small amino acid” or “small residue” refers to an amino acid or residue having a side chain that is composed of a total three or fewer carbon and / or heteroatoms (excluding the a carbon and hydrogens). The small amino acids or residues may be further categorized as aliphatic, non-polar, polar or acidic small amino acids or residues, in accordance with the above definitions. Genetically encoded small amino acids include L-Ala (A), L-Val (V), L-Cys (C), L-Asn (N), L-Ser (S), L-Thr (T), and L-Asp (D).

[0088] "Hydroxyl-containing amino acid” or “hydroxyl-containing residue” refers to an amino acid containing a hydroxyl (-OH) moiety. Genetically encoded hydroxyl-containing amino acids include L-Ser (S), L-Thr (T), and L-Tyr (Y).

[0089] As used herein, “conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids. By way of example and not limitation, in some embodiments, an amino acid with an aliphatic side chain is substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with an hydroxyl side chain is substituted with another amino acid with an hydroxyl side chain (e.g., serine and threonine); an ammo acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another amino acid with an acidic side chain (e.g., aspartic acid and glutamic acid); and / or a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.

[0090] As used herein, “non-conservative substitution” refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Nonconservative substitutions may use amino acids between, rather than within, the defined groups and affects (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine) (b) the charge or hydrophobicity', or (c) the bulk of the side chain. By way of example and not limitation, an exemplary non-conservative substitution can be an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0091] “Mutation” refers to any change in a polypeptide or polynucleotide sequence, and encompasses any number (i.e.. one or more) of substitutions, deletions, insertions, and / or rearrangements present in a sequence compared to a reference sequence.

[0092] As used herein, “deletion” refers to modification to a polypeptide by removal of one or more amino acids relative to a reference polypeptide (e.g., enzy me). Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference polypeptide while retaining activity (e.g., enzymatic activity) and / or retaining the improved properties of an evolved polypeptide. Deletions can be directed to the internal portions and / orterminal portions of the polypeptide. In various embodiments, the deletion can comprise a continuous segment or can be discontinuous. Deletions are typically indicated byin amino acid sequences.

[0093] As used herein, “insertion’" refers to modification to a polypeptide by addition of one or more amino acids relative to a reference polypeptide (e.g., enzyme). Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.

[0094] As used herein with respect to amino acid sequences, a “substitution” refers to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence. In some instances, the present disclosure provides specific amino acid differences denoted by the conventional notation “AnB,” where A is the single letter identifier of the residue in the reference sequence, n is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.

[0095] The term “amino acid substitution set” or “substitution set” refers to a group of amino acid substitutions in a polypeptide sequence, as compared to a reference sequence. For example, a substitution set may include 2, 3, 4, 5, 6. 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions.

[0096] “Corresponding to,” “reference to” or “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refers to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.

[0097] A “functional fragment” and “biologically active fragment” are used interchangeably herein to refer to a polypeptide that has an amino-terminal and / or carboxy -terminal deletion(s) and / or internal deletion(s), but where the remaining amino acid sequence is identical to the corresponding positions in the reference sequence and that retains substantially all of the activity of the full-length polypeptide.

[0098] As used herein, “isolated polypeptide” refers to a composition in which the polypeptide is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides). The term embraces polypeptides that have been removed or purified from their naturally occurring environment or expression system (e g., within a host cell or via in vitro synthesis). The recombinant polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations. As such, in some embodiments, the recombinant polypeptides can be an isolated polypeptide.

[0099] As used herein, “substantially pure polypeptide” or “purified protein” refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight. However, in some embodiments, an enzyme comprising composition comprises enzymes that are less than 50% pure (e.g.. about 10%. about 20%, about 30%, or about 40% pure). Generally, a substantially pure enzy me or polypeptide composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition. In some embodiments, the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, the isolated recombinant polypeptides are substantially pure polypeptide compositions.

[0100] “Improved enzy me property” refers to any property of an enzy me that exhibits an improvement as compared to a reference enzy me. For the enzymes described herein, the comparison is generally made to the wild-type enzyme, although in some embodiments, the reference enzyme can be another improved enzyme. Enzyme properties for which improvement may be desirable include, but are not limited to, enzymatic activity (which may be expressed in terms of percent conversion of the substrate), thermal stability, stability7under high ammonia concentration, soluble expression, pH activity profile, cofactor requirements, refractoriness to inhibitors (e.g.. product inhibition), stereospecificity7, and stereoselectivity (including enanti ©selectivity).

[0101] “Increased enzy matic activity ” refers to an improved property' of the enzymes, which can be represented by an increase in specific activity7(e.g., product produced / time / weight protein)or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of enzyme) as compared to a reference enzyme. Exemplary methods to determine enzyme activity are provided in the Examples. Any property relating to enz me activity may be affected, including the classical enzyme properties of Km. Nmax. or kc«t. changes of which can lead to increased enzymatic activity. Improvements in enzy me activity can be from about 1.5 times the enzymatic activity of the corresponding wild-type enzyme, to as much as 2 times. 5 times, 10 times, 20 times, 25 times, 50 times, 75 times, 100 times, 150 times, 200 times, 500 times, 1000 times, 3000 times, 5000 times. 7000 times or more enzymatic activity than the reference enzyme, e.g., a naturally occurring enzyme or another enzyme from which the polypeptides were derived. In some examples, the enzyme exhibits improved enzymatic activity in the range of 100 to 3000 times, 3000 to 7000 times, or more than 7000 times greater than that of the parent enzy me. It is understood by the skilled artisan that the activity of any enzyme is diffusion limited such that the catalytic turnover rate cannot exceed the diffusion rate of the substrate, including any required cofactors. The theoretical maximum of the diffusion limit, or kcar / K™, is generally about 108to 109 1s"1). Hence, any improvements in the enz me activity will have an upper limit related to the diffusion rate of the substrates acted on by the enzyme. Enzyme activity can be measured by any suitable approach, e.g., an enzyme activity assay or by any of the traditional methods for assaying chemical reactions, including but not limited to high-performance liquid chromatography (HPLC), HPLC-mass spectrometry (MS), ultra-performance liquid chromatography (UPLC), UPLC-MS, thin-layer chromatography (TLC), and nuclear magnetic resonance (NMR). Comparisons of enzyme activities may be made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the numbers of cells and the amount of protein assayed are determined as well as use of identical expression systems and identical host cells to minimize variations in amount of enzy me produced by the host cells and present in the lysates.

[0102] As used herein, a '‘vector’’ is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression of the polypeptide encoded by the polynucleotide (e.g., DNA) sequence in a suitable host. In some embodiments, an “expression vector” has a promoter sequence operably linked to the polynucleotide (e.g., DNA) sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.

[0103] As used herein with respect to polypeptides, the terms “expression” and “production” includes any step involved in the production of a polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.

[0104] As used herein, an amino acid or nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, and the like) is “heterologous” to another sequence with which it is operably linked if the two sequences are not associated in nature. For example, a “heterologous polynucleotide” is any polynucleotide that is introduced into a host cell by laboratory techniques, and the term includes polynucleotides that are removed from a host cell, subjected to laboratory manipulation, and then reintroduced into a host cell.

[0105] As used herein, the terms “host cell” and “host strain” refer to suitable hosts for an expression vector comprising a polynucleotide (e.g., DNA) provided herein (e.g., a polynucleotide encoding a PAL polypeptide disclosed herein). In some embodiments, the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.

[0106] The term “analogue” means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%. 93%. 94%. 95%. 96%. 97%. 98%. or 99% sequence identity) with a reference polypeptide. In some embodiments, “analogues” means polypeptides that contain one or more non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids. In some embodiments, analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0107] “Coding sequence” refers to that portion of a polynucleotide (e g., a gene) that encodes an amino acid sequence of a polypeptide.

[0108] “Naturally occurring” or “wild-type” refers to a form found in nature. For example, a naturally occurring or wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and that has not been intentionally modified by human manipulation, with the sole exception that wild-type polypeptide or polynucleotide sequences as identified herein may include a tag, such as a histidine (His) tag. For example, the PbPAL sequence of SEQ ID NO: 2, which includes an N-terminal His tag (and thus is not found in nature), may be referred to as a wild-type PbPAL herein. Herein, “wild-type” polypeptide or polynucleotide sequences may be denoted “WT.”

[0109] "Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed at a position relative to a polynucleotide sequence (i.e., in a functional relationship) such that the control sequence directs the expression of the polynucleotide and / or a polypeptide encoded by the polynucleotide.

[0110] A “promoter sequence” is a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide. The control sequence may comprise an appropriate promoter sequence. The promoter sequence contains transcriptional control sequences, which mediate the expression of the polynucleotide. The promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.[OHl] The terms “engineered,” “recombinant,” “variant,” and “non-naturally occurring,” when used with reference to, e g., a polynucleotide, polypeptide, or cell, refers to a material, or a material corresponding to the natural or native form of the material, or that has been modified in a manner that would not otherwise exist in nature. Non-limiting examples include, among others, recombinant cells expressing genes that are not found within the native (non-recombinant) form of the cell or express native genes that are otherwise expressed at a different level.

[0112] A “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like. Exemplary selectable markers are described herein.

[0113] “Percentage of sequence identity,” “percent identity,” and “percent identical” are used herein to refer to comparisons between polynucleotide sequences or polypeptide sequences, and are determined by comparing two optimally aligned sequences over a companson window, wherein the portion of the polynucleotide or polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences. The percentage is calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity’. Determination of optimal alignment and percent sequence identity can be performed using the BLAST and BLAST 2.0 algorithms (see e.g., Altschul et al., 1990, J. Mol. Biol. 215: 403-410; and Altschul et al., 1977, Nucleic Acids Res. 3389-3402). Software forperforming BLAST analyses is publicly available through the National Center for Biotechnology Information website.

[0114] Briefly, the BLAST analyses involve first identifying high scoring sequence pairs (HSPs) by identifying short words of length W in the query sequence, which either match or satisfy some positive-valued threshold score T when aligned with a word of the same length in a database sequence. T is referred to as, the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits act as seeds for initiating searches to find longer HSPs containing them. The word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters M (reward score for a pair of matching residues; always >0) and N (penalty score for mismatching residues; always <0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity X from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M = 5, N = -4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see Henikoff and Henikoff, 1989, PROC. NATL. ACAD. SCI. USA 89: 10915).

[0115] Numerous other algorithms are available that function similarly to BLAST in providing percent identify for two sequences. Optimal alignment of sequences for comparison can be conducted, e.g., by the local homology algorithm of Smith and Waterman, 1981, Adv. Appl. Math. 2:482, by the homology7alignment algorithm of Needleman and Wunsch, 1970, J. Mol. Biol. 48:443, by the search for similarity7method of Pearson and Lipman. 1988, Proc. Natl. Acad. Sci. USA 85:2444. by computerized implementations of these algorithms (GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection (see generally, Current Protocols in Molecular Biology7, F. M. Ausubel et al., eds., Current Protocols, ajoint venture between Greene Publishing Associates, Inc. and John Wiley & Sons, Inc., (1995 Supplement) (Ausubel)). Additionally, determination of sequence alignment and percent sequence identify7can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.

[0116] “Stereoselectivity” refers to the preferential formation in a chemical or enzymatic reaction of one stereoisomer over another. Stereoselectivity can be partial, where the formation of one stereoisomer is favored over the other, or it may be complete where only one stereoisomer is formed. When the stereoisomers are enantiomers, the stereoselectivity is referred to as enantioselectivity, the fraction (typically reported as a percentage) of one enantiomer in the sum of both. It is commonly alternatively reported in the art (typically as a percentage) as the enantiomeric excess (EE) calculated therefrom according to the formula [major enantiomer - minor enantiomer] / [major enantiomer + minor enantiomer]. Where the stereoisomers are diastereoisomers, the stereoselectivity is referred to as diastereoselectivity, the fraction (typically reported as a percentage) of one diastereomer in a mixture of two diastereomers, commonly alternatively reported as the diastereomeric excess (DE). Enantiomeric excess and diastereomeric excess are types of stereomeric excess.

[0117] “Conversion” refers to the enz matic transformation of a substrate to the corresponding product. “Percent conversion” refers to the percent of the substrate that is converted to the product within a period of time under specified conditions. Thus, for example, the “enzymatic activity” or “activity7” of a polypeptide can be expressed as “percent conversion” of the substrate to the product.

[0118] “Thermostable” refers to a polypeptide that maintains similar activity (more than 60% to 80%, for example) after exposure to elevated temperatures (e.g., 40 °C to 80 °C) for a period of time (e.g., 0.5 h to 24 h) compared to the untreated enzy me.

[0119] As used herein, the terms “biocatalysis,” “biocatalytic,” “biotransformation,” and “biosynthesis” refer to the use of enzymes to perform chemical reactions on organic compounds.

[0120] The term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.

[0121] The terms “isolated” and “purified” are used to refer to a molecule (e.g., an isolated nucleic acid or polypeptide) or other component that is removed from at least one other component with which it is naturally associated. The term “purified” does not require absolute purity, rather it is intended as a relative definition.

[0122] The terms “PAL enzyme” or “PAL polypeptide” refer to a polypeptide having a capability of catalyzing the non-oxi dative deamination of L-phenylalanine to generate a cinnamic acid and / or catalyzing hydroamination of a cinnamic acid to generate a L-phenylalanine. For example, the PAL polypeptides disclosed herein are capable of converting heterocyclic cinnamic acids (e.g., 3-cyanocinnamic acid) into L-phenylalanines (e.g., 3-cyano-L-phenylalanine). PALpolypeptide as used herein includes naturally occurring (wild-type) PAL polypeptides as well as non-naturally occurring engineered polypeptides generated by human manipulation. In an embodiment, a PAL enzyme as disclosed herein may have an amino acid sequence selected from any one of SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 23. 24. 25, 26, 27, 28, 29, 30, and 31.PAL Polypeptides

[0123] This disclosure provides polypeptides (e.g., PAL polypeptides) capable of converting heterocyclic cinnamic acids (e g., 3-cyanocinnamic acid) into L-phenylalanines (e.g., 3-cyano-L- phenylalanine). See, e.g., FIG. IB.

[0124] In certain embodiments, a polypeptide (e.g., a PAL polypeptide) described herein has an amino acid sequence comprising one or more amino acid differences as compared to a reference amino acid sequence of a wild-type poly peptide that result in an improved enz me property.

[0125] In some embodiments, a polypeptide (e.g., PAL polypeptides) as described herein is a product of directed evolution from a wild-type sequence (e.g., a wild-type PAL polypeptide as set forth in SEQ ID NO: 2, which includes an N-terminal His tag or a wild-type PAL polypeptide as set forth in SEQ ID NO: 21, which lacks an N-terminal His tag).

[0126] In some embodiments, the polypeptide (e.g., the PAL polypeptide) of the disclosure may demonstrate one or more improvements relative the polypeptide of SEQ ID NO: 2 or SEQ ID NO: 21, such as increases in enzyme activity, stereoselectivity (e.g., enantioselectivity), stereospecificity, thermostability', stability under high ammonia concentration, and / or soluble expression (e.g., increased recombinant / soluble expression in E. coli).

[0127] For example, in some embodiments, the polypeptide has one or more of the following properties relative to a reference polypeptide: a) increased activity in converting 3- cyanocinnamic acid into 3-cyano-L-phenylalanine, optionally wherein the polypeptide has at least about 1.1-fold, about 5-fold, about 10-fold, or about 100-fold increased activity; b) increased enantioselectivity; c) increased thermostability; and / or d) increased soluble expression. In some embodiments, the polypeptide r about 100-fold increased activity. In some embodiments, the reference polypeptide comprises the amino acid sequence of SEQ ID NO: 2 or 21.

[0128] In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least about 60% (e.g., 60%, 70%, 80%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher). In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 90%. In some embodiments, such polypeptides are capable of converting thesubstrate to the product with a percent enantiomeric excess of at least 91%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 92%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 93%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 94%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 95%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 96%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 97%. In some embodiments, such polypeptides are capable of converting the substrate to the product with a percent enantiomeric excess of at least 98%. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 99%.

[0129] In some embodiments, the polypeptide (e.g., the PAL polypeptide) is highly enantioselective, wherein the polypeptide can reduce the substrate to the product in greater than about 99%. 99.1%. 99.2%. 99.3%, 99.4%, 99.5%, 99.6%, 99.7%, 99.8%, or 99.9% enantiomeric excess.

[0130] In some embodiments, a polypeptide (e.g., PAL polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 2. In other embodiments, an improved polypeptide (e.g., PAL polypeptide) of the disclosure is a polypeptide that comprises an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 21.

[0131] These differences between these variants and SEQ ID NO:2 or SEQ ID NO: 21 can be amino acid insertions, deletions, substitutions, or any combinations of such changes. In some embodiments, the amino acid sequence differences can comprise non-conservative, conservative, as well as a combination of non-conservative and conservative amino acid substitutions. In some embodiments, the amino acid sequence differences are conservative amino acid substitutions. In other embodiments, the amino acid sequence differences are non-conservative amino acid substitutions.

[0132] For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence having at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to SEQ ID NO: 21, wherein the polypeptide comprises at least one substitution at one or more positions selected from 45. 58. 63. 82. 90. 215, 276. 282, 284, 294, 298, 345. 348, 473. 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0133] In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more positions selected from 45, 58, 63, 82, 90, 215, 276, 282, 284, 294, 298, 345, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0134] In some embodiments, the polypeptide comprises: a) a substitution set at positions 45, 63, 82, 90, 215, 276, 348, 473. 498, and 522; b) a substitution at position 90; c) a substitution at position 294; d) a substitution set at positions 82, 90, 276. and 473; e) a substitution set at positions 45, 82, 90, 276, and 473; 1) a substitution set at positions 82, 90, 276, and 473; g) a substitution set at positions 45, 63, 82, 90, 276, 348, 473, and 498; h) a substitution set at positions 45, 82, 90, 276, 298, 345, and 473; or i) a substitution set at positions 45, 58, 63, 82, 90, 215, 276. 282, 284, 348, 473, 498. and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 45, 63, 82, 90, 215, 276, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution at position 90, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution at position 294, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 82, 90, 276. and 473, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 45, 82, 90, 276, and 473, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 82, 90, 276, and 473, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 45, 63, 82, 90, 276, 348, 473, and 498, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In someembodiments, the polypeptide comprises a substitution set at positions 45, 82, 90, 276. 298, 345, and 473, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises a substitution set at positions 45, 58, 63, 82, 90, 215, 276, 282, 284, 348, 473. 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0135] In some embodiments, the polypeptide comprises one or more substitutions set forth in Table 9.

[0136] In some embodiments, the polypeptide comprises one of the following substitutions or substitution sets: a) R45K, I63V, H82M, L90A, S215A, Y276V, M348T. A473R, E498G, and M522V; b) L90V; c) L294N; d) H82M, L90A, Y276V, and A473R; e) R45K, H82M, L90A, Y276P, and A473R; f) H82M, L90A, Y276P, and A473L; g) R45K, I63V, H82M, L90A, Y276V, M348T, A473R, and E498G; h) R45K, H82M, L90A, Y276V, A298P, Y345S, and A473R; or i) R45K, R58D, I63V, H82M, L90A, S215A, Y276V, K282T, N284D, M348T, A473R, E498G, and M522T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises R45K, I63V, H82M, L90A, S215A, Y276V, M348T, A473R, E498G, and M522V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises an L90V substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises an L294N substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises H82M, L90A, Y276V, and A473R substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises R45K, H82M, L90A, Y276P, and A473R substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises H82M, L90A, Y276P, and A473L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises R45K, I63V, H82M, L90A, Y276V, M348T, A473R, and E498G substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises R45K. H82M, L90A, Y276V, A298P, Y345S, and A473R substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21. In some embodiments, the polypeptide comprises R45K, R58D, I63V, H82M, L90A, S215A, Y276V, K282T, N284D, M348T, A473R, E498G, and M522Tsubstitutions. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

[0137] In some embodiments, the amino acid sequence comprises or consists of any one of SEQ ID NO: 30, 23, 24, 25, 26, 27, 28, 29, or 31.

[0138] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 30. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 30. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30.

[0139] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 23. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 23. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 23. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 23. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 23.

[0140] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 24. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 24. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 24. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 24. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 24.

[0141] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 25. For example, provided herein is a polypeptide (e.g., a PALpolypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 25. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 25. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 25. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 25.

[0142] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%. 94%, 95%. 96%. 97%. 98%. 99%, or higher sequence identity’ to SEQ ID NO: 26. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 26. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 26. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 26. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 26.

[0143] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%. 91%, 92%. 93%. 94%. 95%. 96%, 97%. 98%, 99%, or higher sequence identity’ to SEQ ID NO: 27. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 27. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 27. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 27. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 27.

[0144] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or higher sequence identity’ to SEQ ID NO: 28. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 28. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 28. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 28. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 28.

[0145] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 29. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 29. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 29. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 29. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 29.

[0146] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 30. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 30. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 30. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 30.

[0147] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 31. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 31. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 31. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 31. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 31.

[0148] In some embodiments, the polypeptide further comprises a tag (e g., an epitope tag). Any suitable tag may be used, e.g., a His tag, a FLAG tag, a fluorescent protein tag (e.g., green fluorescent protein (GFP), yellow fluorescent protein (YFP), or red fluorescent protein (RFP)), a hemagglutinin (HA) tag. an ALFA-tag, a V5-tag, a Myc-tag, a SPOT-tag, a T7-tag, or an NE-tag. In some embodiments, the epitope tag is a His tag. In some embodiments, the His tag comprises the amino acid sequence of GSHHHHHHHHSG (SEQ ID NO: 22). In some embodiments, thepolypeptide comprises an N-terminal methionine residue, and the epitope tag is inserted immediately following the N-terminal methionine residue, e.g., relative to a reference sequence.

[0149] In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence having at least 80% (e.g.. 80%. 81%. 82%. 83%. 84%. 85%, 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises at least one substitution at one or more positions selected from 57, 70, 75, 94, 102, 227, 288, 294, 296, 306, 310, 357, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0150] In some embodiments, the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more positions selected from 57, 70, 75, 94, 102, 227, 288, 294, 296, 306, 310, 357, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0151] In some embodiments, the polypeptide comprises: a) a substitution set at positions 57, 75, 94, 102, 227, 288, 360, 485, 510, and 534: b) a substitution at position 102: c) a substitution at position 306; d) a substitution set at positions 94, 102, 288, and 485; e) a substitution set at positions 57, 94, 102, 288, and 485; f) a substitution set at positions 94. 102, 288, and 485; g) a substitution set at positions 57, 75, 94. 102, 288, 360, 485, and 510; h) a substitution set at positions 57, 94, 102, 288, 310, 357, and 485; or i) a substitution set at positions 57, 70, 75, 94, 102, 227, 288, 294, 296, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 57. 75. 94, 102, 227. 288, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution at position 102, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution at position 306, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 94, 102, 288, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 57, 94, 102, 288, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 94, 102, 288, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution setat positions 57. 75. 94. 102, 288. 360, 485, and 510. wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 57, 94, 102, 288, 310, 357, and 485, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises a substitution set at positions 57, 70, 75, 94, 102. 227, 288, 294, 296, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

[0152] In some embodiments, the polypeptide comprises one or more substitutions set forth in any one of Tables 4, 5. 6, 7, or 8.

[0153] In some embodiments, the polypeptide comprises one of the following substitutions or substitution sets: a) R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R, E510G, and M534V; b) L102V; c) L306N; d) H94M, L102A, Y288V, and A485R; e) R57K, H94M, L102A, Y288P, and A485R; f) H94M, L102A, Y288P, and A485L; g) R57K, I75V, H94M, L102A, Y288V, M360T. A485R, and E510G; h) R57K. H94M. L102A, Y288V, A310P. Y357S. and A485R; or i) R57K, R70D, I75V, H94M, L102A, S227A, Y288V, K294T, N296D, M360T, A485R, E510G, and M534T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, I75V, H94M. L102A, S227A, Y288V, M360T, A485R, E510G. and M534V substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an LI 02V substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises an L306N substitution, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises H94M, L102A, Y288V, and A485R substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, H94M, L102A, Y288P, and A485R substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises H94M, L102A, Y288P, and A485L substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, I75V, H94M, L102A, Y288V, M360T, A485R, and E510G substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, H94M, L102A, Y288V, A310P, Y357S, and A485R substitutions, wherein the amino acid positions of the polypeptide are numbered withreference to SEQ ID NO: 2. In some embodiments, the polypeptide comprises R57K, R70D. I75V, H94M, L102A, S227A, Y288V, K294T, N296D, M360T, A485R, E510G, and M534T substitutions, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2

[0154] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 18. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 18. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 18. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 18. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 18.

[0155] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 4. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 4. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 4. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 4. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 4.

[0156] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity' to SEQ ID NO: 6. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 6. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 6. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 6. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 6.

[0157] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 8. For example, provided herein is a poly peptide (e.g., a PALpolypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 8. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 8. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 8.

[0158] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%. 94%, 95%. 96%. 97%. 98%. 99%, or higher sequence identity’ to SEQ ID NO: 10. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 10. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 10. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 10.

[0159] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%. 91%, 92%. 93%. 94%. 95%. 96%, 97%. 98%, 99%, or higher sequence identity’ to SEQ ID NO: 12. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 12. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 12. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 12. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 12.

[0160] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or higher sequence identity’ to SEQ ID NO: 14. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 14. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 14. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 14. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 14.

[0161] Provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher sequence identity to SEQ ID NO: 16. For example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising an amino acid sequence comprising at least 99% sequence identity to SEQ ID NO: 16. In another example, provided herein is a polypeptide (e.g., a PAL polypeptide) comprising the amino acid sequence of SEQ ID NO: 16. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting essentially of the amino acid sequence of SEQ ID NO: 16. Also provided herein is a polypeptide (e.g., a PAL polypeptide) consisting of the amino acid sequence of SEQ ID NO: 16.

[0162] In some embodiments, the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0163] In another embodiment, provided herein is an engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 18, 4, 6, 8, 10, 12, 14, 16, or 20, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

[0164] In some embodiments, the polypeptide is isolated.

[0165] In some embodiments, the polypeptide is a phenylalanine ammonia lyase.

[0166] Also provided herein are functional fragments of any of the polypeptides disclosed herein. Also provided herein are analogues of any of the polypeptides disclosed herein.

[0167] Also provided herein are compositions comprising any of the polypeptides disclosed herein. For example, the composition may include an effective amount of the polypeptide, e.g., for converting a heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). The composition may include one or more carriers or diluents.Polynucleotides Encoding PAL Polypeptides

[0168] In another aspect, the present disclosure provides polynucleotides encoding the polypeptides (e.g., PAL polypeptides) disclosed herein. The polynucleotides may be operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide. Expression vectors containing a heterologous polynucleotide encoding the polypeptide (e.g., PAL polypeptide) can be introduced into appropriate host cells to express the corresponding polypeptide.

[0169] Because of the knowledge of the codons corresponding to the various amino acids, availability of a protein sequence provides a description of all the polynucleotides capable of encoding the subject. The degeneracy of the genetic code, where the same amino acids are encoded by alternative or synonymous codons allows an extremely large number of nucleic acidsto be made, all of which encode the polypeptides (e.g., PAL polypeptides) disclosed herein. Thus, having identified a particular amino acid sequence, those skilled in the art could make any number of different nucleic acids by simply modifying the sequence of one or more codons in a way that does not change the amino acid sequence of the protein. In this regard, the present disclosure specifically contemplates each and every possible variation of polynucleotides that could be made by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide disclosed herein.

[0170] In various embodiments, the codons are preferably selected to be suitable for the host cell in which the polypeptide is being produced. For example, preferred codons used in bacteria are used to express the gene in bacteria; preferred codons used in yeast are used for expression in yeast; and preferred codons used in mammals are used for expression in mammalian cells. By way of example, the polynucleotide of SEQ ID NO: 1 provided herein has been codon optimized for expression in E. coll. In another example, the polynucleotide of SEQ ID NO: 33 provided herein has been codon optimized for expression in E. coli.

[0171] In certain embodiments, all codons need not be replaced to optimize the codon usage of the polypeptide (e.g., PAL polypeptide) since the natural sequence will comprise preferred codons and because use of preferred codons may not be required for all amino acid residues. Consequently, codon optimized polynucleotides encoding the polypeptides (e.g.. PAL polypeptides) may contain preferred codons at about 40%, 50%, 60%, 70%, 80%, or greater than 90% of codon positions of the full-length coding region.

[0172] Provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%. 88%, 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 1, 3, 5. 7, 9, 11, 13, 15, 17, 19, or 33. In some examples, the polynucleotide sequence of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 15, 17, or 19 is modified to no longer code for an N-terminal His tag having the amino acid sequence of SEQ ID NO: 22.

[0173] For example, provided herein is a polynucleotide comprising at least 80% (e.g.. 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 1. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 1. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 1.

[0174] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identify to the polynucleotide sequence of SEQ IDNO: 3. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 3. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 3.

[0175] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%. 91%. 92%. 93%. 94%. 95%. 96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 5. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 5. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 5.

[0176] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%. 82%, 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 7. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 7. In some examples, the poly nucleotide consists of the sequence of SEQ ID NO: 7.

[0177] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 9. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 9. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 9.

[0178] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 11. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 11. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 11.

[0179] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity7to the polynucleotide sequence of SEQ ID NO: 13. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 13. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 13.

[0180] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity' to the polynucleotide sequence of SEQ ID NO: 15. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 15. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 15.

[0181] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%,96%. 97%. 98%. 99%. or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 17. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 17. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 17.

[0182] In another example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%. 82%. 83%. 84%. 85%. 86%. 87%. 88%. 89%. 90%. 91%. 92%. 93%. 94%. 95%. 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 19. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 19. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 19.

[0183] For example, provided herein is a polynucleotide comprising at least 80% (e.g., 80%. 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or 100%) sequence identity to the polynucleotide sequence of SEQ ID NO: 33. In some examples, the polynucleotide comprises the sequence of SEQ ID NO: 33. In some examples, the polynucleotide consists of the sequence of SEQ ID NO: 33.

[0184] In various embodiments, an isolated polynucleotide encoding polypeptide (e.g.. a PAL polypeptide) may be manipulated in a variety of ways to provide for expression of the polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art. For example, guidance is provided in Sambrook et al., 2001, Molecular Cloning: A Laboratory Manual, 3rdEd., Cold Spring Harbor Laboratory' Press; and Current Protocols in Molecular Biology, Ausubel. F. ed., Greene Pub. Associates, 1998, updates to 2006.

[0185] In some embodiments, an isolated polynucleotide encoding any of the polypeptides herein is manipulated in a variety of ways to facilitate expression of the polypeptide. In some embodiments, the polynucleotides encoding the polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the polynucleotides and / or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences utilizing recombinant DNA methods are well known in the art.

[0186] In some embodiments, the control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.

[0187] In some embodiments, the control sequence is a suitable promoter sequence. In some embodiments, suitable promoters are selected based on the host cell selection. For bacterial hostcells, suitable promoters for directing transcnption of the nucleic acid constructs of the present disclosure, include, but are not limited to, promoters obtained from the E. coll lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM). Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA 75: 3727-3731, 1978), as well as the tac promoter (See e.g., DeBoer et al., Proc. Natl Acad. Sci. USA 80: 21-25, 1983). Exemplary promoters for filamentous fungal host cells, include, but are not limited to, promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (See e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triose phosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1). Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (See e.g., Romanos et al., Yeast 8:423-488. 1992).

[0188] In some embodiments, the control sequence is a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription). In some embodiments, the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the enzyme polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary7terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase. Other useful terminators for yeast host cells are known in the art (See e.g., Romanos et al., supra).

[0189] In some embodiments, the control sequence is a suitable leader sequence (i.e.. a nontranslated region of an mRNA that is important for translation by the host cell). In some embodiments, the leader sequence is operably linked to the 5' terminus of the polynucleotide sequence encoding the polypeptide. Any suitable leader sequence that is functional in the host cell of choice find use in the present disclosure. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA, amylase, an Aspergillus nidulans triose phosphate isomerase. Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3- phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP).

[0190] In some embodiments, the control sequence is a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add poly adenosine residues to transcribed mRNA). Any suitable poly adenylation sequence that is functional in the host cell of choice may be used in the present disclosure. Exemplary' polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful poly adenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Biol., 15:5983-5990, 1995).

[0191] In some embodiments, the control sequence is a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell’s secretory pathway). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s). Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to, those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha-amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis betalactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA. Further signal peptides are known in the art (See e.g., Simonen and Palva,Microbiol. Rev.. 57: 109-137, 1993). In some embodiments, effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.

[0192] In some embodiments, regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac. tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or GALI system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.

[0193] In another aspect, the present disclosure provides a recombinant expression vector comprising a polynucleotide encoding a polypeptide (e.g., a PAL polypeptide), and one or more expression regulating regions such as a promoter, a terminator, a replication origin, a leader sequence, a signal peptide, or a regulatory sequence, depending on the type of host into which it is to be introduced. In some embodiments, one or more nucleic acid and control sequences as described herein are joined together to produce recombinant expression vectors that include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the enzyme polypeptide at such sites. Alternatively, in some embodiments, the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of the expression vector, the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.

[0194] The recombinant expression vector may be any suitable vector (e.g.. a plasmid or virus), that can be subjected to recombinant DNA procedures and bring about the expression of the enzyme polynucleotide sequence. The choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector may be a linear or closed circular plasmid.

[0195] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome). The vector may contain any means for assuring self-replication. In some alternative embodiments, the vector is integrated into the genome of the host cell and replicates together with the chromosome(s) into which it has been integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids which together contain the polynucleotide(s) (e.g., DNA) to be introduced into the genome of the host cell, and / or a transposon is utilized.

[0196] In some embodiments, the expression vector contains one or more selectable markers, which permit easy selection of transformed cells. Examples of bacterial selectable markers include, but are not limited to, the dal genes from Bacillus subtilis or Bacillus licheniformis , or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance. Suitable markers for yeast host cells include, but are not limited to. ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3. Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae), argB (ornithine carbamoyltransferases), bar (phosphinothricin acetyltransferase: e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase). niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.

[0197] In another aspect, the present disclosure provides a host cell comprising at least one polynucleotide encoding at least one polypeptide of the present disclosure, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the at least one polypeptide in the host cell. In some examples, the at least one polynucleotide is part of an expression vector. Host cells suitable for use in expressing the polypeptides encoded by the polynucleotide(s) or expression vector(s) of the present disclosure are well known in the art and described herein.

[0198] In some embodiments, the expression vectors of the present disclosure contain one or more element that permits integration of the vector into the host cell’s genome or autonomous replication of the vector in the cell independent of the genome. In some embodiments involving integration into the host cell genome, the vectors rely on the nucleic acid sequence encoding the polypeptide or any other element of the vector for integration of the vector into the genome by homologous or nonhomologous recombination.

[0199] In some alternative embodiments, the expression vectors contain additional nucleic acid sequences for directing integration by homologous recombination into the genome of the host cell. The additional nucleic acid sequences enable the vector to be integrated into the host cell genome at a precise location(s) in the chromosome(s) of the host cell. To increase the likelihood of integration at a precise location, the integrational elements preferably contain a sufficient number of nucleotides, such as 100 to 10,000 base pairs, preferably 400 to 10,000 base pairs, and most preferably 800 to 10,000 base pairs, which are highly homologous with the corresponding target sequence to enhance the probability of homologous recombination. The integrational elements may include any sequence that is homologous with the target sequence in the genome of the host cell. Furthermore, the integrational elements may be non-encoding or encoding nucleic acid sequences. In other examples, the vector may be integrated into the genome of the host cell by non-homologous recombination.

[0200] For autonomous replication, the vector may further comprise an origin of replication enabling the vector to replicate autonomously in the host cell in question. Examples of bacterial origins of replication are P15A ori or the origins of replication of plasmids pBR322, pUC19, pACYC177 (which contains the P15A ori), or pACYC184 (which contains the P15A ori) permitting replication in E. coll, and pUBl 10, pE194, or pTA1060 permitting replication in Bacillus. Examples of origins of replication for use in a yeast host cell are the 2 micron origin of replication, ARS1, ARS4, the combination of ARS1 and CEN3, and the combination of ARS4 and CEN6. The origin of replication may be one having a mutation which makes its functioning temperature-sensitive in the host cell (See e.g., Ehrlich, Proc. Natl. Acad. Sci. USA 75:1433, 1978).

[0201] In some embodiments, more than one copy of a polynucleotide sequence of the present invention is inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0202] Many of the expression vectors for use in the present invention are commercially available. Suitable commercial expression vectors include, but are not limited to. NOVAGEN® pET E. coli T7 expression vectors (Millipore Sigma) and the p3xFLAG® expression vectors (Sigma- Aldrich Chemicals). Other suitable expression vectors include, but are not limited to, pBluescriptll SK(-) and pBK-CMV (Stratagene), and plasmids derived from pBR322 (GibcoBRL), pUC (Gibco BRL). pREP4. pCEP4 (Invitrogen) or pPoly See e.g., Lathe et al.. Gene 57: 193-201, 1987).

[0203] Thus, in some embodiments, a vector comprising a sequence encoding at least one variant polypeptide (e.g., PAL polypeptide) is transformed into a host cell in order to allow propagation of the vector and expression of the variant polypeptide(s). In some embodiments, the transformed host cell described above is cultured in a suitable nutrient medium under conditions permitting the expression of the variant polypeptides(s). Any suitable medium useful for culturing the host cells finds use in the present invention, including, but not limited to minimal or complex media containing appropriate supplements. In some embodiments, host cells are grown in HTP media. Suitable media are available from various commercial suppliers or may be prepared according to published recipes (e.g., in catalogues of the American Type Culture Collection).Host Cells for Expression of PAL Polypeptides

[0204] In another aspect, the present disclosure provides a host cell comprising a polynucleotide encoding a polypeptide (e.g., a PAL polypeptide) disclosed herein, or an expression vector comprising a polynucleotide encoding a polypeptide (e.g., a PAL polypeptide) disclosed herein. In some embodiments, the polynucleotide is operatively linked to one or more control sequences for expression of the polypeptide in the host cell. Suitable host cells for use in expressing the polypeptides described herein are well known in the art and include, but are not limited to, prokaryotic cells (e.g., bacterial cells (e.g., E. coli. B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, B. amyloliquefaciens , Lactobacillus kejir, Lactobacillus brevis. Lactobacillus minor. Streptomyces and Salmonella typhimurium cells)) or eukaryotic cells (e.g., fungal cells (e.g., yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris) or mammalian cells). Appropriate culture mediums and grow th conditions for the above-described host cells are well known in the art.

[0205] Polynucleotides for expression of the polypeptides (e.g.. PAL polypeptides) may be introduced into cells by various methods known in the art. Techniques include among others, electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion. Various methods for introducing polynucleotides into cells will be apparent to the skilled artisan.

[0206] In some embodiments, the host cell is a filamentous fungal host cell. For example, in some embodiments, the filamentous fungal host cells are of any suitable genus and species, including, but not limited to Achlya, Acremonium, Aspergillus, Aureobasidium, Bjerkandera.Ceriporiopsis, Cephalosporium. Chrysosporium, Cochliobolus. Corynascus. Cryphonectria. Cryptococcus, Coprinus, Coriolus, Diplodia, Endothis, Fusarium, Gibberella, Gliocladium, Humicola, Hypocrea, Myceliophthora, Mucor, Neurospora, Penicillium, Podospora, Phlebia, Piromyces, Pyricularia, Rhizomucor, Rhizopus, Schizophyllum. Scytalidium, Sporotrichum, Talaromyces, Thermoascus, Thielavia. Trametes, Tolypocladium. Trichoderma, Verticillium, and / or Volvariella, and / or teleomorphs, or anamorphs, and synonyms, basionyms, or taxonomic equivalents thereof.

[0207] In some embodiments, the host cell is a yeast cell, including but not limited to cells of Candida. Hansenula, Saccharomyces , Schizosaccharomyces , Pichia, Kluyveromyces, or Yarrowia species. In some embodiments of the present invention, the yeast cell is Hansenula polymorpha, Saccharomyces cerevisiae, Saccharomyces carlsber gensis, Saccharomyces diastaticus, Saccharomyces norbensis, Saccharomyces kluyveri, Schizosaccharomyces pombe, Pichia pastoris, Pichia finlandica, Pichia trehalophila, Pichia kodamae, Pichia membranaefaciens, Pichia opuntiae, Pichia thermotolerans, Pichia salictaria, Pichia quercuum. Pichia pijperi, Pichia stipitis, Pichia methanolica, Pichia angusta, Kluyveromyces lactis, Candida cdbicans, or Yarrowia lipolytica.

[0208] In some other embodiments, the host cell is a prokaryotic cell. Suitable prokaryotic cells include, but are not limited to, Gram-positive. Gram-negative and Gram-variable bacterial cells. Any suitable bacterial organism finds use in the present invention, including but not limited to Agrobacterium, Alicyclobacillus , Anabaena, Anacystis, Acinetobacter, Acidothermus, Arthrobacter, Azobacter, Bacillus, Bifidobacterium, Brevibacterium, Butyrivibrio, Buchnera, Campestris, Camplyobacter, Clostridium, Corynebacterium, Chromatium, Coprococcus . Escherichia, Enterococcus, Enterobacter . Erwinia, Fusobacterium. Faecalibacterium, Francisella, Flavobacterium. Geobacillus, Haemophilus, Helicobacter, Klebsiella, Lactobacillus, Lactococcus, Ilyobacter, Micrococcus, Microbacterium, Mesorhizobium, Methylobacterium, Methylobacterium, Mycobacterium, Neisseria, Pantoea, Pseudomonas, Prochlorococcus, Rhodobacter, Rhodopseudomonas, Rhodopseudomonas, Roseburia, Rhodospirillum, Rhodococcus. Scenedesmus, Streptomyces, Streptococcus, Synecoccus, Saccharomonospora, Staphylococcus, Serratia, Salmonella, Shigella, Thermoanaerobacterium, Tropheryma, Tularensis, Temecula, Thermosynechococcus, Thermococcus, Ureaplasma, Xanthomonas, Xylella, Yersinia and Zymomonas. In some embodiments, the host cell is a species of Agrobacterium, Acinetobacter, Azobacter, Bacillus, Bifidobacterium, Buchnera, Geobacillus, Campylobacter, Clostridium, Corynebacterium, Escherichia, Enterococcus, Erwinia, Flavobacterium, Lactobacillus, Lactococcus, Pantoea, Pseudomonas, Staphylococcus,Salmonella, Streptococcus, Streptomyces . or Zymomonas. In some embodiments, the bacterial host strain is non-pathogenic to humans. In some embodiments the bacterial host strain is an industrial strain. Numerous bacterial industrial strains are known and suitable in the present invention. In some embodiments of the present invention, the bacterial host cell is an Agrobacterium species (e.g.. A. radiobacter , A. rhizogenes. and A. rubi). In some embodiments of the present invention, the bacterial host cell is an Arthrobacter species (e.g., A. aurescens, A. citreus,A. globiformis. A. hydrocarboglutamicus, A. mysorens, A. nicotianae,A. paraffineus, A. protophonniae, A. roseoparqffmus, A. sulfur eus, or A. ureafaciens) . In some embodiments of the present invention, the bacterial host cell is a Bacillus species (e.g., B. thuringensis , B. anthracis, B. megaterium, B. subtilis, B. lentus, B. circulans, B. pumilus. B. lautus, B.coagulans, B. brevis, B. firmus, B. alkaophius, B. licheniformis, B. clausii, B. stearothermophilus, B. halodurans, or B. amyloliquefaciens . In some embodiments, the host cell is an industrial Bacillus strain including but not limited to B. subtilis, B. pumilus, B. licheniformis. B. megaterium, B. clausii, B. stearothermophilus, or B. amyloliquefaciens. In some embodiments, the Bacillus host cells are B. subtilis, B. licheniformis, B. megaterium, B. stearothermophilus, and / or B. amyloliquefaciens. In some embodiments, the bacterial host cell is a Clostridium species (e.g., C. acetobutylicum, C. tetani E88, C. lituseburense, C. saccharobutylicum, C. perfringens, and C. beijerinckii). In some embodiments, the bacterial host cell is a Corynebacterium species (e.g., C. glutamicum and C. acetoacidophilum). In some embodiments the bacterial host cell is an Escherichia species (e.g., E. coli). In some embodiments, the host cell is Escherichia coli W3110. In some embodiments the host is Escherichia coli BL21 or BL21(DE3). In some embodiments, the bacterial host cell is an Erwinia species (e.g., E. uredovora, E. carotovora, E. ananas. E. herbicola. E. punctata, and E. terreus). In some embodiments, the bacterial host cell is aPantoea species (e.g., P. citrea or P. agglomerans). In some embodiments the bacterial host cell is a Pseudomonas species (e.g., P. putida, P. aeruginosa, P. mevalonii, and P. sp. D-01 10). In some embodiments, the bacterial host cell is a Streptococcus species (e.g., S. equisimiles, S. pyogenes, and S. uberis). In some embodiments, the bacterial host cell is a Streptomyces species (e.g.. S. ambofaciens, S. achromogenes, S. avermitilis, S. coelicolor, S. aureofaciens, S. aureus, S. fungicidicus, S. griseus, or S. lividans). In some embodiments, the bacterial host cell is a Zymomonas species (e.g., Z. mobilis, or Z. lipolytica).

[0209] Many prokary otic and eukaryotic strains that find use in the present disclosure are readily available to the public from a number of culture collections such as American Type Culture Collection (ATCC), Deutsche Sammlung von Mikroorganismen und Zellkulturen GmbH(DSM), Centraalbureau Voor Schimmelcultures (CBS), and Agricultural Research Service Patent Culture Collection, Northern Regional Research Center (NRRL).

[0210] In some embodiments, host cells are genetically modified to have characteristics that improve protein secretion, protein stability and / or other properties desirable for expression and / or secretion of a protein. Genetic modification can be achieved by genetic engineering techniques and / or classical microbiological techniques (e.g., chemical or UV mutagenesis and subsequent selection). Indeed, in some embodiments, combinations of recombinant modification and classical selection techniques are used to produce the host cells. Using recombinant technology', nucleic acid molecules can be introduced, deleted, inhibited or modified, in a manner that results in increased yields of PAL variant(s) within the host cell and / or in the culture medium. In one genetic engineering approach, homologous recombination is used to induce targeted gene modifications by specifically targeting a gene in vivo to suppress expression of the encoded protein. In alternative approaches, siRNA, antisense and / or ribozyme technology find use in inhibiting gene expression. A variety of methods are known in the art for reducing expression of protein in cells, including, but not limited to deletion of all or part of the gene encoding the protein and site-specific mutagenesis to disrupt expression or activity' of the gene product. (See e.g., Chaveroche et al., Nucl. Acids Res., 28:22 e97, 2000; Cho et al., Molec. Plant Microbe Interact., 19:7-15, 2006; Maruyama and Kitamoto, Biotechnol. Lett., 30: 1811-1817, 2008;Takahashi et al., Mol. Gen. Genom, 272: 344-352, 2004; and You et al., Arch.Microbiol. ,191 :615-622, 2009, all of which are incorporated by7reference herein). Random mutagenesis, followed by screening for desired mutations may also be used (See e.g., Combier et al., FEMS Microbiol. Lett.. 220: 141-8, 2003; and Firon et al.. Eukary. Cell 2:247-55, 2003, both of which are incorporated by reference).

[0211] Introduction of a vector or polynucleotide (e.g., DNA) construct into a host cell can be accomplished using any suitable method known in the art, including but not limited to calcium phosphate transfection, diethylaminoethyl (DEAE)-dextran mediated transfection, polyethylene glycol (PEG)-mediated transformation, electroporation, or other common techniques known in the art.

[0212] In some embodiments, the engineered host cells of the present disclosure are cultured in conventional nutrient media modified as appropriate for activating promoters, selecting transformants, or amplifying the polynucleotide(s). Culture conditions, such as temperature, pH and the like, may be those previously used with the host cell selected for expression, and are well-known to those skilled in the art. As noted, many standard references and texts are availablefor the culture and production of many cells, including cells of bacterial, plant, animal (especially mammalian) and archaebacterial origin.

[0213] In some embodiments, cells expressing a PAL polypeptide of the present disclosure are grown under batch or continuous fermentations conditions. Classical batch fermentation is a closed system, wherein the compositions of the medium are set at the beginning of the fermentation and is not subject to artificial alternations during the fermentation. A variation of the batch system is a fed-batch fermentation that also finds use in the present disclosure. In this variation, the substrate is added in increments as the fermentation progresses. Fed-batch systems are useful when catabolite repression is likely to inhibit the metabolism of the cells and where it is desirable to have limited amounts of substrate in the medium. Batch and fed-batch fermentations are common and well known in the art. Continuous fermentation is an open system where a defined fermentation medium is added continuously to a bioreactor and an equal amount of conditioned medium is removed simultaneously for processing. Continuous fermentation generally maintains the cultures at a constant high density where cells are primarily in log phase growth. Continuous fermentation systems strive to maintain steady state growth conditions. Methods for modulating nutrients and growth factors for continuous fermentation processes as w ell as techniques for maximizing the rate of product formation are w ell known in the art of industrial microbiology.

[0214] More than one copy of a polynucleotide sequence of the present disclosure may be inserted into the host cell to increase production of the gene product. An increase in the copy number of the nucleic acid sequence can be obtained by integrating at least one additional copy of the sequence into the host cell genome or by including an amplifiable selectable marker gene with the nucleic acid sequence where cells containing amplified copies of the selectable marker gene, and thereby additional copies of the nucleic acid sequence, can be selected for by cultivating the cells in the presence of the appropriate selectable agent.

[0215] In some embodiments, cell-free transcription and translation systems find use in producing the polypeptide(s) (e.g.. PAL polypeptide(s)). Several systems are commercially available, and the methods are well-known to those skilled in the art.Methods of Evolving PALs

[0216] Evolution (e.g., directed evolution) may be used to identify polypeptides (e.g., PAL polypeptides) of the present disclosure. For example, in some embodiments, to make a PAL polypeptide of the present disclosure, a PAL polypeptide may be obtained (or derived) from any suitable bacterial species, e.g., Planctomyces brasiliensis . In some embodiments, the parentpolynucleotide sequence is codon optimized to enhance expression of the PAL polypeptide in a specified host cell (e.g., E. coh). For example, as is described in Example 1, a parental polynucleotide sequence, designated as SEQ ID NO: 1, was codon optimized for expression in E. coli and the codon-optimized polynucleotide was cloned into an expression vector, placing the expression of the PAL gene under the control of the T7 promoter. The T7 polymerase needed to express the gene of interest may be under control of the lac promoter, and both the gene of interest and the T7 polymerase may be subject to lacl repression. In this example, the presence of IPTG activates the T7 polymerase production and eliminates the repression, resulting in production of the PAL gene. Clones expressing the active PAL in E. coli may be identified and the genes sequenced to confirm their identity.

[0217] The polypeptides (e.g., PAL polypeptides) of the disclosure may be obtained by subjecting a polynucleotide encoding a parent sequence to mutagenesis and / or directed evolution methods. An exemplary directed evolution technique is mutagenesis and / or DNA shuffling as described in Stemmer. 1994. Proc. Natl. Acad. Sci. USA 91:10747-10751; WO 95 / 22625; WO 97 / 20078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767 and U.S. Pat. No. 6,537,746. Other directed evolution procedures that can be used include, among others, staggered extension process (StEP), in vitro recombination (Zhao et al., 1998. Nat. Biotechnol. 16:258- 261). mutagenic polymerase chain reaction (PCR) (Caldwell et al., 1994, PCR Methods Appl.3: S 136-S 140), and cassette mutagenesis (Black et al., 1996, Proc. Natl. Acad. Sci. USA 93:3525- 3529).

[0218] The clones obtained following mutagenesis treatment may be screened for PAL polypeptides having a desired improved enzyme property. Measuring enzyme activity from the expression libraries can be performed using standard chemistry analytical techniques for measuring substrates and products such as UPLC-MS. For example, a PAL activity assay as described in Example 1 may be used.

[0219] Where the improved enzyme property desired is thermal stability, enzyme activity may be measured after subjecting the enzyme preparations to a defined temperature and measuring the amount of enzy me activity remaining after heat treatments. Any suitable approach may be used, e.g., differential scanning colorimetry (DSC) a biochemical assay, or spectroscopy. Clones containing a polynucleotide encoding a PAL may then be isolated, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.

[0220] Where the sequence of the polypeptide is known, the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individuallysynthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence. For example, polynucleotides and oligonucleotides of the disclosure can be prepared by chemical synthesis using, e.g., the classical phosphoramidite method described by Beaucage et al., 1981, Tet. Lett. 22: 1859-69, or the method described by Matthes et al., 1984. EMBO J. 3:801-05, e.g.. as it is typically practiced in automated synthetic methods. According to the phosphoramidite method, oligonucleotides are synthesized, e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors. In addition, essentially any nucleic acid can be obtained from any of a variety of commercial sources, such as Integrated DNA Technologies, Coralville, IA, The Midland Certified Reagent Company, Midland, Tex., GENEART® Gene Synthesis (Thermo Fisher Scientific), and many others.

[0221] Polypeptides (e.g., PAL polypeptides) expressed in a host cell can be recovered from the cells and or the culture medium using any one or more of the well-known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out. ultra-centrifugation, and chromatography. Suitable solutions for lysing and the high efficiency extraction of proteins from bacteria, such as E. coll, are commercially available under the trade name CELLYTIC B® from Sigma- Aldrich.

[0222] Chromatographic techniques for isolation of the polypeptide (e.g.. PAL polypeptide) include, among others, reverse phase chromatography high performance liquid chromatography, ion exchange chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme will depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to those having skill in the art.

[0223] In some embodiments, affinity techniques may be used to isolate the improved polypeptides (e.g., PAL polypeptides). For affinity chromatography purification, the protein sequence can be tagged with a recognition sequence to enable purification. Common tags include cellulose-binding domains, poly His-tags, di-His chelates, FLAG-tags and many others that will be apparent to those having skill in the art. Antibodies can also be used as affinity purification reagents. Any antibody that specifically binds the PAL polypeptide may be used.Methods of Using PAL Polypeptides

[0224] Also provided herein are methods of using a polypeptide (e.g., a PAL polypeptide) of the disclosure. In some examples, a polypeptide (e.g., a PAL polypeptide) of the disclosure isused in a method of converting heterocyclic cinnamic acids (e.g.. 3-cyanocinnamic acid) into L- phenylalanines (e g., 3-cyano-L-phenylalanine).

[0225] For example, provided herein is a method of converting a heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine), the method comprising contacting or incubating the heterocyclic cinnamic acid with an ammonia source in the presence of any one of the polypeptides (e.g., PAL polypeptides) disclosed herein. In some embodiments, the heterocyclic cinnamic acid is 3-cyanocinnamic acid.

[0226] Any suitable ammonia source can be used. In some embodiments, the ammonia source is ammonium carbonate, ammonium carbamate, ammonium sulfate, ammonium acetate, or ammonium formate. In some embodiments, the ammonia source is ammonium carbonate. In some embodiments, the ammonia source is ammonium carbamate. In some embodiments, the ammonia source is ammonium sulfate. In some embodiments, the ammonia source is ammonium acetate. In some embodiments, the ammonia source is ammonium formate.

[0227] In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) comprises contacting or incubating the heterocyclic cinnamic acid with a polypeptide (e.g., a PAL polypeptide) as disclosed herein under reaction conditions suitable for converting the heterocyclic cinnamic acid into 3-cyano-L-phenylalanine.

[0228] Any polypeptide (e.g., PAL polypeptide) or combination of polypeptides disclosed herein may be used in the methods for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). As noted herein, in some embodiments, the polypeptide (e.g., PAL polypeptide) can comprise an amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %. 92%. 93%. 94%. 95%, 96%, 97%, 98%, or 99% identical as compared a reference sequence comprising the sequence of SEQ ID NO: 2 or 21. In some embodiments, these polypeptides can have one or more modifications to the amino acid sequence of SEQ ID NO: 2 or 21. The modifications can include substitutions, deletions, and insertions. The substitutions can be non-conservative substitutions, conservative substitutions, or a combination of nonconservative and conservative substitutions.

[0229] In some embodiments, the polypeptide (e.g., PAL polypeptide) comprises an amino acid sequence that is at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91 %. 92%. 93%. 94%. 95%, 96%, 97%, 98%, or 99% identical as compared to SEQ ID NO: 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 24, 25, 26, 27, 28, 29, 30, or 31. In some examples, the polypeptide comprises the aminoacid sequence of SEQ ID NO: 18. In some examples, the polypeptide comprises the ammo acid sequence of SEQ ID NO: 30.

[0230] In some embodiments, the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) with a percent enantiomeric excess of at least about 60%. 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or higher. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher). In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 95% (e.g., at least about 95%, 96%, 97%, 98%, 99%, or higher). In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 96%. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 97%. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 98%. In some embodiments, such polypeptides are also capable of converting the substrate to the product with a percent enantiomeric excess of at least about 99%.

[0231] In some embodiments, at least about 60%, 65%, 70%, 75%, 80%, 85%, 90%, 95%, 96%, 97%, 98%, 99%, or more of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 90% (e.g., at least about 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or higher) of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L- phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 91% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 92% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano- L-phenylalanine). In some embodiments, at least about 93% of the heterocyclic cinnamic acid (e g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L- phenylalanine). In some embodiments, at least about 94% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 95% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 96% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, atleast about 97% of the heterocyclic cinnamic acid (e.g.. 3 -cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 98% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine). In some embodiments, at least about 99% of the heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) is converted into L-phenylalanine (e.g., 3-cyano-L- phenylalanine).

[0232] In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed at a pH of less than about 11, e.g., less than about 11, 10.9, 10.8, 10.7, 10.6, 10.5. 10.4. 10.3, 10.2, 10.1, or 10.0.

[0233] In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed at a pH of from about 8 to about 11, e.g., 8.0, 8.1, 8.2, 8.3, 8.4, 8.5, 8.6, 8.7, 8.8, 8.9, 9.0. 9.1, 9.2, 9.3, 9.4, 9.5, 9.6. 9.7, 9.8, 9.9, 10.0, 10.1, 10.2, 10.3, 10.4, 10.5. 10.6. 10.7. 10.8. 10.9, or 11.0. In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed at a pH of from 9 to 11, from 9 to 10.5, from 9 to 10, from 9 to 9.5, from 9.5 to 11, from 9.5 to 10.5, from 9.5 to 10. from 10 to 11, or from 10 to 10.5.

[0234] In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed at a temperature of from about 15 °C to about 45 °C, e.g., about 15 °C, 16 °C, 17 °C, 18 °C, 19 °C, 20 °C, 21 °C, 22 °C, 23 °C, 24 °C. 25 °C, 26 °C, 27 °C. 28 °C, 29 °C, 30 °C, 31 °C, 32 °C, 33 °C. 34 °C, 35 °C, 36 °C. 37 °C, 38 °C. 39 °C. 40 °C, 41 °C. 42 °C, 43 °C, 44 °C. or 45 °C.

[0235] In some embodiments, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed at a temperature of from about 20 °C to about 30 °C, e.g., about 20 °C, 21 °C, 22 °C, 23 °C, 24 °C, 25 °C, 26 °C, 27 °C, 28 °C, 29 °C. or 30 °C. In some embodiments, the method for converting a heterocyclic cinnamic acid (e g., 3-cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano- L-phenylalanine) is performed at a temperature of from about 25 °C to about 30 °C, e.g., about 25 °C, 26 °C, 27 °C, 28 °C, 29 °C, or 30 °C.

[0236] In some examples, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) comprises contacting or incubating from about 25 g*L-1to about 400 g*L-1(e.g., about 25 g*L-1, 50 g*L-1, 75 g*L , 100 g*L , 125 g*L4, 150 g*L , 175 g*L , 200 g*L4, 225 g’T’1, 250 g*L , 275 g*L ,300 g*L-1, 325 g*L-1, 350 g*L-1. 375 g*L-1, or 400 g*L-1) of 3-cyanocinnamic acid with any one of the polypeptides (e.g., PAL polypeptides) disclosed herein or any combination of the polypeptides disclosed herein. In some examples, the method for converting a heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) into an L-phenylalanine (e.g.. 3-cyano-L- phenylalanine) comprises contacting or incubating from about 50 g*L-1to about 150 g*L-1(e.g., about 50 g*L-1, 75 g*L-1, 100 g*L-1, 125 g*L-1, or 150 g*L-1) of 3-cyanocinnamic acid with any one of the polypeptides (e.g., PAL polypeptides) disclosed herein or any combination of the polypeptides disclosed herein.

[0237] In some examples, the method for converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed in the presence of from about 1% w / w to about 15% w / w (e.g., about 1% w / w, 2% w / w, 3% w / 2, 4% w / w, 5% w / w, 6% w / w, 7% w / w, 8% w / w, 9% w / w, 10% w / w, 11% w / w, 12% w / w, 13% w / w, 14% w / w or 15% w / w) of any one of the polypeptides (e.g., PAL polypeptides) disclosed herein or any combination of the polypeptides disclosed herein. In some examples, the method for converting a heterocyclic cinnamic acid (e.g., 3-cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) is performed in the presence of from about 2.5% w / w to about 7.5% w / w (e.g., about 2.5% w / w, 3% w / w, 3.5% w / w, 4% w / w, 4.5% w7w, 5% w / w, 5.5% w / w, 6% w / w, 6.5% w / w, 7% w / w, or 7.5% w / w) of any one of the polypeptides (e.g.. PAL polypeptides) disclosed herein or any combination of the polypeptides disclosed herein.

[0238] Any of the methods disclosed herein may further comprise filtering the product, e.g., 3- cyano-L-phenylalanine. Any suitable approach may be used for filtration. In some embodiments, the pH is adjusted to from about 6 to about 8 (e.g., about 6.0, 6.1, 6.2, 6.3, 6.4, 6.5. 6.6, 6.7, 6.8, 6.9, 7.0, 7.1, 7.2. 7.3, 7.4, 7.5, 7.6. 7.7, 7.8, 7.9. or 8.0).

[0239] In particular examples, the method for converting a heterocyclic cinnamic acid (e g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) comprises contacting or incubating about 100 g*L-1of 3-cyanocinnamic acid at pH 10±0.5 and about 25-30 °C using about 5 w / w% of the polypeptide (e.g., PAL polypeptide). Any of the polypeptides disclosed herein may be used. In some embodiments, the polypeptide comprises or consists of the ammo acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 16, 18, 20, 23, 24, 25, 26, 27, 28, 29, 30, or 31. In some embodiments, the polypeptide comprises the amino acid sequence of SEQ ID NO: 18. In some embodiments, the polypeptide consists of the amino acid sequence of SEQ ID NO: 18.

[0240] In some examples, the method of converting a heterocyclic cinnamic acid (e.g., 3- cyanocinnamic acid) into an L-phenylalanine (e.g., 3-cyano-L-phenylalanine) comprisescontacting or incubating the heterocyclic cinnamic acid (e.g.. 3-cyanocinnamic acid) with the polypeptide (e.g., PAL polypeptide) for any suitable period of time, e.g., about 1 min, 5 min, 10 min, 15 min, 20 min, 25 min, 30 min, 1 h, 2 h, 3 h, 4 h, 5 h, 6 h, 7 h, 8 h, 9 h, 10 h, 11 h, 12 h, 13 h, 14 h. 15 h, 16 h, 17 h, 18 h, 19 h, 20 h, 21 h, 22 h, 23 h, 24 h, 25 h. 26 h, 27 h, 28 h, 29 h, 30 h, 31 h, 32 h, 33 h, 34 h, 35 h, 36 h, 37 h, 38 h, 39 h. 40 h. 41 h. 42 h, 43 h, 44 h, 45 h, 46 h, 47 h, 48 h, 36 h, 60 h, or longer. In some embodiments, the period of time is about 1 h to about 36 h, about 1 h to about 24 h, about 1 h to 23 h, about 1 h to about 22 h, about 1 h to about 21 h, about1 h to about 20 h, about 1 h to about 19 h, about 1 h to about 18 h, about 1 h to about 17 h, about1 h to about 16 h, about 1 h to about 15 h, about 1 h to about 14 h, about 1 h to about 13 h, about1 h to about 12 h, about 1 h to about 11 h, about 1 h to about 10 h, about 1 h to about 9 h, about 1 h to about 8 h, about 1 h to about 7 h, about 1 h to about 6 h, about 1 h to about 5 h, about 1 h to about 4 h, about 1 h to about 3 h, about 1 h to about 2 h, about 5 h to about 36 h, about 5 h to about 24 h. about 5 h to 23 h, about 5 h to about 22 h, about 5 h to about 21 h, about 5 h to about20 h. about 5 h to about 19 h, about 5 h to about 18 h. about 5 h to about 17 h, about 5 h to about1 h, about 5 h to about 15 h, about 5 h to about 14 h, about 5 h to about 13 h, about 5 h to about12 h, about 5 h to about 11 h, about 5 h to about 10 h, about 5 h to about 9 h, about 5 h to about 8 h, about 5 h to about 7 h. about 5 h to about 6 h, about 10 h to about 36 h, about 10 h to about 24 h, about 10 h to 23 h, about 10 h to about 22 h, about 10 h to about 21 h, about 10 h to about 20 h, about 10 h to about 19 h, about 10 h to about 18 h, about 10 h to about 17 h, about 10 h to about 16 h, about 10 h to about 15 h, about 10 h to about 14 h, about 10 h to about 13 h, about 10 h to about 12 h, about 10 h to about 11 h, about 12 h to about 36 h, about 12 h to about 24 h, about 12 h to 23 h, about 12 h to about 22 h, about 12 h to about 21 h, about 12 h to about 20 h, about 12 h to about 19 h. about 12 h to about 18 h, about 12 h to about 17 h, about 12 h to about16 h, about 12 h to about 15 h, about 12 h to about 14 h, about 12 h to about 13 h, about 18 h to about 36 h, about 18 h to about 24 h, about 18 h to 23 h, about 18 h to about 22 h, about 18 h to about 21 h, about 18 h to about 20 h, or about 18 h to about 19 h.EXAMPLES

[0241] The following examples are meant to be illustrative and should not be construed as further limiting. The contents of the figures and all references, patents, and published patent applications cited throughout this application are expressly incorporated herein by reference.Example 1: Synthesis, optimization, and assay of phenylalanine ammonia lyase (PAL) enzymes with activity of conversion of 3-cyanophenylalanine from 3-cyanocinnamic acid

[0242] This example describes methods to synthesize, optimize, and assay the activity of PAL enzymes and the composition of optimized PAL enzymes.

[0243] PALs are a versatile class of enzymes capable of catalyzing deamination of phenylalanine to generate cinnamic acid. At high ammonia concentrations, PALs can also convert cinnamic acids to phenylalanines through hydroamination, which represents a highly efficient approach for the synthesis of chiral phenylalanine amino acids. This synthesis route benefits from using inexpensive starting materials in 100% atom economy and excluding the need for exogenous co-factors or recycling systems.

[0244] We evaluated synthetic approaches for the non-canonical amino acid 3-cyano-L- phenylalanine (3 in FIG. 1A). In prior approaches, 3-cyano-L-phenylalanine is prepared via enzymatic resolution of the corresponding racemic ester or N-acyl derivative (2 in FIG. 1A), or by palladium-catalyzed cross-coupling reactions of alanine-derived organozinc reagents with ar l bromides (FIG. 1A). These methods suffer from high step count, low atom economy in case of the racemate resolution or they require expensive catalysts.

[0245] In contrast, we envisioned synthesizing 3-cyano-L-phenylalanine (“substrate’' in Table 1) via an enantioselective hydroamination of readily accessible 3-cyanocinnamic acid (4 in FIG. 1A) catalyzed by a PAL enzyme. While it has been reported that some organisms can catalyze this transformation, these reports utilized whole cell biocatalysts at very high loadings that are unsuitable for industrial scale manufacturing (FIG. IB).

[0246] Herein, we describe our development efforts toward identifying and engineering a PAL enzyme that ultimately allowed for the efficient and scalable synthesis of 3-cyano-L- phenylalanine in >99% ee and was demonstrated on up to 100-gram scale.Gene synthesis and optimization

[0247] The wild-type Planctomyces brasiliensis PAL (PbPAL) polypeptide (SEQ ID NO: 21) and wild-type Streptomyces rimosus PAL (SrPAL) polypeptide (SEQ ID NO: 34) are described in Weise, Sci. Rep. 2017, 7 (1), 13691. We engineered N-terminal His-tagged versions of the wild-type PbPAL and SrPAL polypeptides. The amino acid sequence of the N-terminal His tag was GSHHHHHHHHSG (SEQ ID NO: 22). The sequence for PbPAL polypeptide having the wild- type sequence with the N-terminal His tag is provided as SEQ ID NO: 2. We optimized the polynucleotide sequence encoding SEQ ID NO: 2 based on the frequencies of codon usage for expression in E. coli and synthesized a polynucleotide with the codon-optimized sequence (seeSEQ ID NO: 1). SEQ ID NO: 1 was cloned into pET30 vector under the control of T7 promoter in 7’. coli strain (BL21(DE3)).

[0248] The amino acid sequence for SrPAL having the wild-type sequence containing the N- terminal His tag is shown in SEQ ID NO: 34. We optimized the polynucleotide sequence encoding SEQ ID NO: 34 based on the frequencies of codon usage for expression in E. coli and synthesized a polynucleotide with the codon-optimized sequence (see SEQ ID NO: 33).Enzyme screening

[0249] We designed DNA sequences optimized for expression in E. coli for two PAL enzymes reported in literature with broad substrate scopes for their activity in the hydroamination of 3- cyanocinnamic acid (4 in FIG. IB) in ammonium carbamate buffer at two different pHs. This substrate was not previously reported in literature. PbPAL (SEQ ID NO: 2) was 10 times more active than the SrPAL (SEQ ID NO: 34) in converting 3 -cyanocinnamic acid into 3-cyano-L- phenylalanine at higher pH (FIG. 2A). However, recombinant production of the PbPAL (SEQ ID NO: 2) in E. coli suffered from poor soluble expression (FIG. 2B). Even under very high lysate loading of 270 v / v%, we could only reach 14% conversion of 3-cyanocinnamic acid to 3-cyano- L-phenylalanine with PbPAL (SEQ ID NO: 2), which is not feasible for industrial applications. Therefore, we decided to initiate protein engineering to improve the activity and soluble expression of PbPAL (SEQ ID NO: 2).Identification of starting point for evolution

[0250] Activity was detected for SEQ ID NO: 2. 6.3% of 3-cyanocinnamic acid was converted to 3-cyano-L-phenylalanine in the assay described below in the sub-section “Assay method for PAL activity.”High-throughput (HTP) grow th, expression, and lysate preparation

[0251] The enzyme-encoding DNA plasmid library variants were picked from single colonies on the LB agar plates and grown in liquid culture with Luria-Bertani (LB) Broth medium with kanamycin (50 pg / ml) in 96-well plates with shaking (400 rpm) at 30°C overnight. Subsequently, the overnight cultures were diluted in 1 : 100 with Terrific Broth medium containing antibiotics (kanamycin 30 pg / ml) and grown until the cellular optical density reached 0.5, measured at 600 nm (OD600). Protein production was induced by adding isopropyl [3-D-l -thiogalactopyranoside (IPTG) to 0.02 mM final concentration for 19 hours at 30 °C. After induction, cells were centrifuged and resuspended in lysis buffer (25 mM Tris-HCl buffer (pH 8.0), 1 mg / mLlysozyme, 0.5 mg / mL polymixin B sulfate, 3 U / mL DNase I. 4 mM magnesium sulfate) at 25 °C with shaking at 800 rpm for 2 hours. The cell lysate was collected by centrifugation (4000 x g, 15 minutes). The cell lysates were used in the following well-plate enzymatic reactions.Production of shake flask powders (SFP)

[0252] PAL enzyme plasmids were transformed into BL21(DE3). Freshly transformed colonies were picked clonally and inoculated in the Terrific Broth medium with 30 pg / ml kanamycin at 37 °C with shaking (250RPM). When the growth reaches cellular optical density of 0.6, measured at 600 nm (OD600), 0.02 mM of IPTG was added to the culture to induce protein production for 19 hours at 30 °C. Cells were then collected by centrifugation and resuspended in 37.5 mL lysis solution per 250 ml of growth cultures (1 g / L lysozyme. 0.5 mg / mL polymixin B sulfate, 3 U / mL DNase I, 4 mM magnesium sulfate, 25 mM Tris-HCl Buffer, pH 8.0) at 25oC with shaking (800 rpm) for 1 hour. Cells were further lysed by ultrasonication at 500 W for 15 min on ice (performed at 2 seconds of sonication with 4 seconds of intervals). The resulting lysate was clarified and centrifuged at 4°C, and the supernatant was subsequently frozen and lyophilized to generate the enzyme powders.Assay method for PAL activity

[0253] For a 200 pL volume reaction, 20 pL of the substrate (3-cyanocinnamic acid, 300 mg / mL dissolved in dimethyl sulfoxide (DMSO)) was mixed with 180 pL of a mixture of 4 M ammonium carbamate buffer (pH 10.00) and cellular lysate. To generate the 90 v / v% lysate loading, 120 pL ammonium buffer was mixed with 60 pL concentrated cellular lysate (concentrated 3 times before adding). Reaction mixtures were incubated at 30 °C with shaking at 600 rpm for 48 hours.Analytical method for PAL activity (High-performance liquid chromatography (HPLC))

[0254] The conversion in the enzymatic reaction was determined using an Agilent ZORBAX® Rapid Resolution High Definition (RRHD) Eclipse Plus C18 column (1.8 pm particle size, 2.1 x 50 mm). Mobile phase A (0. 1% formic acid in water) / B (0. 1% formic acid in acetonitrile), flow rate 0.75 mL min-1, detection wavelength 210 nm at 30 °C column temperature. Retention time of the substrate (3-cyanocinnamic acid): 1.97 minutes; Retention time of the product (3-cyano-L- phenylalanine): 0.48 minutes. Table 2 shows the gradient for the achiral HPLC method.Table 2. Gradient for achiral HPLC method.Directed evolution strategy and summary of results

[0255] Engineering of PALs has been demonstrated to improve the properties of various biocatalysts, including activity and substrate selectivity. Nonetheless, the engineering efforts reported in the literature often focused solely on the active-site residues, partly due to limitations in high throughput screening capabilities. We planned to explore the entire protein sequence of PbPAL (SEQ ID NO: 2; 569 amino acids) through mutagenesis to improve the activity, stability under high ammonia concentration, and soluble expression. To prepare for high-throughput screening of enzyme variant libraries, we first optimized protein expression conditions to maximize soluble protein expression. Different temperatures and concentrations of inducers were tested during induction for PbPAL (SEQ ID NO: 2) expression. 0.02 mM IPTG and 30 °C were chosen for high throughput expression. We also scaled dow n the enzymatic reactions to the 96- well microtiter plate format and developed a 4-minute UHPLC method to quantify the conversions to enable library-scale PAL, as described further above.

[0256] In the directed evolution campaign, single-site-saturation mutagenesis (SSM) libraries were built and screened for increased conversion relative to the starting enzyme (Table 1). SSM libraries in each round of evolution were generated using splicing by overlap extension (SOEing) PCR methods described in Ho et al. Gene 1989, 77(1), 51-59. In short, mutations at the designated positions were incorporated by degenerative NNK oligos through overlapping PCRs. The full-length gene-of-interest region was further amplified and assembled into the expression vector (pET30a) by Gibson Assembly (Gibson et al. Nat. Methods 2009, 6(5), 343-345). The combinatorial libraries w ere built following the instructions from the QuiKCHANGE® Lightning Multi Site-Directed Mutagenesis kit (Agilent Technologies). Both mutagenesis libraries were transformed into the BL21(DE3) E.coli strains by electroporation and plated on LB agar plates with selection (1% glucose, 30 pg / ml Kanamycin).Table 1. Summary of PAL evolution

[0257] To prioritize the sites for SSM library designs, we built a structural model of PbPAL (SEQ ID NO: 2) using the Protein Data Bank (PDB) template 5LTM3, which had 62% sequence similarity. We employed Schrodinger toolbox to design the tetramer homology model, which was further refined by adding hydrogens using PROPKA (Olsson et al. J. Chem. Theory Comput. 2011, 7(2). 525-537) at pH 10 and running a restrained minimization to converge heavy atoms to a maximum root mean square deviation of 0.30A. Substrate docking was performed using Glide (Friesner et al. J. Med. Chem. 2004, 47(7), 1739-1749) and the best pose was selected after visually inspecting a pool of docked poses rank ordered by their docking energy scores and filtering them based on the distances between the reaction site and the catalytic residues.

[0258] We partitioned the model into three regions: active site, surface, and core. The active site shell residues were selected using a distance criterion, i.e., all residues within 12A of the docked substrate, resulting in 96 sites. The remaining residues were rank ordered by their solvent accessibilities and grouped into sets of 96 sites. The top two sets were classified as surface sites, consisting of high surface accessibility’ sites, whereas the remaining were classified as the core sites. The initial rounds targeted the active site and the surface residues, whereas the later rounds targeted the core (see Table 1). Beneficial mutations identified from these SSM libraries were recombined into combinatorial libraries for subsequent screening in alternate rounds. The best variant served as the backbone for the next round of mutagenesis. Additional details regarding the rounds of evolution are provided below in Examples 2-5.

[0259] In round 1.1 of evolution (Table 1), we identified a mutation at position 102 (L102V) relative to SEQ ID NO: 2, which improved product formation under screening conditions by more than 4-fold (FIG. 3A). For the second round (round 2.1, Table 1), we recombined other beneficial mutations from round 1, to identify a quadruple mutant (H94M, L102A. Y288V and A485R) with enhanced enzyme activity under screening conditions and soluble expression (FIG. 3A and FIG. 3D). Continuing round 3 and 4 of directed evolution, we increased the selective pressure by increasing the ammonia concentration and decreasing the volume of E. coli lysate added to screen for PAL variants with improved soluble expression and activity at high pH (Table 1). We eventually identified the PALRSBB variant (SEQ ID NO: 18), which had improved soluble expression and activity- while retaining high enantioselectivity (>99% enantiomeric excess (ee)) (FIG. 3A and FIG. 3D). At equivalent catalyst loadings of lyophilized enzyme, PALRSBB (SEQ ID NO: 18) had >100-fold conversion of the desired products in comparison to the wild-type parent (SEQ ID NO: 2) .Example 2: Round 1 evolution of PALEnzyme variants of SEQ ID NO: 2

[0260] In this example, directed evolution of SEQ ID NO: 2 for improved activity against 3- cyanocinnamic acid and the resulting improved variant SEQ ID NO: 4 are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with active sites of the enzyme were subjected to mutagenesis. These libraries were plated to form single colonies, which were grow n and screened using the high-throughput growth, expression, and SFP production methods described in Example 1 and the HTP assay described below.HTP assay for PAL activity

[0261] In a 500 pL volume reaction, 50 pL of the substrate (3-cyanocinnamic acid, 300 mg / mL dissolved in DMSO) was mixed with 360 pL 5.5 M ammonium carbamate buffer (pH 10.2) and 90 pL of cellular lysate (final concentration 30 g / L substrate, 4 M ammonia, pH 10). The reaction was incubated at 30°C with shaking at 600 rpm for 20 hours.Analytical method for PAL activity (achiral high-throughput ultra HPLC (UHPLC) analysis)

[0262] The conversion in the enzymatic reaction was determined using a BEH C18 column (2. 1 x 30 mm, 1.7 pm particle size) with a mobile phase of water (0.1% formic acid) (A) / acetonitrile (0.1% formic acid) (B), a flow rate of 1.5 mL min'1, a detection w avelength of 230 nm, at a 30 °C column temperature. Retention time of the substrate (3-cyanocinnamic acid): 0.51 minutes; Retention time of the product (3-cyano-L-phenylalanine): 0.21 minutes. The gradient for the achiral UHPLC method is shown in Table 3.Table 3. Gradient for achiral HPLC methodChiral liquid chromatography (LC) method

[0263] The enantiomeric excess was determined using a AZYP TEICOSHELL teicoplanin column (2.7 pm particle size, 4.6 x 50 mm). The mobile phase was 20 mM ammonium formatepH 4 (A) / MeOH (B). and a flow rate of 0.8 mL min’1, a 30 °C column temperature, and a detection wavelength of 210 nm were used. Retention time of the substrate (3-cyanocinnamic acid): 2.7 minutes; Retention time of the product (3-cyano-L-phenylalanine): 1.3 minutes; Retention time of the enantiomer (3-cyano-D-phenylalanine): 1.9 minutes. Table 4 shows the gradient for the chiral-LC method.Table 4. Gradient for the chiral-LC methodIdentification of more active PAL variants and selection for next directed evolution

[0264] Candidates with improved activity from the HTP screening were identified and submitted for SFP production. Assays were repeated with the SFP samples at 20% (w / w) enzyme loading. Engineered polypeptides with >2-fold conversion relative to the parent polypeptide are listed in Table 5.Table 5. Variants and Conversion(a) Levels of increased conversion were determined relative to the reference polypeptide ofSEQ ID NO: 2,(b) "‘+” = conversion at least 2-fold relative to reference polypeptide, but less than 3-fold; and(c) “++” = conversion more than 4-fold as compared to the reference polypeptide.

[0265] Variants with mutations LI 02V and L306N (relative to SEQ ID NO: 2) produced more 3-cyano-L-phenylalanine from 3-cyanocinnamic acid, and these engineered PAL enzymes provide new biocatalytic reagents for the synthesis of non-canonical phenylalanine derivatives. The variant (SEQ ID NO: 4; also referred to as PbPALR2BB) with mutation L102V relative to SEQ ID NO: 2 had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 3) for SEQ ID NO: 4 was selected for further directed evolution.Example 3: Round 2 evolution of PALEnzyme variants of SEQ ID NO: 4

[0266] In this example, directed evolution of SEQ ID NO: 4 for improved soluble expression and activity against 3 -cyanocinnamic acid and the resulting improved variants, including SEQ ID NO: 8, are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with active sites and surface residues of the enzyme were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the high-throughput growth, expression and shake flask powder production methods described in Example 1. The high-throughput analytical methods were described in Example 2.HTP assay for PAL activity:

[0267] In a 250 pL volume reaction, 25 pL of the substrate (3-cyanocinnamic acid. 300 mg / mL dissolved in DMSO) was mixed with 205 pL 7.3 M ammonium carbamate buffer (pH 10.2) and 20 pL of cellular lysate (final concentration 30 g / L substrate, 6 M ammonia, pH 10). The reaction was incubated at 30°C with shaking at 600 rpm for 42 hours.Identification of more active PAL variants and selection for next directed evolution

[0268] Candidates with the improved activity from the HTP screening were identified and submitted for shake flask powder production. Assays were repeated with the SFP samples at 20% (w / w) enzyme loading. The SFP samples were also examined using sodium dodecyl sulfatepolyacrylamide gel electrophoresis (SDS-PAGE) to quantify soluble expression of PAL enzymes. Engineered polypeptides with >5-fold conversion relative to the parent polypeptide are listed in Table 6.Table 6. Variants and Conversion(a) Levels of increased conversion were determined relative to the reference polypeptide ofSEQ ID NO: 4: = conversion at least 5-fold relative to reference polypeptide.(b) “+” = increased soluble expression by >2-folds relative to reference polypeptide.

[0269] Variants with mutations (R57K, H94M, V 102A. Y288P, Y288V, A485R. A485L) relative to SEQ ID NO: 4 produced 3-cyano-L-phenylalanine from 3-cyanocinnamic acid, and these engineered PAL enzymes provide new biocatalytic reagents for the synthesis of non- canonical phenylalanine derivatives. The variant (SEQ ID NO: 8; also referred to as PbPALR3BB) with mutations H94M. V102A, Y288V, and A485R relative to SEQ ID NO: 4 had the highest activity. Thus, the encoding polynucleotide (SEQ ID NO: 7) for SEQ ID NO: 8 was selected for further directed evolution.Example 4: Round 3 evolution of PALEnzyme variants of SEQ ID NO: 8

[0270] In this example, directed evolution of SEQ ID NO: 8 for improved soluble expression and activity against 3-cyanocinnamic acid and the resulting improved variants, including SEQ ID NO: 14, are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with active sites, surface and core residues of the enzyme were subjected to mutagenesis. These libraries were plated to form single colonies, which were grown and screened using the HTP growth and expression method described in Example 1. The HTP analytical methods were described in Example 2.HTP assay for PAL activity:

[0271] In a 250 pL volume reaction, 25 pL of the substrate (3-cyanocinnamic acid, 300 mg / mL dissolved in DMSO) was mixed with 215 pL 7.78 M ammonium carbamate buffer (pH 10.2) and 10 pL of the cellular lysate (final concentration 30 g / L substrate, 6.7 M ammonia, pH 10). The reaction was incubated at 30°C with shaking at 600 rpm for 24 hours.Identification of more active PAL variants and selection for next directed evolution:

[0272] Candidates with improved activity from the HTP screening were identified and submitted for SFP production. Assays were repeated with the SFP samples at 5 % (w / w) enzyme loading. The shake flask powder samples were also examined using SDS-PAGE to quantify soluble expression of PAL enzymes. Engineered polypeptides with >2.5-fold conversion relative to the parent polypeptide w ere listed in Table 7.Table 7. Variants and Conversion(a) Levels of increased conversion were determined relative to the reference polypeptide ofSEQ ID NO: 8; “+” = conversion at least 2.5-fold relative to reference polypeptide.(b) “+” = increased soluble expression by >2-folds relative to reference polypeptide.

[0273] Variants with mutations (R57K, I75V, A310P, Y357S, M360T, E510G) relative to SEQ ID NO: 8 produced 3-cyano-L-phenylalanine from 3-cyanocinnamic acid, and these engineered PAL enzymes provide new biocatalytic reagents for the synthesis of non-canonical phenylalanine derivatives. The variant (SEQ ID NO: 14; also referred to as PbPALR4BB) with mutations R57K, I75V, M360T, and E510G relative to SEQ ID NO: 8, had the highest activity7. Thus, the encoding polynucleotide (SEQ ID NO: 13) for SEQ ID NO: 14 was selected for further directed evolution.Example 5: Round 4 evolution of PALEnzyme variants of SEQ ID NO: 14

[0274] In this example, directed evolution of SEQ ID NO: 14 for improved activity against 3- cyanocinnamic acid and the resulting improved variants, including SEQ ID NO: 18, are described. Directed evolution was carried out by constructing libraries of variant genes in which positions associated with active sites, surface and core residues of the enzyme were subjected to mutagenesis. These libraries were plated to form single colonies, which w ere grown and screened using the HTP growth, expression, and SFP production methods described in Example 1. The HTP analytical methods were described in Example 2. The thermostability of the evolved variants was examined as described below-.HTP assay for PAL activity7:

[0275] In a 250 pL volume reaction. 25 pL of the substrate (3-cyanocinnamic acid. 300 mg / mL dissolved in DMSO) was mixed with 220 pL 7.78 M ammonium carbamate buffer (pH 10.2) and 5 pL of the cellular lysate (final concentration 30 g / L substrate, 6.8 M ammonia, pH 10). The reaction was incubated at 30 °C with shaking at 600 rpm for 24 hours.Thermostability test on the evolved variants:

[0276] Variants which showed improved activity in the HTP screening were identified, grown, and expressed as describe above. Post cell lysis, cell lysates were incubated at 60 °C for 2 hours before being used for assays under the same conditions as the HTP screening. The residual activity was quantified by comparing the PAL activity with or without the heat treatment.Identification of more active PAL variants and selection for next directed evolution:

[0277] Candidates with the improved activity from the HTP screening were identified and submitted for SFP production. Assays were repeated with the SFP samples at 2.5 % (w / w) enzyme loading. Engineered polypeptides with >1.5-fold conversion relative to the parent polypeptide were listed in Table 8. The thermostability of each variant was examined as described above.Table 8. Variants and Conversion(a) Levels of increased conversion were determined relative to the reference polypeptide of SEQ ID NO: 14; “+’■ = conversion at least 1.5-fold relative to reference polypeptide.

[0278] Variants with mutations (R70D, S227A, K294T, N296D, M534V, M534T) relative to SEQ ID NO: 14 produced 3-cyano-L-phenylalanine from 3-cyanocinnamic acid, and these engineered PAL enzymes provide new biocatalytic reagents for the synthesis of non-canonical phenylalanine derivatives. The variant (SEQ ID NO: 18; also referred to as PbPALRSBB) with mutations S227A and M534V relative to SEQ ID NO: 14 was chosen as the final variant to conclude the evolution in this example and demonstrated for scale-up evaluation.Scale-up of PAL reaction

[0279] To showcase the scalability of engineered PAL variants, SEQ ID NO: 18 was selected for demonstration at up to 100-gram scale. The reaction was performed at 3% (w / w) of PALenzyme powder with 100 grams of 3-cyanocinnamic acid (100 g / L) in the aqueous ammonium carbonate buffer at 30 °C for 21 hours, resulting in conversion of 94% and >99% ee. The product (3-cyanophenylalanine) could be further isolated via filtration in 84% yield, demonstrating the synthetic utility of the engineered PAL variants at scale.Discussion

[0280] Biocatalysts have emerged as promising tools for sustainable and green manufacturing of chemicals and medicines in the future. Nonetheless, there are challenges in utilizing enzymes for industrial applications, such as scalability, robustness, and identifying enzymes that can tolerate relevant process conditions.

[0281] The wild-type PAL enzyme (SEQ ID NO: 2) exhibits very low activity towards the desired reactions, which limits its practical applications. However, through multiple rounds of protein engineering, improved PAL variants, including the PbPALRSBB variant having the polypeptide sequence of SEQ ID NO: 18 selected from the fourth round of evolution, have been developed with the improved activity, enantioselectivify, and thermostability for the conversion of 3-cyanocinnamic acid to 3-cyanophenylalanine at scale.

[0282] To enhance the enzyme's performance, ten mutations were introduced during directed evolution to yield the PAL variant PALRSBB (SEQ ID NO: 18) selected from fourth round of evolution. The incorporation of these mutations significantly improved the enzyme's activity (>100-fold increase) and soluble expression in E. coll. Importantly, the perfect enantioselectivify of this enzy me was maintained throughout the engineering process. These characteristics are important for successful large-scale manufacturing of non-canonical phenylalanine amino acids.

[0283] Table 9 below shows a summary of mutations identified across all four rounds of evolution. In Table 9, the positions and mutations relative to SEQ ID NO: 2 (starting PbPAL based on wild-type sequence containing an N-terminal His tag) and relative to SEQ ID NO: 21 (wild-type PbPAL) are provided.Table 9. Summary of positions and mutations identified across four rounds of evolutionExample 6: In silico analysis of mutations

[0284] PAL enzymes function as a homo-tetramer comprised of four separate active sites. To rationalize the effect of the 10 mutations (i.e., R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R, E510G, and M534V) introduced across four rounds of directed evolution that are present in PbPALRSBB, we constructed a model of PALRSBB using the same homology template 5LTM and superimposed it on the wild type. Six of these mutations are located on the surface of the protein (FIG. 3B), which is consistent with the improvement we observed in soluble expression of the protein and tolerance toward high ammonia concentrations (FIG. 3D). These solvent accessible mutations are R57K, H94M, Y288V, A485R, E510G, and M534V. Of the remaining 4 mutations, two mutations are in the active site (L102A. 175 V), one in the core of each monomer (S227A) and one at the interface of two monomers (M360T).

[0285] To examine how the mutations impact the interactions between the enzyme and the substrate and the stabi 1 i ty of the enzyme, we ran molecular dynamics (MD) simulations on the wild type and PbPALR5BB. The most significant difference in the dynamics of the enzyme was observed in the loop from positions 284 to 305 (FIG. 4A and FIG. 4B), where the mutation Y288V is located. In the model, this loop is in the active-site region (~5A from the docked substrate) and is more flexible in PbPALRSBB (FIG. 4A and FIG. 4B) than in PbPALwr, which, without wishing to be bound by any particular theory, could contribute to improving the substrate accessibility of the active site. When simulating the molecular interactions between the substrate and PbPALR5BB relative to PbPALwr, we also observed increased simulated molecular interactions toward the substrate in many of the residues in the active-site region, including G83, S166, Q301, R307 and N337 (FIG. 4C). The active site mutation L102V / A is located at a distance of 4.6A from the docked substrate and may reduce steric hindrance against the substrate by substituting the bulkier leucine for the smaller valine or alanine. The mutations S227A and M360T were found in the core and at the interface of the tetramer complex. Without wishing to be bound by any particular theory, these buried mutations could be improving the solubility ofthe enzyme by stabilizing the core and improving the interfacial contacts of the tetramer complex.

[0286] The majority of the mutations we identified are not located in the active-site region and do not directly participate in substrate binding based on modeling. This emphasizes the advantage of an unbiased mutagenesis approach we took relative to the active site targeted approach used for previous PAL engineering efforts.Example 7: Reaction optimization and scale-up

[0287] To develop an enzymatic PAL process that would be amenable to scale up, additional optimization of the reaction conditions was performed. In order to drive the reversible reaction towards the desired formation of 3 in FIG. IB and achieve a high equilibrium conversion, an excess of ammonia is required. This was initially achieved by carrying out the reaction at relatively high dilution (30 g*L-1in 3-cyanocinnamic acid) and utilizing saturated aqueous ammonium carbamate solution as the reaction buffer, a relatively expensive ammonia source. The use of unbuffered ammonia led to poor conversion, which was attributed to a reduced stability of the enzyme at high pH. Among various ammonium salts, ammonium carbonate and carbamate gave the highest conversion (95%) to 3-cyano-L-phenylalanine in >99% ee (FIG. 5A). In contrast, other ammonia sources (sulfate, acetate and formate) gave lower conversions of 79- 82%. As carbonate and carbamate rapidly equilibrate in aqueous solution, ammonium carbonate was chosen for further optimization as the more economical ammonia source.

[0288] Optimal enzy me activity7was observed with 100 g*L-13-cyanocinnamic acid at pH 10+0.5 and 25-30 °C using 5 w / w% enzyme, at which an equilibrium conversion of 94% was achieved (FIG. 5B, entry 1). At high pH (11-11.5) the conversion decreased to 79%, while the impact of lower pH (8-8.5) was less pronounced (FIG. 5B, entry 2+3). An increased reaction temperature (40 °C) resulted in lower conversion of 93%, while a lower temperature (20 °C) provided a slightly higher conversion of 97% (FIG. 5B, entry 4+5). Finally, the effect of substrate concentration on the reaction was evaluated. A lower concentration of 3-cyanocinnamic acid provided a higher conversion to 3-cyano-L-phenylalanine (96-97%), likely due to the larger excess of ammonia relative to of 3-cyanocinnamic acid (FIG. 5B, entry 6). Notably, increasing the concentration of 3-cyanocinnamic acid to up to 200 g*L_|. corresponding to approximately 13 equivalents of ammonia, still provided 84% conversion (FIG. 5B, entry 7+8). The optimal reaction conditions at 100 g*L-1concentration of 3-cyanocinnamic acid were next evaluated for scale-up. Utilizing 100 g of 3-cyanocinnamic acid with an enzyme loading of 3 w / w% of the evolved PbPAL variant, 94% equilibrium conversion and >99% ee was achieved within 21 h at30 °C (FIG. 5B. entry 9). The product was subsequently isolated via filtration after adjusting the pH to 7 with sulfuric acid in 84% isolated yield and >99% ee.

[0289] The disclosed subject matter is not to be limited in scope by the specific embodiments and examples described herein. Indeed, various modifications of the disclosure in addition to those described will become apparent to those skilled in the art from the foregoing description and accompanying figures. Such modifications are intended to fall within the scope of the appended claims.

[0290] All references (e.g., publications or patents or patent applications) cited herein are incorporated herein by reference in their entirety and for all purposes to the same extent as if each individual reference (e.g.. publication or patent or patent application) was specifically and individually indicated to be incorporated by reference in its entirety for all purposes. Other embodiments are within the following claims.

Claims

WHAT IS CLAIMED IS:

1. An engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, wherein the polypeptide comprises at least one substitution at one or more amino acid positions selected from 57.

70. 75, 94, 102, 227. 288, 294. 296, 306, 310, 357, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

2. The polypeptide of claim 1, wherein the polypeptide comprises 2. 3, 4, 5, 6, 7, 8, 9.

10. 11, 12, 13, 14, 15, or 16 substitutions at two or more amino acid positions selected from 57, 70, 75, 94, 102, 227, 288, 294, 296, 306, 310, 357, 360, 485, 510, and 534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

3. The polypeptide of claim 1 or 2, wherein the polypeptide comprises: a) a substitution set at positions 57, 75, 94, 102, 227, 288, 360, 485, 510, and 534; b) a substitution at position 102; c) a substitution at position 306; d) a substitution set at positions 94, 102. 288, and 485; e) a substitution set at positions 57, 94, 102, 288, and 485;1) a substitution set at positions 94, 102, 288, and 485; g) a substitution set at positions 57, 75, 94, 102, 288, 360, 485, and 510; h) a substitution set at positions 57, 94, 102, 288, 310. 357, and 485; or i) a substitution set at positions 57.

70.

75.

94. 102, 227. 288, 294, 296. 360, 485. 510, and534, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 2.

4. The polypeptide of any one of claims 1-3, wherein the polypeptide comprises one or more substitutions set forth in any one of Tables 5, 6, 7, 8, or 9.

5. The polypeptide of any one of claims 1-4, wherein the polypeptide comprises one of the following substitutions or substitution sets: a) R57K, I75V, H94M, L102A, S227A, Y288V, M360T, A485R, E510G, and M534V; b) LI 02V;c) L306N; d) H94M, L102A, Y288V, and A485R; e) R57K, H94M, L102A, Y288P, and A485R; f) H94M, L102A, Y288P, and A485L; g) R57K. I75V, H94M, L102A, Y288V. M360T, A485R. and E510G; h) R57K, H94M, L102A, Y288V, A310P, Y357S, and A485R; or i) R57K, R70D, I75V, H94M, L102A, S227A, Y288V, K294T, N296D, M360T, A485R,E510G, and M534T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ IDNO: 2.

6. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 18.

7. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 4.

8. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 6.

9. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 8.

10. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 10.

11. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 12.

12. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 14.

13. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprisesSEQ ID NO: 16.

14. The polypeptide of any one of claims 1-5, wherein the amino acid sequence comprises SEQ ID NO: 20.

15. An engineered polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 21, wherein the polypeptide comprises at least one substitution at one or more amino acid positions selected from 45, 58, 63, 82, 90, 215, 276, 282, 284, 294, 298, 345, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

16. The polypeptide of claim 15, wherein the polypeptide comprises 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or 16 substitutions at two or more amino acid positions selected from 45, 58, 63, 82, 90, 215, 276, 282, 284. 294, 298, 345, 348, 473, 498, and 522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

17. The polypeptide of claim 15 or 16, wherein the polypeptide comprises: a) a substitution set at positions 45, 63, 82, 90, 215, 276, 348, 473, 498, and 522; b) a substitution at position 90; c) a substitution at position 294; d) a substitution set at positions 82, 90, 276, and 473; e) a substitution set at positions 45, 82, 90. 276, and 473;1) a substitution set at positions 82, 90. 276, and 473; g) a substitution set at positions 45, 63, 82, 90.

276. 348, 473. and 498; h) a substitution set at positions 45, 82, 90, 276, 298, 345, and 473; or i) a substitution set at positions 45, 58, 63, 82, 90, 215, 276, 282, 284, 348, 473, 498, and522, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

18. The polypeptide of any one of claims 15-17, wherein the polypeptide comprises one or more substitutions set forth in Table 9.

19. The polypeptide of any one of claims 15-18, wherein the polypeptide comprises one of the following substitutions or substitution sets:a) R45K. I63V. H82M, L90A. S215A. Y276V, M348T. A473R, E498G, and M522V; b) L90V; c) L294N; d) H82M, L90A, Y276V, and A473R; e) R45K. H82M. L90A, Y276P, and A473R; f) H82M, L90A, Y276P, and A473L; g) R45K, I63V, H82M, L90A, Y276V, M348T, A473R, and E498G; h) R45K, H82M, L90A, Y276V, A298P, Y345S, and A473R; or i) R45K, R58D, I63V, H82M, L90A, S215A, Y276V. K282T, N284D, M348T, A473R,E498G, and M522T, wherein the amino acid positions of the polypeptide are numbered with reference to SEQ ID NO: 21.

20. The polypeptide of any one of claims 15-19. wherein the amino acid sequence comprises any one of SEQ ID NO: 30, 23, 24, 25, 26, 27, 28, 29, or 31.

21. The polypeptide of claim 20, wherein the amino acid sequence comprises SEQ ID NO: 30.

22. The polypeptide of any one of claims 15-21, further comprising an epitope tag.

23. The polypeptide of claim 22. wherein the epitope tag is a His tag.

24. The polypeptide of claim 23, wherein the His tag comprises the amino acid sequence of GSHHHHHHHHSG (SEQ ID NO: 22).

25. The polypeptide of any one of claims 22-24. wherein the polypeptide comprises an N- terminal methionine residue, and the epitope tag is inserted immediately following the N- tenninal methionine residue.

26. The polypeptide of any one of claims 1-25, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

27. An engineered polypeptide comprising at least 98% sequence identity to any one of SEQ ID NO: 18, 4, 6, 8, 10, 12, 14, 16, or 20, wherein the polypeptide does not comprise the sequence of SEQ ID NO: 35 or SEQ ID NO: 36.

28. A polypeptide comprising at least 99% sequence identity to any one of SEQ ID NO: 18, 4, 6, 8, 10, 12, 14, 16, or 20.

29. The polypeptide of any one of claims 1-28, which is isolated.

30. The polypeptide of any one of claims 1-29, which is a phenylalanine ammonia lyase.

31. The polypeptide of any one of claims 1-30, wherein the polypeptide has one or more of the following properties relative to a reference polypeptide comprising the amino acid sequence of SEQ ID NO: 2: a) increased activity in converting 3-cyanocinnamic acid into 3-cyano-L-phenylalanine, optionally wherein the polypeptide has at least about 1.1 -fold, about 5-fold, about 10-fold, or about 100-fold increased activity; b) increased enantioselectivity; c) increased thermostability; and / or d) increased soluble expression.

32. A polynucleotide encoding at least one polypeptide set forth in any one of claims 1-31.

33. The polynucleotide of claim 32, wherein the polynucleotide is codon-optimized.

34. The polynucleotide of claim 32 or 33, wherein the polynucleotide comprises SEQ ID NO:

17.

3. 5, 7, 9.

11.

13.

15. or 19.

35. A polynucleotide comprising at least 80% sequence identity to any one of SEQ ID NO:

17.

3. 5, 7, 9, 11, 13, 15, or 19.

36. An expression vector comprising at least one polynucleotide sequence of any one of claims 32-35.

37. The expression vector of claim 36. wherein the polynucleotide sequence is operably linked to a control sequence.

38. The expression vector of claim 37, wherein the control sequence is a promoter.

39. The expression vector of claim 38, wherein the promoter is a heterologous promoter.

40. A host cell comprising the polynucleotide of any one of claims 32-35 or the expression vector of any one of claims 36-39.

41. The host cell of claim 40, wherein the host cell is prokaryotic or eukaryotic.

42. A method of producing a polypeptide, the method comprising culturing the host cell of claim 40 or 41 under conditions that the polypeptide encoded by the polynucleotide is produced.

43. The method of claim 42, further comprising the step of recovering the polypeptide.

44. The method of claim 43, further comprising the step of purifying the polypeptide.

45. A method of converting 3 -cyanocinnamic acid into 3-cyano-L-phenylalanine, the method comprising contacting 3-cyanocinnamic acid with an ammonia source in the presence of the polypeptide of any one of claims 1-31.

46. The method of claim 45, wherein the ammonia source is ammonium carbonate, ammonium carbamate, ammonium sulfate, ammonium acetate, or ammonium formate.

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