Engineered leucine decarboxylase

Abstract

The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, and polynucleotides encoding engineered leucine decarboxylase polypeptides. In some embodiments, the engineered leucine decarboxylase polypeptides exhibit enhanced catalytic activity, as well as reduced susceptibility to proteolysis and / or increased tolerance to low pH environments. In some embodiments, the engineered leucine decarboxylase polypeptides are optimized to provide improved storage stability. The present invention also provides methods of using compositions comprising engineered leucine decarboxylase polypeptides for therapeutic and industrial purposes.

Description

[Technical field]

[0001] CROSS-REFERENCE TO RELATED APPLICATIONS This application claims the benefit under 35 U.S.C. § 119(e) of U.S. Provisional Application No. 63 / 274,395, filed November 1, 2021, the entire contents of which are incorporated herein by reference.

[0002] Reference to a sequence listing, table, or computer program The sequence listing concurrently submitted via EFS-Web under the file name CX7-221WO2_ST26.xml (created on Oct. 30, 2022, file size 2.08 MB) is a part of and incorporated herein by reference.

[0003] Technical Field The present disclosure relates to engineered leucine decarboxylases (LDCs), compositions thereof, and polynucleotides encoding engineered leucine decarboxylase polypeptides. The present disclosure also relates to methods of using engineered leucine decarboxylase polypeptides for therapeutic and industrial purposes. [Background technology]

[0004] 2. Background of the Invention Maple syrup urine disease (MSUD), also known as "leucinuria," "branched-chain alpha-leucine dehydrogenase deficiency," and "BCKD deficiency," is a rare inherited disorder of amino acid metabolism secondary to a malfunction of the branched-chain ketoacid dehydrogenase (BCKDH) complex, which is involved in the catabolic pathways of leucine, isoleucine, and valine (i.e., branched-chain amino acids). It was first described in 1954 by Menkes et al., Pediatrics, 1954, 14:462-467, and was so named because the urine of affected newborns has a distinctive sweet odor. It is also characterized by poor feeding, vomiting, lethargy, abnormal behavior (e.g., hypertonia or hypotonia), and developmental delay. If untreated, the disease can progress to encephalopathy, seizures, coma, permanent neurological damage, and death. Later in life, developmental delay, learning disabilities, seizures, and movement disorders are common. Based on the signs and symptoms of the disease, it is classified into four common forms. The most common and severe type is the "classic" type, which becomes evident within the first two weeks of life. The other types are intermediate MSUD, intermittent MSUD, and thiamine-responsive MSUD. In the classical form, the disease becomes evident after the newborn consumes milk containing protein. This results in an increase in isoleucine, leucine, and valine in the body, which is toxic to the brain. In the intermittent form, brain damage occurs during physical stress (e.g., infection, fever, prolonged fasting, etc.), leading to metabolic decompensation.

[0005] Diagnostic testing for MSUD in newborns includes blood and urine amino acid testing to measure the concentrations of leucine, isoleucine, alloisoleucine, and valine in these fluids. When MSUD is confirmed, signs of ketosis and acidosis are present. After diagnosis and during symptoms, treatment involves the ingestion of a protein-free diet and correction of metabolic consequences with increased amino acid levels. The use of special intravenous solutions reduces leucine levels (the most toxic) and corrects the energy deficiency.

[0006] In addition to MSUD, other disorders of leucine metabolism and branched amino acids include isovaleric acidemia and 3-methylcrotonylglycinuria (also called 3-methylcrotonyl CoA carboxylase deficiency). Current treatments for these types of disorders include dietary restriction of leucine and other branched chain amino acids (BCAAs) and / or protein intake restriction. Deficient levels of enzymes involved in BCAA metabolism result in toxic accumulation of BCAAs and their associated metabolites in cerebrospinal fluid, blood, and tissues. Without treatment or ongoing careful care, this leads to a number of serious side effects (e.g., neurological dysfunction, seizures, and infant death). Although some of the BCAAs are metabolized by renal clearance (resulting in the typical sweet maple syrup odor of the urine of affected patients), this is not sufficient to alleviate the accumulation of toxic amino acid levels in the body (see Schadewalt and Wendel, Eur. J. Pediatr., 1997, 156(Suppl. 1):S62-66; and Skvorak, J. Inherit. Metab. Dis., 2009, 32(2):229-46). Summary of the Invention

[0007] Summary of the Invention The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, and polynucleotides encoding engineered leucine decarboxylase polypeptides. In some embodiments, the leucine decarboxylase polypeptides are engineered to have improved properties, including enhanced catalytic activity, reduced susceptibility to proteolysis, and / or increased tolerance to low pH. In some embodiments, the engineered leucine decarboxylase polypeptides are engineered to exhibit improved storage stability. The present invention also provides methods of using engineered leucine decarboxylase polypeptides and compositions thereof for therapeutic and industrial purposes.

[0008] In one aspect, the present invention relates to engineered leucine decarboxylase polypeptides, and biologically active fragments and analogs thereof, having improved properties when compared to a wild-type leucine decarboxylase or a reference leucine decarboxylase polypeptide under essentially the same conditions.

[0009] In some embodiments, the engineered leucine decarboxylase polypeptide comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.

[0010] In some embodiments, the engineered leucine decarboxylase polypeptide comprises at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO:828, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:828.

[0011] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0012] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises a substitution at at least two amino acid positions: amino acid positions 33, 55, 64, 126, 270, and 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises a substitution at amino acid position 33, 55, 64, 126, 270, and / or 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the substitutions at amino acid positions 33, 55, 64, 126, 270, and / or 357 are selected from 33L, 55I, 64N, 126A, 270L, and 357S.

[0013] In some further embodiments, the engineered leucine decarboxylase comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 828, wherein the amino acid sequence is at or near amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 141 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 272 / 383, 41 / 141 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 126 / 267, 64 / 267 / 353 / 3 84, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 19 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 19 / 55 / 64 / 1 26 / 267 / 270 / 353, 19 / 33 / 126 / 270 / 353 / 357 / 384, 19 / 33 / 64 / 267 / 353, 126 / 353 / 384, 126 / 270 / 312 / 353 / 384, 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0014] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:888.

[0015] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:888, wherein the amino acid sequence contains one or more substitutions relative to a reference sequence corresponding to SEQ ID NO:888.

[0016] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888.

[0017] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises amino acid residues 5V, 19L, 33L, 41D, 47F, 51E, 55I, 64S / N, 141P, 170P, 173I, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 290I, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0018] In some further embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence is at or near amino acid positions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 202 / 357, 202 / 353, 5 / 5 1 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 353, 357, 64, 5 / 41 / 141, 41 / 141 / 173 / 202 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 64 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 353 / 357, 5 / 41 / 64 / 173 / 353 / 357, 64 / 173 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 202 / 27 2 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 353, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / 173 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202,or 5 / 173 / 272, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0019] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least a substitution or set of substitutions of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO:12.

[0020] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least a substitution or set of substitutions of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO: 828 or 888.

[0021] In some embodiments, the engineered leucine decarboxylase polypeptide comprises the amino acid sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.

[0022] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO:854-1064.

[0023] In some embodiments, the engineered leucine decarboxylase polypeptides described herein exhibit one or more improved properties compared to a wild-type Planctomycetaceae species leucine decarboxylase or an engineered leucine decarboxylase having a sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptides described herein exhibit an improved property selected from (i) increased activity towards leucine, (ii) increased resistance to proteolysis, (iii) increased tolerance to low pH environments, or (iv) increased thermostability, or any combination thereof, compared to a wild-type Planctomycetaceae species leucine decarboxylase or a leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12.

[0024] In further aspects, the present disclosure also provides recombinant polynucleotides encoding at least one engineered leucine decarboxylase polypeptide described herein. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a polynucleotide sequence corresponding to an odd-numbered SEQ ID NO: 853-1063, wherein the recombinant polynucleotide encodes a polypeptide having leucine decarboxylase activity. In some embodiments, the recombinant polynucleotide comprises a polynucleotide sequence comprising any of the odd-numbered SEQ ID NOs: 15-851.

[0025] In some additional embodiments, the recombinant polynucleotide sequence is operably linked to a regulatory sequence, hi some embodiments, the recombinant polynucleotide sequence is codon optimized.

[0026] In another aspect, the present disclosure also provides an expression vector comprising at least one recombinant polynucleotide sequence provided herein.In some embodiments, the expression vector further comprises at least one control sequence.In some embodiments, the control sequence comprises a promoter.In some embodiments, the promoter is a heterologous promoter.

[0027] In another aspect, the present invention further provides a host cell transformed with at least one polynucleotide sequence and / or comprising an expression vector provided herein. In some embodiments, the host cell is transformed with a polynucleotide sequence provided herein. In some further embodiments, the host cell comprises an expression vector provided herein. In some embodiments, the host cell is E. coli.

[0028] In another aspect, the present disclosure also provides a method for producing an engineered leucine decarboxylase polypeptide in a host cell, the method comprising culturing a host cell comprising at least one expression vector provided herein under suitable culture conditions to produce at least one leucine decarboxylase polypeptide. In some embodiments, the method further comprises recovering the at least one engineered leucine decarboxylase polypeptide from the culture and / or the host cell. In some additional embodiments, the method further comprises purifying the at least one engineered leucine decarboxylase polypeptide.

[0029] In another aspect, the disclosure provides a composition comprising at least one engineered leucine decarboxylase polypeptide. In some embodiments, the composition is a pharmaceutical composition comprising at least one engineered leucine decarboxylase polypeptide. In some embodiments, the pharmaceutical composition comprises at least one pharma- ceutically acceptable excipient and / or carrier. In some embodiments, the pharmaceutical composition comprising at least one engineered leucine decarboxylase polypeptide is suitable for oral or parenteral administration. In some embodiments, the composition is in the form of a pill, tablet, capsule, gel capsule, liquid, or emulsion. In some additional embodiments, the composition is suitable for oral administration to a mammal, particularly a human patient. In some embodiments, the composition comprises at least one additional therapeutically effective compound.

[0030] In some embodiments, the present disclosure also provides a composition comprising at least one polynucleotide encoding an engineered leucine decarboxylase polypeptide disclosed herein, wherein the polynucleotide is suitable for use in gene therapy. In some embodiments, the composition comprises an expression vector comprising at least one polynucleotide encoding an engineered polypeptide disclosed herein, wherein the expression vector is a gene therapy vector suitable for use in treating a disease or condition associated with elevated levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid in plasma. In some embodiments, the composition is suitable for use in mRNA therapy.

[0031] In a further aspect, the present disclosure provides a method for treating and / or preventing symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels. In some embodiments, the method for treating and / or preventing symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels comprises administering to a subject in need thereof an effective amount of engineered leucine decarboxylase to reduce the subject's plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels. In some embodiments, the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels is maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.

[0032] Also provided herein in some embodiments is a method of reducing plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid in a subject, comprising administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase. In some embodiments, the subject in need thereof suffers from maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.

[0033] In some embodiments of the methods of treating and / or preventing symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels, or of reducing plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels in a subject, the engineered leucine decarboxylase is administered at a dose of 1 mg / kg to 500 mg / kg, 1 mg / kg to 400 mg / kg, or 1 mg / kg to 200 mg / kg. In some embodiments, the engineered leucine decarboxylase is administered at a dose of 1 mg / kg to less than 25 mg / kg. In some embodiments, the engineered leucine decarboxylase is administered at a dose of 5 mg / kg to less than 25 mg / kg. In some embodiments, the engineered leucine decarboxylase is administered at a dose of about 6.25 mg / kg to about 12.5 mg / kg.

[0034] In some embodiments, the engineered leucine decarboxylase is administered to a subject in need thereof in an amount effective to reduce plasma levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and / or ketoisocaproic acid. In some embodiments, the engineered leucine decarboxylase is administered to a subject in need thereof in an amount effective to reduce plasma levels of leucine, ketoisocaproic acid, and methionine. In some embodiments, the engineered leucine decarboxylase is administered to a subject for at least two or more consecutive days. In some embodiments, the engineered leucine decarboxylase is administered to a subject for at least three or more consecutive days. In some embodiments, the engineered leucine decarboxylase is administered to a subject continuously as needed. In some embodiments, the engineered leucine decarboxylase is administered immediately before, simultaneously with, and / or immediately after ingestion of a meal containing protein.

[0035] In some embodiments, the subject for treatment with engineered leucine decarboxylase suffers from maple syrup urine disease, and wherein the symptoms of said maple syrup urine disease are alleviated. In some embodiments, the subject for treatment with engineered leucine decarboxylase suffers from isovaleric acidemia, and wherein the symptoms of said isovaleric acidemia are alleviated. In some embodiments, the subject for treatment with engineered leucine decarboxylase suffers from 3-methylcrotonyl-CoA carboxylase deficiency, and wherein the symptoms of said 3-methylcrotonyl-CoA carboxylase deficiency are alleviated. In some embodiments, the subject can consume a diet with less restricted leucine, isoleucine, and / or valine content compared to the diet required by subjects suffering from the disease. In some embodiments, the subject is an infant, a child, a young adult, or an adult. In some embodiments, the subject is an infant. In some embodiments, the subject is a child.

[0036] In some embodiments, for the methods and uses described herein, including methods of treatment or reducing plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid, an engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, and / or 888.

[0037] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.

[0038] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.

[0039] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12.

[0040] In some embodiments of the method, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 2, 3, 5, 12, 14, 16, 19, 33, 34, 38, 39, 41, 47, 48, 51, 55, 63, 64, 66, 69, 76, 77, 80, 87, 89, 91, 92, 102, 106, 109, 118, 123, 126, 127, 132, 134, 135, 139, 140, 141, 156, 161, 164, 168, 170, 173, 181, 187, 189, 193, 194, 196, 198, 200, 201, 202, 211, 223, 228, 245, 255, 256, 259, 26 2, 263, 265, 267, 270, 272, 275, 290, 296, 299, 300, 303, 304, 312, 317, 319, 324, 328, 331, 338, 339, 340, 343, 349, 350, 352, 353, 357, 364, 365, 365, 366, 379, 380, 381, 382, ​​383, 384, 386, 388, 389, 390, 391, 393, 394, 395, 397, 398, 401, 404, or 405, or combinations thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12.

[0041] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 5, 14, 14 / 34 / 38 / 39 / 102 / 267 / 275 / 350 / 357, 14 / 39 / 102 / 127 / 245 / 267 / 275 / 3 49 / 350, 34 / 38 / 39 / 102 / 127 / 275 / 357, 34 / 38 / 39 / 102 / 275 / 357, 34 / 38 / 39 / 127 / 245 / 349 / 350 / 357, 34 / 38 / 39 / 127 / 245 / 350 / 357, 34 / 39 / 102 / 127 / 26 4 / 275 / 357, 34 / 39 / 102 / 127 / 275 / 349 / 357, 34 / 39 / 102 / 264 / 275 / 350 / 357, 34 / 39 / 275 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 264 / 267 / 350 / 357, 38 / 39 / 102 / 127 / 267 / 275 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 350, 38 / 39 / 102 / 127 / 350 / 357, 38 / 39 / 127 / 245 / 267 / 357, 38 / 39 / 127 / 2 64 / 275, 38 / 39 / 127 / 264 / 350 / 357, 38 / 39 / 127 / 350 / 357, 38 / 39 / 127 / 357, 38 / 39 / 245 / 275 / 357, 38 / 39 / 264 / 267 / 275 / 350, 38 / 39 / 264 / 275 / 357, 38 / 39 / 275, 38 / 39 / 275 / 350, 39, 39 / 102 / 127 / 264 / 275 / 357, 39 / 102 / 264 / 275 / 357, 39 / 102 / 267 / 275 / 357, 39 / 127 / 245 / 264 / 267 / 275 / 350, 39 / 127 / 245 / 26 4 / 275 / 350 / 357, 39 / 127 / 245 / 357, 39 / 127 / 267 / 275 / 350 / 357, 39 / 127 / 267 / 350 / 357, 39 / 127 / 357, 39 / 245 / 264 / 267 / 275 / 357, 39 / 264 / 267 / 275 / 350,39 / 275 / 350 / 357, 48, 139, 164, 196, 255, 299, 318, 324, 339, 343, 350, 353, 357, 364, 365, 379, 381, 386, 389, 391, 393, 394, 395, 397, 398, or 405, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 12.

[0042] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:38, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 48 / 64 / 164 / 324 / 343 / 35 3 / 357 / 364, 48 / 64 / 164 / 324 / 343 / 364, 48 / 64 / 164 / 353 / 357 / 364, 48 / 64 / 357 / 364, 64 / 164 / 324 / 343 / 353 / 357 / 364, 64 / 164 / 324 / 343 / 357 / 364, 64 / 164 / 353 / 357, 64 / 318 / 324 / 357 / 364, 64 / 324 / 353 / 357 / 364, 132 / 255 / 339 / 379 / 395, 164 / 196 / 324 / 357 / 364, 164 / 318 / 324 / 343 / 353 / 357, 164 / 318 / 324 / 357 / 364, 164 / 324 / 343 / 353 / 357 / 364, 164 / 324 / 357 / 364, 164 / 353 / 357 / 364, 164 / 364, 196 / 318 / 324 / 353 / 357 / 364, 318 / 343 / 357, 324 / 343 / 357 / 364, 324 / 353 / 357 / 364, 324 / 357 / 364, 339 / 379 379 / 386, 379 / 394 / 395 / 397 / 404 / 405, 379 / 394 / 395 / 397 / 405, 389 / 394 / 395 / 397 / 405, or 394 / 397, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:38.

[0043] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:234, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 2, 3, 33, 48 / 64 / 255, 48 / 255 / 339, 48 / 255 / 379, 64, 64 / 255, 69, 161, 193, 255, 255 / 318 / 379, 259, 263, 318 / 339 / 379, 324, 324 / 389 / 394, 324 / 389 / 394 / 395, 324 / 389 / 394 / 397, 324 / 394, 324 / 394 / 395, 324 / 394 / 395 / 397, 324 / 395, 339, 340, 380, 382, ​​389, 389 / 394, 389 / 394 / 395, 389 / 394 / 395 / 397, 389 / 394 / 397, 389 / 395, 389 / 397, 390, 394, 394 / 395, 394 / 395 / 397, 395, 395 / 397, 397, 401, or 405, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:234.

[0044] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:284, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 2 / 64 / 69 / 324 / 380 / 382 / 388 / 389, 3 / 64 / 69 / 263 / 339 / 380 / 388, 3 / 64 / 69 / 38 9, 3 / 64 / 69 / 390, 3 / 64 / 379 / 380 / 390, 3 / 69 / 263 / 380, 3 / 69 / 324, 3 / 69 / 324 / 380 / 382 / 389 / 390, 12 / 135 / 259 / 263, 12 / 135 / 263 / 382, 12 / 259 / 263 / 304 , 48 / 64 / 255, 64 / 69, 64 / 69 / 189 / 259 / 263 / 304, 64 / 69 / 189 / 259 / 263 / 304 / 339 / 340 / 379, 64 / 69 / 223 / 388, 64 / 69 / 223 / 388 / 389 / 390, 64 / 69 / 304 / 379 / 382, 64 / 69 / 324, 64 / 69 / 324 / 339 / 380 / 389 / 390, 64 / 69 / 339, 64 / 69 / 339 / 382 / 388 / 389, 64 / 69 / 339 / 389 / 390, 64 / 69 / 379 / 380, 64 / 69 / 380 / 388 / 390, 64 / 69 / 389, 64 / 69 / 390, 64 / 255 / 263, 64 / 263, 64 / 324 / 339 / 389 / 390, 69 / 223 / 263 / 324 / 382 / 388 / 390, 69 / 223 / 324 / 379 / 380 / 382 / 388 / 390, 69 / 263, 6 9 / 263 / 324, 69 / 263 / 339, 69 / 263 / 388, 69 / 263 / 389 / 390, 69 / 324 / 379 / 380 / 388, 69 / 324 / 380, 69 / 339 / 390, 69 / 382 / 390, 259 / 263 / 304, 259 / 263 / 304 / 339 / 340 / 379, 263 / 339 / 389 / 390, 263 / 390, or 304 / 340 / 379 / 380 / 382, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:284.

[0045] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:484, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 3 / 194 / 304, 3 / 25 9 / 263 / 304, 3 / 259 / 304, 3 / 259 / 304 / 324 / 339, 3 / 259 / 304 / 324 / 382, 3 / 259 / 304 / 382, 3 / 263 / 304 / 324, 3 / 263 / 304 / 324 / 339, 3 / 263 / 304 / 324 / 382, 3 / 304, 3 / 304 / 324, 16, 63, 77, 80, 87 / 270, 87 / 270 / 365, 87 / 328 / 365, 91, 92, 126, 140, 156, 168 / 270 / 328 / 338, 181, 194, 201, 256, 259, 259 / 263, 259 / 263 / 304, 259 / 263 / 304 / 324, 259 / 263 / 304 / 324 / 382, 259 / 263 / 304 / 379, 259 / 263 / 304 / 382, 259 / 304, 259 / 304 / 324, 259 / 304 / 324 / 339, 259 / 304 / 324 / 339 / 382, 259 / 304 / 382, 262, 263 / 3 04, 263 / 304 / 324, 263 / 304 / 324 / 339, 263 / 304 / 324 / 382, 263 / 324, 270, 270 / 319, 270 / 328 / 338, 270 / 328 / 338 / 365, 304, 304 / 324, 324, 328, 352, 365, 366, or 382, ​​wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484.

[0046] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:594, and wherein the engineered leucine decarboxylase polypeptide The amino acid sequence of the peptide is shown in Table 1. The amino acid sequence of the peptide is shown in Table 1. 68 / 324, 16 / 80 / 168 / 366, 16 / 80 / 324, 16 / 91 / 126 / 168 / 324 / 366, 16 / 126 / 168 / 366, 16 / 168 / 259 / 366, 16 / 168 / 270 / 324 / 366, 16 / 168 / 324 / 328 / 366, 16 / 168 / 324 / 366, 16 / 168 / 366, 16 / 259 / 263 / 328, 16 / 32 and / or 168 / 366, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.

[0047] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 686, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 66 / 76 / 118 / 141 / 201 / 300, 66 / 76 / 198 / 200 / 296 / 303, 66 / 76 / 198 / 200 / 300, 66 / 118 / 200 / 296 / 303 / 317, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 200, 76 / 118 / 141 / 200 / 296, 76 / 141 / 198 / 200 / 201 / 300, 80 / 201 / 270, 80 / 270, 80 / 270 / 324, 89 / 118 / 200, 106 / 270 / 324 / 352, 118 / 141 / 200, 126, 126 / 201 / 270 / 324, 12 6 / 270, 141 / 144 / 198 / 200 / 300, 156 / 270, 156 / 270 / 324, 201 / 270, 201 / 270 / 352, 270, or 270 / 324, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:686.

[0048] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:686, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:686.

[0049] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:688, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 19 / 109 / 123 / 141 / 170 / 198 / 200 / 211 / 270 / 312, ...312, 19 / 109 / 123 / 141 / 170 / 198 / 400 / 411 / 470 / 500 / 600 / 700 / 800 / 900 / 1000 / 1100 211, 19 / 109 / 123 / 141 / 170 / 198 / 211 / 270 / 312, 19 / 109 / 123 / 170 / 211 / 270 / 312, 19 / 109 / 123 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 170 / 173 / 211 / 270 / 312, 19 / 109 / 211 / 270 / 312, 109 / 170 / 211 / 270 / 312, or 109 / 211 / 270 / 312, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:688.

[0050] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:766, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 5 / 41, 5 / 41 / 228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766.

[0051] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 766, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one amino acid sequence identical to SEQ ID NO: 766, , 66 / 118, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 118 / 300, 66 / 296, 66 / 296 / 300, 66 / 300, 118, 118 / 296, 118 / 296 / 300, 118 / 300, 296, 296 / 300, or 300, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766.

[0052] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 14 1 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 272 / 383, 41 / 141 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 12 6 / 267, 64 / 267 / 353 / 384, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 19 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 1 9 / 55 / 64 / 126 / 267 / 270 / 353, 19 / 33 / 126 / 270 / 353 / 357 / 384, 19 / 33 / 64 / 267 / 353, 126 / 353 / 384, 126 / 270 / 312 / 353 / 384, 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0053] In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 888, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 20 2 / 357, 202 / 353, 5 / 51 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 3 53, 357, 64, 5 / 41 / 141, 41 / 141 / 173 / 202 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 6 4 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 353 / 357, 5 / 41 / 64 / 1 73 / 353 / 357, 64 / 173 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 2 02 / 272 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 353, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / 17 3 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202,or 5 / 173 / 272, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0054] In some embodiments of the method, the engineered leucine decarboxylase polypeptide exhibits one or more improved properties compared to a wild-type Planctomycetaceae species' leucine decarboxylase or an engineered leucine decarboxylase having a sequence corresponding to SEQ ID NO: 12. In some embodiments, the improved properties are selected from (i) increased activity towards leucine, (ii) increased resistance to proteolysis, (iii) increased tolerance to low pH environments, or (iv) increased thermostability, or any combination thereof, compared to a wild-type Planctomycetaceae species' leucine decarboxylase or an engineered leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12.

[0055] In some embodiments of the method, the engineered leucine decarboxylase comprises at least one leucine decarboxylase polypeptide provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and / or 12-2. In some embodiments of the method, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO:2-1064. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO:16-1064. [Brief description of the drawings]

[0056] [Figure 1A]Figures 1A and 1B show the results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylase in an intermediate MSUD mouse model. Treatment with engineered leucine decarboxylase after administration of a whey protein diet suppresses plasma leucine levels. Figure 1A shows the time course of plasma leucine levels in animals treated with vehicle, LDC polypeptide of SEQ ID NO: 484, LDC polypeptide of SEQ ID NO: 686, or LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 1B shows plasma leucine AUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post-hoc test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 1B] Figures 1A and 1B show the results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylase in an intermediate MSUD mouse model. Treatment with engineered leucine decarboxylase after administration of a whey protein diet suppresses plasma leucine levels. Figure 1A shows the time course of plasma leucine levels in animals treated with vehicle, LDC polypeptide of SEQ ID NO: 484, LDC polypeptide of SEQ ID NO: 686, or LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 1B shows plasma leucine AUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey's post-hoc test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 2A]Figures 2A and 2B show the results of a pharmacodynamic (PD) dose-response study of the LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model. Treatment with different amounts of the engineered LDC polypeptide of SEQ ID NO: 766 after administration of a whey protein meal results in a dose-dependent suppression of plasma leucine levels. Figure 2A shows the time course of plasma leucine levels in animals treated with vehicle or three different doses of LDC of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 2B shows plasma leucine AUC. One-way ANOVA compared to vehicle: **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 2B] Figures 2A and 2B show the results of a pharmacodynamic (PD) dose-response study of the LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model. Treatment with different amounts of the engineered LDC polypeptide of SEQ ID NO: 766 after administration of a whey protein meal results in a dose-dependent suppression of plasma leucine levels. Figure 2A shows the time course of plasma leucine levels in animals treated with vehicle or three different doses of LDC of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 2B shows plasma leucine AUC. One-way ANOVA compared to vehicle: **, p<0.01; ***, p<0.001; ****, p<0.0001. [Figure 3A]Figures 3A and 3B show the results of a pharmacodynamic (PD) dose-response study comparing engineered LDC polypeptides of SEQ ID NO: 828 and SEQ ID NO: 766 in an iMSUD mouse model. Figure 3A shows the plasma leucine time course of treatment with LDC polypeptides of SEQ ID NO: 766 or LDC polypeptides of SEQ ID NO: 828 after a whey protein meal. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. Figure 3B shows the corresponding plasma leucine AUC; one-way ANOVA compared to vehicle: *, p<0.05. [Figure 3B] Figures 3A and 3B show the results of a pharmacodynamic (PD) dose-response study comparing engineered LDC polypeptides of SEQ ID NO: 828 and SEQ ID NO: 766 in an iMSUD mouse model. Figure 3A shows the plasma leucine time course of treatment with LDC polypeptides of SEQ ID NO: 766 or LDC polypeptides of SEQ ID NO: 828 after a whey protein meal. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. Figure 3B shows the corresponding plasma leucine AUC; one-way ANOVA compared to vehicle: *, p<0.05. [Figure 4A]4A-4D show pharmacodynamic (PD) dose-response studies of engineered leucine decarboxylase in healthy cynomolgus monkeys. FIG. 4A shows the time course of plasma leucine levels after administration of three different doses of engineered LDC polypeptide of SEQ ID NO: 484. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4B shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 686. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4C shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 766. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4D shows the corresponding plasma leucine AUC of engineered leucine decarboxylase administered at different doses. Data are shown as mean ± SEM. One-way ANOVA compared with vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 4B] 4A-4D show pharmacodynamic (PD) dose-response studies of engineered leucine decarboxylase in healthy cynomolgus monkeys. FIG. 4A shows the time course of plasma leucine levels after administration of three different doses of engineered LDC polypeptide of SEQ ID NO: 484. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4B shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 686. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4C shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 766. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4D shows the corresponding plasma leucine AUC of engineered leucine decarboxylase administered at different doses. Data are shown as mean ± SEM. One-way ANOVA compared with vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 4C]4A-4D show pharmacodynamic (PD) dose-response studies of engineered leucine decarboxylase in healthy cynomolgus monkeys. FIG. 4A shows the time course of plasma leucine levels after administration of three different doses of engineered LDC polypeptide of SEQ ID NO: 484. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4B shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 686. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4C shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 766. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4D shows the corresponding plasma leucine AUC of engineered leucine decarboxylase administered at different doses. Data are shown as mean ± SEM. One-way ANOVA compared with vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 4D] 4A-4D show pharmacodynamic (PD) dose-response studies of engineered leucine decarboxylase in healthy cynomolgus monkeys. FIG. 4A shows the time course of plasma leucine levels after administration of three different doses of engineered LDC polypeptide of SEQ ID NO: 484. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4B shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 686. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4C shows the time course of plasma leucine levels after administration of three different doses of LDC polypeptide of SEQ ID NO: 766. Data are shown as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 4D shows the corresponding plasma leucine AUC of engineered leucine decarboxylase administered at different doses. Data are shown as mean ± SEM. One-way ANOVA compared with vehicle: *, p<0.05; **, p<0.01; ***, p<0.001. [Figure 5A]5A-5D show the results of a pharmacodynamic (PD) dose-response study and a 3-day repeat-dose study with an engineered LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys. FIG. 5A shows the time course of plasma leucine after treatment with three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 5B shows the corresponding plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: *, p<0.05; **, p<0.01. FIG. 5C shows the time course of plasma ketoisocaproic acid (KIC) for three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01. Figure 5D shows the corresponding plasma KIC iAUC for different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: **, p<0.01. [Figure 5B] 5A-5D show the results of a pharmacodynamic (PD) dose-response study and a 3-day repeat-dose study with an engineered LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys. FIG. 5A shows the time course of plasma leucine after treatment with three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 5B shows the corresponding plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: *, p<0.05; **, p<0.01. FIG. 5C shows the time course of plasma ketoisocaproic acid (KIC) for three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01. Figure 5D shows the corresponding plasma KIC iAUC for different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: **, p<0.01. [Figure 5C] 5A-5D show the results of a pharmacodynamic (PD) dose-response study and a 3-day repeat-dose study with an engineered LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys. FIG. 5A shows the time course of plasma leucine after treatment with three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 5B shows the corresponding plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: *, p<0.05; **, p<0.01. FIG. 5C shows the time course of plasma ketoisocaproic acid (KIC) for three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01. Figure 5D shows the corresponding plasma KIC iAUC for different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: **, p<0.01. [Figure 5D] 5A-5D show the results of a pharmacodynamic (PD) dose-response study and a 3-day repeat-dose study with an engineered LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys. FIG. 5A shows the time course of plasma leucine after treatment with three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05. FIG. 5B shows the corresponding plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: *, p<0.05; **, p<0.01. FIG. 5C shows the time course of plasma ketoisocaproic acid (KIC) for three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test compared to vehicle: *, p<0.05; **, p<0.01. Figure 5D shows the corresponding plasma KIC iAUC for different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. One-way ANOVA vs. vehicle: **, p<0.01. [Figure 6A] Figures 6A and 6B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on leucine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge dosing. Figure 6A shows the time course of plasma leucine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6B shows the corresponding plasma leucine AUC. Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. Figure 6C shows the time course of baseline-subtracted plasma leucine. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6D shows the corresponding plasma leucine iAUC (baseline subtracted). Data are presented as mean ± SEM. Unpaired t-test, vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 6B]Figures 6A and 6B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on leucine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge dosing. Figure 6A shows the time course of plasma leucine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6B shows the corresponding plasma leucine AUC. Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. Figure 6C shows the time course of baseline-subtracted plasma leucine. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6D shows the corresponding plasma leucine iAUC (baseline subtracted). Data are presented as mean ± SEM. Unpaired t-test, vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 6C] Figures 6A and 6B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on leucine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge dosing. Figure 6A shows the time course of plasma leucine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6B shows the corresponding plasma leucine AUC. Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. Figure 6C shows the time course of baseline-subtracted plasma leucine. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6D shows the corresponding plasma leucine iAUC (baseline subtracted). Data are presented as mean ± SEM. Unpaired t-test, vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 6D] Figures 6A and 6B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on leucine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge dosing. Figure 6A shows the time course of plasma leucine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6B shows the corresponding plasma leucine AUC. Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. Figure 6C shows the time course of baseline-subtracted plasma leucine. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001; ****, p<0.0001. Figure 6D shows the corresponding plasma leucine iAUC (baseline subtracted). Data are presented as mean ± SEM. Unpaired t-test, vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 7A] Figures 7A and 7B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on methionine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge. Figure 7A shows the time course of baseline-subtracted plasma methionine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001. Figure 7B shows the corresponding plasma methionine iAUC (baseline-subtracted). Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 7B]Figures 7A and 7B show the results of a study investigating the efficacy of an engineered LDC polypeptide of SEQ ID NO: 766 on methionine in a whey protein diet in healthy cynomolgus monkeys over three consecutive days of challenge. Figure 7A shows the time course of baseline-subtracted plasma methionine following treatment with the engineered LDC polypeptide of SEQ ID NO: 766. Data are presented as mean ± SEM. Multiple t-test *, p<0.05; **, p<0.01; ***, p<0.001. Figure 7B shows the corresponding plasma methionine iAUC (baseline-subtracted). Data are presented as mean ± SEM. Unpaired t-test; vehicle vs. treatment *, p<0.05; **, p<0.01. [Figure 8A] Figures 8A and 8B show the results of a pharmacodynamic (PD) dose-response study of the effect of engineered LDC polypeptide in an intermediate MSUD mouse model. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine levels. Figure 8A shows the time course of normalized plasma leucine levels in mice treated with vehicle and 25, 50, and 100 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 8B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: **p<0.01; ***p<0.001. [Figure 8B]Figures 8A and 8B show the results of a pharmacodynamic (PD) dose-response study of the effect of engineered LDC polypeptide in an intermediate MSUD mouse model. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine levels. Figure 8A shows the time course of normalized plasma leucine levels in mice treated with vehicle and 25, 50, and 100 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): *p<0.05; **p<0.01; ***p<0.001; ****p<0.0001. Figure 8B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: **p<0.01; ***p<0.001. [Figure 9A] Figures 9A and 9B show the results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine levels. Figure 9A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 6.25, 12.5, and 25 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): *p<0.05; **p<0.01. Figure 9B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: *p<0.05; ***p<0.001. [Figure 9B]Figures 9A and 9B show the results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine levels. Figure 9A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 6.25, 12.5, and 25 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): *p<0.05; **p<0.01. Figure 9B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: *p<0.05; ***p<0.001. [Figure 10A] Figures 10A, 10B, and 10C show the results of a pharmacodynamic (PD) study of the effects of engineered LDC in healthy cynomolgus monkeys. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine and KIC levels. Figure 10A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 3.125, 6.25, 12.5, and 25 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): **p<0.01; ***p<0.001; ****p<0.0001. Figure 10B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test (compared to vehicle): *p<0.05; **p<0.01. Figure 10C shows plasma KIC iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: *p<0.05. [Figure 10B]Figures 10A, 10B, and 10C show the results of a pharmacodynamic (PD) study of the effects of engineered LDC in healthy cynomolgus monkeys. Treatment with three doses of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine and KIC levels. Figure 10A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 3.125, 6.25, 12.5, and 25 mg / kg of the LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle): **p<0.01; ***p<0.001; ****p<0.0001. Figure 10B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test (compared to vehicle): *p<0.05; **p<0.01. Figure 10C shows plasma KIC iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared to vehicle: *p<0.05. [Figure 11A] Figures 11A, 11B, and 11C show the results of a pharmacodynamic (PD) 3-day repeat dose study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys. Treatment with low doses (6.25 mg / kg) of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine and KIC levels. Figure 11A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 6.25 mg / kg LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle). Figure 11B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test (compared to vehicle): *p<0.05; **p<0.01. Figure 11C shows plasma KIC iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared with vehicle: *p<0.05. [Figure 11B]Figures 11A, 11B, and 11C show the results of a pharmacodynamic (PD) 3-day repeat dose study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys. Treatment with low doses (6.25 mg / kg) of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine and KIC levels. Figure 11A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 6.25 mg / kg LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle). Figure 11B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test (compared to vehicle): *p<0.05; **p<0.01. Figure 11C shows plasma KIC iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared with vehicle: *p<0.05. [Figure 11C] Figures 11A, 11B, and 11C show the results of a pharmacodynamic (PD) 3-day repeat dose study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys. Treatment with low doses (6.25 mg / kg) of engineered LDC (SEQ ID NO: 828) after administration of a whey protein meal suppresses plasma leucine and KIC levels. Figure 11A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and 6.25 mg / kg LDC polypeptide of SEQ ID NO: 828. Data are presented as mean ± SEM. Unpaired multiple t-test with Welch's correction (compared to vehicle). Figure 11B shows plasma leucine iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test (compared to vehicle): *p<0.05; **p<0.01. Figure 11C shows plasma KIC iAUC. Data are presented as mean ± SEM. One-way ANOVA with Tukey post-hoc test compared with vehicle: *p<0.05. DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

[0057] Description of the invention The present disclosure provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, and polynucleotides encoding engineered leucine decarboxylase polypeptides. In some embodiments, the engineered leucine decarboxylase polypeptides are engineered to provide enhanced catalytic activity, reduced susceptibility to proteolysis, increased tolerance to low pH environments, and / or increased thermostability. In some embodiments, the engineered leucine decarboxylase polypeptides are evolved to provide improved storage stability. The present disclosure also provides methods of using engineered leucine decarboxylase polypeptides and compositions thereof for therapeutic and industrial purposes.

[0058] Abbreviations and Definitions Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by one of ordinary skill in the art to which the present invention pertains. Generally, the nomenclature used herein and the laboratory procedures of cell culture, molecular genetics, microbiology, organic chemistry, analytical chemistry, and nucleic acid chemistry described below are well known and commonly used in the art. Such techniques are well known and described in numerous textbooks and references familiar to those skilled in the art. Standard techniques or modifications thereof are used for chemical synthesis and chemical analysis. All patents, patent applications, articles, and publications mentioned herein, both above and below, are expressly incorporated herein by reference.

[0059] Although any suitable method and material similar or equivalent to those described herein can be used to practice the present invention, some methods and materials are described herein. It is understood that the present invention is not limited to the specific methods, protocols, and reagents described, as these may vary according to the circumstances used by those skilled in the art. Thus, the terms defined immediately below are more fully defined by reference to the entire application. All patents, patent applications, articles, and publications mentioned herein, both above and below, are expressly incorporated herein by reference.

[0060] Additionally, as used herein, the singular forms "a," "an," and "the" include plural references unless the context clearly indicates otherwise.

[0061] Numeric ranges are inclusive of the numerical values ​​defining the range. Accordingly, every numerical range disclosed herein is intended to include every narrower numerical range that falls within that broader numerical range, as if such narrower numerical ranges were all expressly written herein. Also, every maximum (or minimum) numerical limitation disclosed herein is intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitation were all expressly written herein.

[0062] The term "about" refers to a tolerance for a particular value. In some cases, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a range of a particular value. In some cases, "about" means within 1, 2, 3, or 4 standard deviations of a particular value.

[0063] Moreover, the headings provided herein are not intended to limit the various aspects or embodiments of the invention, which can be had by reference to the entire application. Accordingly, the terms defined immediately below are more fully defined by reference to the entire application. Nonetheless, in order to facilitate understanding of the invention, certain terms are defined below.

[0064] Unless otherwise indicated, nucleic acids are written left to right in 5' to 3' orientation; amino acid sequences are written left to right in amino to carboxy orientation, respectively.

[0065] As used herein, the term "comprising" and its cognates are used in their inclusive sense (i.e., equivalent to the term "including" and its corresponding cognates).

[0066] The "EC" numbers refer to the International Union of Biochemistry and Molecular Biology Commission's (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0067] "ATCC" refers to the American Type Culture Collection, which holds biorepository collections containing genes and strains.

[0068] "NCBI" refers to the National Center for Biological Information and the sequence databases it provides.

[0069] As used herein, the term "leucine decarboxylase polypeptide" or "LDC" refers to members of the valine decarboxylase class (EC 4.1.1.14). These enzymes use pyridoxal 5'-phosphate (PLP) cofactor to decarboxylate amino acids such as valine and leucine to produce 2-methylpropanamine and isopentylamine, respectively, with the concomitant release of carbon dioxide.

[0070] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently joined by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0071] As used herein, "polynucleotide" refers to a polymer containing at least two nucleotides, either deoxyribonucleotides or ribonucleotides.

[0072] As used herein, "amino acids" may be referred to by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise, may be referred to by their commonly accepted single-letter codes as indicated. 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), aspartic acid (Asp or D), cysteine ​​(Cys or C), glutamic acid (Glu or E), glutamine (Gln or Q), glycine (Gly or G), histidine (His or H), isoleucine (Ile 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), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).

[0073] The terms "engineered," "recombinant," "non-naturally occurring," and "variant," when used with respect to a cell, polynucleotide, or polypeptide, refer to a material that corresponds to a natural or native form that has been modified in such a way that it is not naturally occurring in nature, or a material that is identical but produced from synthetic materials and / or by manipulation using recombinant techniques. In some embodiments, a "recombinant LDC polypeptide" (also referred to herein as an "engineered LDC polypeptide," "variant LDC enzyme," and "LDC variant") is a leucine decarboxylase that has been created using recombinant techniques.

[0074] As used herein, "wild-type" and "naturally-occurring" refer to forms found in nature. For example, a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a natural source and has not been intentionally modified by human manipulation.

[0075] "Coding sequence" refers to a portion of a nucleic acid (such as a gene) that codes for the amino acid sequence of a protein.

[0076] The term "percent (%) sequence identity" is used herein to refer to comparisons between polynucleotides and polypeptides, and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the comparison window of the polynucleotide or amino acid sequence may contain additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage can be calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percent sequence identity. Alternatively, the percentage can be calculated by determining the number of positions where identical nucleic acid bases or amino acid residues are present in both sequences, or where the nucleic acid bases or amino acid residues are aligned with gaps, to calculate the number of matched positions, dividing the number of matched positions by the total number of positions in the comparison window, and multiplying the result by 100 to calculate the percent sequence identity. Those skilled in the art will appreciate that there are many established algorithms available for aligning two sequences. Optimal alignment of sequences for comparison can be carried out (e.g., by the local homology algorithm of Smith and Waterman; Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the Needleman and Wunsch homology alignment algorithm (Needleman and Wunsch, J. Mol. Biol., 1970, 48:443), by the similarity search method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA, 1988, 85:2444), by computerized implementations of these algorithms (e.g., GAP, BESTFIT, FASTA, and TFASTA in the GCG Wisconsin Software Package), or by visual inspection as known in the art.Examples of algorithms suitable for determining percent sequence identity and sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms (see, e.g., Altschul et al., J. Mol. Biol., 1990, 215: 403-410; and Altschul et al., Nucleic Acids Res., 1977, 3389-3402). Software for performing BLAST analyses is publicly available from the National Center for Biotechnology Information website. This algorithm involves identifying high-scoring sequence pairs (HSPs) by first identifying short words "W" in the query sequence that match or meet some positive threshold score "T" when aligned with words of the same length in a database sequence. T is referred to as the neighborhood word score threshold (see 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. For nucleotide sequences, the cumulative score is calculated using 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. The extension of the word hits in each direction is stopped if: the cumulative alignment score falls by an amount "X" from the maximum achieved; if the cumulative score falls below 0 due to the accumulation of one or more negative scoring residue alignments; or if 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 wordlength (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, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89:10915). For exemplary determination of sequence alignment and percent sequence identity, the BESTFIT or GAP programs of the GCG Wisconsin software package (Accelrys, Madison WI) can be used using the default parameters provided.

[0077] "Reference sequence" refers to a defined sequence used as a basis for sequence comparison. A reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or amino acid sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues long, at least 25 residues long, at least 50 residues long, at least 100 residues long, or the entire length of a nucleic acid or polypeptide. Since two polynucleotides or polypeptides each may (1) contain a sequence that is similar between the two sequences (i.e., a portion of the complete sequence), and (2) further contain a sequence that differs between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is usually performed by comparing the sequences of the two polynucleotides or polypeptides over a "comparison window" to identify and compare local regions of sequence similarity. In some embodiments, a "reference sequence" may be based on a primary amino acid sequence, where the reference sequence is a sequence that may have one or more changes in the primary sequence. For example, the phrase "a reference sequence based on SEQ ID NO: 686 having a valine at the residue corresponding to "X123"" refers to a reference sequence in which the corresponding residue at position X123 of SEQ ID NO: 686 (e.g., tyrosine) has been changed to a valine.

[0078] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues, where a sequence is compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and where the portion of a sequence within the comparison window can include no more than 20 percent additions or deletions (i.e., gaps) compared to the reference sequence (no additions or deletions) in order to optimally align the two sequences. A comparison window can be longer than 20 contiguous residues, and optionally includes windows of 30, 40, 50, 100, or more.

[0079] "Corresponding to," "referring to," and "with respect to," as used in the context of numbering a given amino acid or polynucleotide sequence, refer to the numbering of residues in a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence. In other words, the residue numbers or residue positions of a given polymer are specified relative to the reference sequence, not the actual numerical positions of the residues in the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered leucine decarboxylase, can be aligned with a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although gaps are present, the numbering of residues in a given amino acid or polynucleotide sequence is done with respect to the reference sequence to which it is aligned.

[0080] "Amino acid difference" and "residue difference" refer to a difference between an amino acid residue at a position in an amino acid sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of an amino acid difference is generally referred to herein as "Xn," where n refers to the corresponding position in the reference sequence that is the basis for the residue difference. For example, "a residue difference at position X123 compared to SEQ ID NO:686" refers to an amino acid residue difference at a polypeptide position that corresponds to position 123 in SEQ ID NO:686. Thus, if a reference polypeptide of SEQ ID NO:686 has a tyrosine at position 123, then "a residue difference at position X123 compared to SEQ ID NO:686" refers to an amino acid substitution of any residue other than tyrosine at the polypeptide position that corresponds to position 123 in SEQ ID NO:686. In most cases herein, a particular amino acid residue difference at a position is designated as "XnY," where "Xn" designates the corresponding residue and position in the reference polypeptide (as described above), and "Y" is a one-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs from the reference polypeptide). In some cases, the original amino acid is not indicated (e.g., 123F). In some instances (e.g., Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and 12-2), the disclosure also provides specific amino acid differences, designated with the conventional designation "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 engineered polypeptide sequence. In some instances, the polypeptides of the disclosure can include one or more amino acid residue differences relative to the reference sequence, which are indicated by a listing of the specific positions at which the residue difference relative to the reference sequence occurs. In some embodiments, when more than one amino acid can be used at a particular residue position in a polypeptide, the various amino acid residues that can be used are separated by a " / " (e.g., X123F / X123M / X123V or X123F / M / V or 123F / M / V). The present disclosure includes engineered polypeptide sequences that contain one or more amino acid differences, including either or both conservative and non-conservative amino acid substitutions.

[0081] The terms "amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within an amino acid sequence. In some embodiments, a substitution set includes 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions. In some embodiments, a substitution set refers to a set of amino acid substitutions present in any of the variant leucine decarboxylase polypeptides listed in any of the Tables in the Examples and Appendix (i.e., Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and 12-1).

[0082] "Conservative amino acid substitution" refers to the replacement of a residue with a different residue having a similar side chain, and thus typically includes the replacement of an amino acid in a polypeptide with an amino acid within the same or similar defined amino acid class. By way of example, and not limitation, an amino acid with an aliphatic side chain can be replaced with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be replaced with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid with an aromatic side chain can be replaced with another amino acid with an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be replaced with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain can be replaced with another amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid can be replaced with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions include replacing A, L, V, or I with other aliphatic residues (e.g., A, L, V, I) or other non-polar residues (e.g., A, L, V, I, G, M); replacing G or M with other non-polar residues (e.g., A, L, V, I, G, M); replacing D or E with other acidic residues (e.g., D, E); replacing K or R with other basic residues (e.g., K, R); replacing N, Q, S, or T with other polar residues (e.g., N, Q, S, T); replacing H, Y, W, or F with other aromatic residues (e.g., H, Y, W, F); or replacing C or P with other non-polar residues (e.g., C, P).

[0083] "Non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid that has significantly different side chain properties. Non-conservative substitutions use amino acids between, rather than within, defined groups and affect (a) the structure of the peptide backbone in the region of substitution (e.g., glycine for proline), (b) the charge or hydrophobicity, and / or (c) the bulk of the side chain. By way of example, and without limitation, examples of non-conservative substitutions include an acidic amino acid replaced with a basic or aliphatic amino acid; an aromatic amino acid replaced with a small amino acid; and a hydrophilic amino acid replaced with a hydrophobic amino acid.

[0084] "Deletion" refers to modifying a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removal of one or more amino acids, two or more amino acids, five or more amino acids, ten or more amino acids, fifteen or more amino acids, or twenty 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 that make up the reference enzyme, while maintaining the enzymatic activity and / or maintaining improved properties of the engineered leucine decarboxylase. Deletions can be directed to internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include contiguous segments or can be discontinuous.

[0085] "Insertion" refers to modifying a polypeptide by the addition of one or more amino acids from a reference polypeptide. Insertions can be made internal to the polypeptide, or at the carboxy- or amino-terminus. As used herein, insertions include fusion proteins, which are known in the art. Insertions can be contiguous segments of amino acids or can be separated by one or more amino acids in the native polypeptide.

[0086] The terms "functional fragment" and "biologically active fragment" are used interchangeably herein and refer to a polypeptide that has amino- and / or carboxy-terminal deletions and / or internal deletions, but the remaining amino acid sequence is identical to the corresponding positions in the compared sequence (e.g., a full-length engineered LDC of the invention), and that retains substantially all of the activity of the full-length polypeptide. In some embodiments, substantially all of the activity of the full-length polypeptide refers to at least 90% of the activity of the recombinant polypeptide from which it is derived.

[0087] An "isolated polypeptide" refers to a polypeptide that has been substantially separated from other contaminants (e.g., proteins, lipids, and polynucleotides) that naturally accompany it. The term encompasses a polypeptide that has been removed or purified from its naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant leucine decarboxylase polypeptides can be present within a cell, present in cell culture medium, or prepared in a variety of forms, such as a lysate or isolated preparation. Thus, in some embodiments, the recombinant leucine decarboxylase polypeptides provided herein are isolated polypeptides.

[0088] "Substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant molecular species present (i.e., on a molar or weight basis, the polypeptide species is more abundant than other individual macromolecular species in the composition), and is generally a substantially purified composition when the molecular species of interest constitutes at least about 50 percent, by molar or weight percent, of the molecular species present. Generally, a substantially pure leucine decarboxylase composition constitutes about 60 percent or more, about 70 percent or more, about 80 percent or more, about 90 percent or more, about 95 percent or more, and about 98 percent or more, by molar or weight percent, of all the molecular species present in the composition. In some embodiments, the molecular species of interest is purified to essential homogeneity (i.e., contaminant molecular species cannot be detected in the composition by conventional detection methods), and the composition consists essentially of a single macromolecular species. Solvent molecular species, small molecules (<500 Daltons), and elemental ion molecular species are not considered to be macromolecular species. In some embodiments, an isolated recombinant leucine decarboxylase polypeptide is a substantially pure polypeptide composition.

[0089] "Improved enzymatic properties" in the context of an engineered leucine decarboxylase polypeptide refers to any improved enzymatic property compared to a reference leucine decarboxylase polypeptide, such as a wild-type leucine decarboxylase polypeptide (e.g., wild-type LDC having SEQ ID NO:2) or another engineered leucine decarboxylase polypeptide. Improved properties include, but are not limited to, properties such as increased protein production, increased serum stability, increased in vivo serum half-life, increased thermal activity, increased thermostability, increased pH activity, increased stability, increased enzymatic activity, increased substrate specificity and / or affinity, increased specific activity, increased activity towards substrate and / or end-product inhibition, increased chemical stability, improved chemical selectivity, improved solvent stability, increased tolerance to acidic pH, increased tolerance to proteolytic activity (i.e., reduced susceptibility to proteolytic degradation), reduced aggregation, increased solubility, reduced immunogenicity (i.e., reduced ability to induce and / or elicit an immune response), and altered temperature profile.

[0090] "Increased enzymatic activity" and "enhanced catalytic activity" refer to improved properties of an engineered leucine decarboxylase polypeptide, which may be expressed by an increase in specific activity (e.g., product produced / time / weight of protein) and / or an increase in the rate of conversion of substrate to product (e.g., the rate of conversion of a starting amount of substrate to product in a specified time using a specified amount of leucine decarboxylase) compared to a reference leucine decarboxylase (e.g., a wild-type leucine decarboxylase and / or another engineered leucine decarboxylase). Exemplary methods for determining enzyme activity are provided in the Examples. Any property associated with enzyme activity may be affected, including classical enzyme properties of Km, Vmax, or kcat, which may be altered to increase enzyme activity. Improvements in enzymatic activity can range from about 1.1-fold the enzymatic activity of the corresponding wild-type enzyme to 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more, the enzymatic activity of the native leucine decarboxylase or other engineered leucine decarboxylase from which the leucine decarboxylase polypeptide is derived.

[0091] In some embodiments, the engineered leucine decarboxylase polypeptide has a specific activity of at least 0.01 μmol / min-mg, at least 0.02 / μmol / min-mg, at least 0.03 / μmol / min-mg, at least 0.05 / μmol / min-mg, at least 1.0 / μmol / min-mg, and in some preferred embodiments, greater than 2.0 / μmol / min-mg. In some embodiments, the Km is in the range of about 1 μm to about 5 mM, in the range of about 5 μm to about 2 mM, in the range of about 10 μm to about 2 mM, or in the range of about 10 μm to about 1 mM. In some specific embodiments, the engineered leucine decarboxylase has improved enzymatic activity in the range of 1.5-10 fold, 1.5-25 fold, 1.5-50 fold, 1.5-100 fold or more over a reference leucine decarboxylase. Leucine decarboxylase activity can be measured by any standard assay known in the art (e.g., by tracking reactant depletion or product generation). In some embodiments, the amount of product produced or the amount of substrate consumed is measured by high performance liquid chromatography (HPLC) separation combined with UV absorption or mass spectrometry detection. In some embodiments, the comparison of enzyme activity is performed using a defined enzyme preparation, a defined assay under set conditions, and one or more defined substrates, as described in more detail herein. In general, when comparing lysates, the cell number and the amount of protein assayed are determined, and the same expression system and the same host cells are used to minimize the variation in the amount of enzyme produced by the host cells and present in the lysate.

[0092] The phrase "improved storage stability" means that the engineered leucine decarboxylase polypeptides of the invention retain more activity in standard assays (e.g., as described in the Examples) compared to a reference leucine decarboxylase after being produced in a dried form (e.g., lyophilized or spray dried) and stored at temperatures above room temperature (e.g., 30° C., 37° C., 45° C., 55° C., etc.) for periods ranging from several days to several months.

[0093] "Conversion" refers to the enzymatic conversion (or biotransformation) of a substrate to a corresponding product. "Percent conversion" refers to the percent of a substrate that is converted to a product in a given period of time under specified conditions. Thus, the "enzyme activity" or "activity" of a leucine decarboxylase polypeptide can be expressed as the "percent conversion" of substrate converted to product in a particular period of time.

[0094] "Stringency of hybridization" refers to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under lower stringency conditions, followed by varying but higher stringency washing. The term "moderate stringency hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60%, preferably about 75%, about 85% identity to the target DNA and have more than about 90% identity to the target polynucleotide. Exemplary medium stringency conditions are those that correspond to hybridization in 50% formamide, 5x Denhart's solution, 5x SSPE, 0.2% SDS at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS at 42°C. "Stringency hybridization" generally refers to conditions that are about 10°C or less from the thermal melting temperature Tm determined under solution conditions of a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only those nucleic acid sequences that form stable hybrids at 65°C in 0.018M NaCl (i.e., if a hybrid is not stable at 65°C in 0.018M NaCl, it is not stable under high stringency conditions as envisaged herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS at conditions equivalent to 42°C, followed by washing in 0.1x SSPE, 0.1% SDS at 65°C. Another high stringency condition is hybridization in 5x SSC with 0.1% (w:v) SDS at 65°C, followed by washing in 0.1x SSC with 0.1% SDS at 65°C. Other high stringency hybridization conditions, as well as moderate stringency conditions, are described in the references cited above. In some embodiments, a polynucleotide encoding a leucine decarboxylase hybridizes under high stringency conditions to a recombinant polynucleotide disclosed herein encoding an engineered decarboxylase polypeptide.

[0095] "Codon optimization" refers to changing the codons of a polynucleotide encoding a protein to those that are preferentially used in a particular organism, so that the encoded protein is more efficiently expressed in that organism. Although the genetic code is degenerate, with most amino acids being represented by multiple codons, referred to as "synonymous" or "synonymous" codons, it is well known that codon usage by a particular organism is not random, but is biased toward certain codon triplets. This bias in codon usage can be high for certain genes, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and aggregated protein-coding regions of an organism's genome. In some embodiments, the polynucleotide encoding leucine decarboxylase is codon-optimized for optimal production from the host organism selected for expression. "Control sequence" is used herein to mean to include all components necessary or advantageous for expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, a polyadenylation sequence, a propeptide sequence, a promoter sequence, a signal peptide sequence, an initiation sequence, and a transcription terminator. Control sequences include at least a promoter, and transcriptional and translational stop signals. In some embodiments, the control sequences are provided with linkers for the purpose of introducing specific restriction sites that facilitate ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.

[0096] "Operably linked" is defined herein as a configuration in which a control sequence is appropriately positioned (i.e., in functional relationship) with a polynucleotide of interest such that the control sequence directs or controls the expression of a polynucleotide encoding a polypeptide of interest.

[0097] "Promoter sequence" refers to a nucleic acid sequence recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence includes transcriptional control sequences that mediate expression of the polynucleotide of interest. A promoter is any nucleic acid sequence that exhibits transcriptional activity in a selected host cell, including mutant, truncated, and hybrid promoters, and can be obtained from genes encoding extracellular or intracellular polypeptides that are homologous or heterologous to the host cell.

[0098] A "substrate" in an enzymatic conversion reaction process refers to a compound or molecule on which a leucine decarboxylase polypeptide acts. A "product" in an enzymatic conversion process refers to a compound or molecule that is produced by the action of a leucine decarboxylase polypeptide on a substrate.

[0099] As used herein, the term "culturing" refers to the propagation of a population of cells under appropriate conditions using any appropriate medium (eg, liquid, gel, or solid).

[0100] Recombinant polypeptides (e.g., LDC enzyme variants) can be generated using any suitable method known in the art. For example, there are a wide variety of mutagenesis techniques well known to those of skill in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for making specific substitutions at specific amino acids (site-directed), specific or random mutations in local regions of a gene (region-directed), or random mutagenesis throughout the gene (e.g., saturation mutagenesis). Many suitable methods for generating enzyme variants are known to those of skill in the art and include, but are not limited to, site-directed mutagenesis of single- or double-stranded DNA using PCR, cassette mutagenesis, gene synthesis, error-prone PCR, shuffling, and chemical saturation mutagenesis, or other suitable methods known in the art. Non-limiting examples of methods used in DNA and protein engineering are provided in the following patents: U.S. Patent No. 6,117,679, U.S. Patent No. 6,420,175, U.S. Patent No. 6,376,246, U.S. Patent No. 6,586,182, U.S. Patent No. 7,747,391, U.S. Patent No. 7,747,393, U.S. Patent No. 7,783,428, and U.S. Patent No. 8,383,346. After variants are generated, they can be screened for any desired property (e.g., high or increased activity, or low or decreased activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.).

[0101] The term "suitable reaction conditions" as used herein refers to conditions in an enzymatic conversion reaction solution (e.g., enzyme loading, substrate loading, temperature, pH, buffer, co-solvent ranges) that allow the leucine decarboxylase polypeptide of the present application to convert a substrate into a desired product compound. Exemplary "suitable reaction conditions" are provided in the present application and illustrated by the Examples. "Load," such as "compound loading" or "enzyme loading," refers to the concentration or amount of a component in a reaction mixture at the start of the reaction. "Substrate" in the context of an enzymatic conversion reaction process refers to a compound or molecule that is acted upon by a leucine decarboxylase polypeptide. "Product" in the context of an enzymatic conversion process refers to a compound or molecule that is generated by the action of a leucine decarboxylase polypeptide on a substrate.

[0102] As used herein, "vector" refers to a DNA construct for introducing DNA sequence into cells.In some embodiments, vector is an expression vector that is operably linked to a suitable control sequence that can express the polypeptide encoded by the DNA sequence in a suitable host.In some embodiments, "expression vector" has a promoter sequence operably linked to a DNA sequence (e.g., a transgene) to drive expression in a host cell, and in some embodiments, also includes a transcription terminator sequence.

[0103] As used herein, the term "expression" includes all steps 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 includes secretion of the polypeptide from the cell.

[0104] As used herein, an amino acid or nucleotide sequence (e.g., promoter sequence, signal peptide, terminator sequence) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature.

[0105] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA provided herein (e.g., a polynucleotide sequence encoding at least one LDC variant). In some embodiments, a host cell is a prokaryotic or eukaryotic cell that is transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0106] The term "analog" refers to a polypeptide having greater than 70% sequence identity, but less than 100% sequence identity (e.g., greater than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide. In some embodiments, analog refers to unnatural amino acid residues, including but not limited to homoarginine, ornithine, norvaline, as well as natural amino acids. In some embodiments, analogs also include one or more D-amino acid residues and non-peptide bonds between two or more amino acid residues.

[0107] The term "therapeutic" refers to a compound that is administered to a subject who exhibits signs or symptoms of a medical condition to produce a beneficial or desired medical effect.

[0108] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., a human) comprising a pharma- ceutical effective amount of an engineered leucine decarboxylase polypeptide encompassed by the present invention and an acceptable carrier.

[0109] The term "gene therapy" is used to refer to the use of genes (i.e., genetic material) to treat and / or prevent disease in a mammalian subject (e.g., a human). In some embodiments, the genetic material is directly introduced into at least some cells of the mammalian subject. It is not intended that the present invention be limited to particular methods or compositions useful for gene therapy.

[0110] The term "mRNA therapy" is used in reference to the use of messenger RNA (mRNA) to treat and / or prevent disease in a mammalian subject (e.g., a human). In some embodiments, the genetic material is directly introduced into at least some cells of the mammalian subject. It is not intended that the present invention be limited to a particular method or composition useful for mRNA therapy.

[0111] The term "effective amount" means an amount sufficient to produce a desired result. Those skilled in the art can determine the effective amount in light of the guidance of the specification.

[0112] The terms "isolated" and "purified" are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that has been separated from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but is intended as a relative definition.

[0113] The term "subject" includes mammals, such as humans, non-human primates, farm animals, pet animals, and laboratory animals (e.g., rodents and lagomorphs). The term is intended to include both males and females.

[0114] As used herein, the term "patient" refers to any subject being evaluated for, treated for, or experiencing a disease.

[0115] The term "infant" refers to a child from 1 month to approximately 1 year of age. As used herein, the term "newborn" refers to a child from birth to 28 days after birth. The term "premature infant" refers to an infant born after the 20th week of gestation but before full term, typically weighing between about 500 grams and about 2499 grams at birth. A "very low birth weight infant" is an infant weighing less than 1500 grams at birth.

[0116] The term "child" as used herein refers to a person who has not reached the legal age to consent to a therapeutic or research procedure. In some embodiments, the term refers to a person from birth to adolescence.

[0117] As used herein, the term "adult" refers to a person who has reached the legal age of the relevant jurisdiction (e.g., 18 years old in the United States). In some embodiments, the term refers to a fully grown, mature adult. In some embodiments, the term "young adult" refers to a person who is under 18 years old but is sexually mature.

[0118] As used herein, "composition" and "formulation" encompass products comprising at least one engineered leucine decarboxylase of the invention and intended for any suitable use (e.g., pharmaceutical compositions, dietary / nutritional supplements, feeds, etc.).

[0119] The terms "administration" and "administering" a composition refer to providing a composition of the invention to a subject (eg, a person suffering from the effects of an MSUD).

[0120] The term "carrier" as used with respect to pharmaceutical compositions means any of the standard pharmaceutical carriers, buffers, excipients such as stabilizers, preservatives, adjuvants, and the like.

[0121] The term "pharmaceutical acceptable" means a material that may be administered to a subject without causing undesired biological effects or interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0122] As used herein, the term "excipient" refers to a pharma- ceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API, e.g., an engineered leucine decarboxylase polypeptide of the invention). Excipients are typically included for formulation and / or administration purposes.

[0123] The term "therapeutically effective amount" as used in reference to symptoms of a disease / condition refers to an amount and / or concentration of a compound (e.g., an engineered LDC polypeptide) that relieves, reduces, or eliminates one or more symptoms of a disease / condition, or prevents or delays the onset of a symptom (e.g., MSUD). In some embodiments, the term is used in reference to the amount of a composition that elicits a biological (e.g., medical) response by a tissue, system, or animal subject that is desired by a researcher, physician, veterinarian, or other clinician.

[0124] The term "therapeutically effective amount" as used in reference to a disease / condition refers to an amount and / or concentration of a composition that alleviates, reduces, or eliminates the disease / condition.

[0125] The terms "treating," "treat," and "treatment" are intended to include preventive (eg, prophylactic) and palliative treatment.

[0126] As used herein, the term "at least one" does not limit the invention to a particular number of items, but is intended to encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more items, as appropriate.

[0127] Engineered LDC Polypeptides The present disclosure provides engineered leucine decarboxylases (LDCs) and their uses as pharmaceuticals, nutraceuticals, and / or industrial applications. In this disclosure, when a particular leucine decarboxylase variant (i.e., an engineered LDC polypeptide) is referred to in connection with a modification of a particular amino acid residue in the sequence of a wild-type leucine decarboxylase or a reference leucine decarboxylase, it is understood that a variant of another leucine decarboxylase modified at the equivalent position (as determined from an alignment of the amino acid sequences between the respective amino acid sequences) is also included herein.

[0128] In some embodiments, the parent leucine decarboxylase polypeptide from which the engineered leucine decarboxylase polypeptides of the invention are derived includes bacterial strains, such as Planctomycetaceae bacteria. Leucine decarboxylases from various sources applicable for use herein are shown in Tables 1-2.

[0129] In some embodiments, an engineered leucine decarboxylase polypeptide is produced by culturing a cell or microorganism comprising at least one polynucleotide sequence encoding at least one engineered leucine decarboxylase polypeptide under conditions suitable for the production of the engineered leucine decarboxylase polypeptide, in some embodiments, the engineered leucine decarboxylase polypeptide is then recovered from the resulting culture medium and / or cells.

[0130] In some embodiments, an engineered leucine decarboxylase polypeptide of the disclosure comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828 or 888, wherein the amino acid sequence comprises one or more substitutions in the amino acid sequence.

[0131] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO:828, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:828.

[0132] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828.

[0133] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0134] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitutions 5V, 19L, 33L, 41D, 47F, 51E, 55I, 64S / N, 141P, 170P, 173I, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 290I, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0135] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0136] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitutions 33L, 55I, 64N, 126A, 270L, or 357S, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0137] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least substitutions at positions 33, 55, 64, 126, 270, and 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0138] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitutions 33L, 55I, 64N, 126A, 270L, and 357S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0139] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 141 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 272 / 383, 41 / 141 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 126 / 267, 64 / 267 / 353 / 384, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 19 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 19 / 55 / 64 / 126 / 267 / 270 / 353, 19 / 33 / 126 / 2 and / or a substitution or set of substitutions at 70 / 353 / 357 / 384, 19 / 33 / 64 / 267 / 353, 126 / 353 / 384, 126 / 270 / 312 / 353 / 384, 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0140] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 170P / 270L / 383S, 270L, 41D / 173I, 272A, 5V / 141P / 272A / 383S, 41D / 383S, 41D / 141P / 187L / 272A / 290I, 41D / 141P / 173I / 290I, 5V / 272A / 383S, 5V / 41D / 173I / 272A / 383S, 41D / 141P, 141P / 272A, 353E / 384W, 272A / 383S, 41D / 141P / 173I, 41D / 272A / 383S, 41D / 141P / 187L / 200S / 202H / 272A, 33L / 55I / 64N / 126A / 270L / 357S, 33L / 126A / 353E / 357S , 55I / 64N / 267L / 353E / 384W, 33L / 64N / 357S, 126A / 267L, 64N / 267L / 353E / 384W, 33L / 55I / 64N / 357S, 19L / 64N / 126A / 267L, 55I / 267L, 33 L / 126A / 267L / 270T / 312T / 357S, 19L / 33L / 55I / 353E / 357S / 384W, 19L / 33L / 126A, 126A / 312T, 126A / 198G / 202H / 267L / 312T, 126A / 353E, 55I / 126A, 126A / 270T / 384W, 33L / 64N / 353E / 357S, 19L / 267L, 51E / 55I / 267L / 270T / 353E, 33L / 126A / 267L / 270T, 19L / 55I / 64N / 126A / 26 and 7L / 270T / 353E, 19L / 33L / 126A / 270T / 353E / 357S / 384W, 19L / 33L / 64N / 267L / 353E, 126A / 353E / 384W, 126A / 270T / 312T / 353E / 384W, 19L / 33L / 55I / 126A, 33L / 357S, 47F / 51E / 64N / 126A / 353E / 384W, 126A, or 126A / 270T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0141] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions A170P / R270L / A383S, R270L, H41D / F173I, T272A, K5V / R141P / T272A / A383S, H41D / A383S, H41D / R141P / V187L / T272A / V290I, H41D / R141P / F173I / V290I, K5V / T272A / A383S, K5V / H41D / F173I / T272A / A383S, H41D / R141P, R141P / T272A, D3 53E / P384W, T272A / A383S, H41D / R141P / F173I, H41D / T272A / A383S, H41D / R 141P / V187L / H200S / S202H / T272A, F33L / V55I / S64N / D126A / R270L / C357S, F 33L / D126A / D353E / C357S, V55I / S64N / I267L / D353E / P384W, F33L / S64N / C3 57S, D126A / I267L, S64N / I267L / D353E / P384W, F33L / V55I / S64N / C357S, I19 L / S64N / D126A / I267L, V55I / I267L, F33L / D126A / I267L / R270T / A312T / C35 7S, I19L / F33L / V55I / D353E / C357S / P384W, I19L / F33L / D126A, D126A / A312T , D126A / A198G / S202H / I267L / A312T, D126A / D353E, V55I / D126A, D126A / R2 70T / P384W, F33L / S64N / D353E / C357S, I19L / I267L, L51E / V55I / I267L / R270 T / D353E, F33L / D126A / I267L / R270T, I19L / V55I / S64N / D126A / I267L / R270 T / D353E, I19L / F33L / D126A / R270T / D353E / C357S / P384W, I19L / F33L / S64N / I267L / D353E, D126A / D353E / P384W, D126A / R270T / A312T / D353E / P384W, I1 9L / F33L / V55I / D126A, F33L / C357S, L47F / L51E / S64N / D126A / D353E / P384W,D126A, or D126A / R270T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828.

[0142] In some embodiments, the engineered leucine decarboxylase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence contains one or more substitutions relative to a reference sequence corresponding to SEQ ID NO: 888.

[0143] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888.

[0144] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises amino acid residues 5V, 19L, 33L, 41D, 47F, 51E, 55I, 64S / N, 141P, 170P, 173I, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 290I, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0145] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 202 / 357, 202 / 353, 5 / 51 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 3 53, 357, 64, 5 / 41 / 141, 41 / 141 / 173 / 202 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 64 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 3 53 / 357, 5 / 41 / 64 / 173 / 353 / 357, 64 / 173 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 202 / 272 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 3 53, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / 173 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202, or 5 / 173 / 272, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888.

[0146] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 64S / 173I / 202H / 353E / 384W, 41D / 141P / 272A / 353E, 141P / 202H / 272A / 353E / 357C, 173I / 202H / 357C, 202H / 353E, 5V / 51E / 173I / 272A / 353E / 384W, 51E / 202H / 272A / 357C, 141P / 173I / 272A, 272A, 41D / 173I / 384W, 41D / 64S / 141P / 353E / 35 7C / 383S, 141P / 173I / 202H, 5V / 51E / 64S / 202H / 353E, 357C, 64S, 5V / 41D / 14 1P, 41D / 141P / 173I / 202H / 353E, 353E, 202H / 357C, 51E / 141P / 202H / 272A / 35 3E, 202H, 51E / 141P / 173I / 353E / 384W, 41D / 141P / 173I / 202H / 272A / 353E / 3 83S / 384W, 64S / 202H / 357C, 5V / 64S / 353E / 383S / 384W, 41D / 272A / 353E / 383S , 41D / 173I / 272A / 353E / 357C, 51E / 141P / 272A / 353E / 357C / 383S / 384W, 41D / 353E / 357C, 173I / 272A / 353E / 357C, 5V / 41D / 64S / 173I / 353E / 357C, 64S / 17 3I / 357C, 51E / 272A, 51E / 64S / 357C / 384W, 51E / 141P / 173I / 272A / 353E, 64S / 202H / 272A / 353E / 357C / 384W, 51E / 272A / 357C, 51E / 173I / 272A / 353E / 384W , 353E / 384W, 202H / 272A / 357C, 64S / 141P / 173I / 202H / 353E / 357C, 5V / 41D / 51E / 202H / 357C / 383S, 5V / 51E / 173I / 272A / 383S, 41D / 141P / 272A, 51E / 173I , 5V / 353E, 41D / 64S / 173I / 272A / 353E / 383S, 5V / 64S / 173I / 272A / 353E, 51E / 64S, 41D / 357C / 383S, 41D / 173I / 353E / 357C, 202H / 272A / 383S, 202H / 272A,353E / 357C, 41D / 173I / 202H / 272A / 357C, 141P / 173I / 202H / 272A / 353E / 357C, 64S / 141P / 202H, 5V / 173I / 272A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0147] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions N64S / F173I / S202H / D353E / P384W, H41D / R141P / T272A / D353E, R141P / S202H / T272A / D353E / S357C, F173I / S202H / S357C, S202H / D353E, K5V / L51E / F173I / T272A / D353E / P384W, L51E / S202H / T272A / S357C, R141P / F173I / T272A, T272A, H41D / F173I / P384W, H41D / N64S / R141P / D353E / S357C / A383S, R141P / F173I / S202H, K5V / L51E / N64S / S202H / D353E, S357C, N64S, K5V / H41D / R141P, H41 D / R141P / F173I / S202H / D353E, D353E, S202H / S357C, L51E / R141P / S202H / T 272A / D353E, S202H, L51E / R141P / F173I / D353E / P384W, H41D / R141P / F173I / S202H / T272A / D353E / A383S / P384W, N64S / S202H / S357C, K5V / N64S / D353E / A383S / P384W, H41D / T272A / D353E / A383S, H41D / F173I / T272A / D353E / S357 C, L51E / R141P / T272A / D353E / S357C / A383S / P384W, H41D / D353E / S357C, F1 73I / T272A / D353E / S357C, K5V / H41D / N64S / F173I / D353E / S357C, N64S / F17 3I / S357C, L51E / T272A, L51E / N64S / S357C / P384W, L51E / R141P / F173I / T27 2A / D353E, N64S / S202H / T272A / D353E / S357C / P384W, L51E / T272A / S357C, L 51E / F173I / T272A / D353E / P384W, D353E / P384W, S202H / T272A / S357C, N64S / R141P / F173I / S202H / D353E / S357C, K5V / H41D / L51E / S202H / S357C / A383S,K5V / L51E / F173I / T272A / A383S, H41D / R141P / T272A, L51E / F173I, K5V / D353E, H41D / N64S / F173I / T272A / D35 3E / A383S, K5V / N64S / F173I / T272A / D353E, L51E / N64S, H41D / S357C / A383S, H41D / F173I / D353E / S357C, S202H / T272A / A383S, S202H / T272A, D353E / S357C, H41D / F173I / S202H / T272A / S357C, R141P / F173I / S202H / T272A / D353E / S357C, N64S / R141P / S202H, K5V / F173I / T272A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0148] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence that comprises a variant substitution or set of substitutions provided in Tables 12-1 and 12-2, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO:12.

[0149] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising a substitution at at least one amino acid position provided in Tables 12-1 and 12-2.

[0150] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least one substitution provided in Tables 12-1 and 12-2.

[0151] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence that includes at least a substitution or set of substitutions of a variant provided in Tables 12-1 and 12-2, wherein the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO: 828 or 888.

[0152] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising the sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.

[0153] In some embodiments, the engineered leucine decarboxylase polypeptide is an even-numbered SEQ ID NO: 854-1064 (e.g., 854, 856, 858, 860, 862, 864, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 90 2, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980 , 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046 , 1048, 1050, 1052, 1054, 1056, 1058, 1060, 1062, or 1064). In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence that has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO:854-948. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO:950-1604.

[0154] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 854-1064. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 854-1064, wherein the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 854-948. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 854-948, wherein the polypeptide optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 950-1604. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even numbered SEQ ID NO: 950-1604, wherein the polypeptide optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence. In some embodiments, the engineered leucine decarboxylase polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence. In some embodiments, the engineered leucine decarboxylase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence. In some embodiments, the substitutions comprise non-conservative or conservative substitutions. In some embodiments, the substitutions comprise conservative substitutions. In some embodiments, the substitutions comprise non-conservative and conservative substitutions. In some embodiments, guidance regarding non-conservative and conservative substitutions is provided by the variants disclosed herein.

[0155] In some embodiments, the engineered leucine decarboxylase polypeptides of the disclosure exhibit one or more improved properties compared to a wild-type Planctomycetaceae species leucine decarboxylase or a leucine decarboxylase having a sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptides exhibit an improved property selected from: (i) increased activity towards leucine, (ii) increased resistance to proteolysis, increased tolerance to low pH environments, and (iii) increased thermostability compared to a wild-type Planctomycetaceae species leucine decarboxylase or a leucine decarboxylase having a sequence corresponding to SEQ ID NO: 12.

[0156] It should be apparent that the examples provided in Tables 12-1 and 12-2, and Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and 11-2, as well as the related activities disclosed in International Publication No. WO2021158686 (herein incorporated by reference), provide sequence / structure information correlating specific amino acid sequence features with functional activity of engineered leucine decarboxylase polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences relative to a reference engineered polypeptide of SEQ ID NO: 12, as well as in the form of associated experimentally determined activity data of exemplary engineered leucine decarboxylase polypeptides, thereby providing guidance for the preparation of engineered leucine decarboxylase polypeptides having the enzymatic properties described herein.

[0157] In some embodiments, the engineered leucine decarboxylase polypeptide is purified. In some embodiments, the engineered leucine decarboxylase polypeptide has leucine decarboxylase activity, particularly with improved or enhanced properties as described herein.

[0158] In some embodiments, the present invention provides functional or biologically active fragments of engineered leucine decarboxylase polypeptides. In some embodiments, a functional fragment comprises at least about 90%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the activity of the engineered leucine decarboxylase polypeptide from which it is derived (i.e., the parent engineered LDC). In some embodiments, the functional fragment comprises at least about 90%, at least about 91%, at least about 92%, at least about 93%, at least about 94%, at least about 95%, at least about 96%, at least about 97%, at least about 98%, or at least about 99% of the parent sequence of engineered leucine decarboxylase. In some embodiments, the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.

[0159] In some embodiments, the functional fragment comprises at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered leucine decarboxylase. In some embodiments, the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.

[0160] In some embodiments, a functional or biologically active fragment of an engineered leucine decarboxylase polypeptide described herein comprises at least one mutation or set of mutations in the amino acid sequence of an engineered leucine decarboxylase described herein. Thus, in some embodiments, a functional or biologically active fragment of an engineered leucine decarboxylase exhibits enhanced or improved properties associated with the mutation or set of mutations in the parent leucine decarboxylase.

[0161] Polynucleotides encoding engineered polypeptides, expression vectors, and host cells In another aspect, the disclosure provides engineered or recombinant polynucleotides encoding the engineered leucine decarboxylase polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences capable of controlling gene expression to express the polypeptide. In some embodiments, an expression construct comprising at least one heterologous polynucleotide encoding an engineered leucine decarboxylase polypeptide is introduced into a suitable host cell to express the corresponding leucine decarboxylase polypeptide.

[0162] As will be apparent to one of skill in the art, the availability of protein sequences and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding a polypeptide of interest. The degeneracy of the genetic code, in which the same amino acid is coded for by alternative or synonymous codons, allows for the creation of an extremely large number of nucleic acids, all of which code for engineered leucine decarboxylase polypeptides. Thus, the present invention provides methods and compositions for producing each and every variation of leucine decarboxylase polynucleotides that can be made that encode the leucine decarboxylase polypeptides described herein by selecting combinations based on possible codon choices, and all such variations of polynucleotides are considered to be specifically disclosed for any polypeptide described herein, including the amino acid sequences shown in the Examples (e.g., Tables 12-1 and / or 12-2).

[0163] In some embodiments, the codons are preferably optimized for utilization by the host cell selected for protein production. For example, preferred codons used in bacteria are typically used for bacterial expression. As a result, a codon-optimized polynucleotide encoding an engineered leucine decarboxylase polypeptide contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions within the full-length coding region.

[0164] In some embodiments, the recombinant polynucleotide encodes an engineered polypeptide having leucine decarboxylase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein the amino acid sequence contains one or more substitutions relative to the amino acid sequence of the reference sequence corresponding to SEQ ID NO: 828 or 888 or any variant disclosed in the Examples.

[0165] In some embodiments, a recombinant polynucleotide encodes an engineered polypeptide having leucine decarboxylase activity having the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence SEQ ID NO:888.

[0166] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence, SEQ ID NO:828, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:828.

[0167] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO:888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:828.

[0168] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0169] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0170] In some embodiments, the recombinant polynucleotide is selected from the group consisting of amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 141 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 27 2 / 383, 41 / 141 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 126 / 267, 64 / 267 / 353 / 384, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 19 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 19 / 55 / 64 / 126 / 267 / 270 / 353, 19 / 33 / 126 / 270 / 353 / 357 / 384, 19 / 33 / 64 / 267 / 35 3, 126 / 353 / 384, 126 / 270 / 312 / 353 / 384, 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0171] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:888, wherein the amino acid sequence is relative to a reference sequence corresponding to SEQ ID NO:888.

[0172] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase comprising an amino acid sequence comprising at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888.

[0173] In some embodiments, the recombinant polynucleotide is selected from the group consisting of amino acid positions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 202 / 357, 202 / 353, 5 / 51 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 353, 357, 64, 5 / 41 / 141, 4 1 / 141 / 173 / 202 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 64 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 353 / 357, 5 / 41 / 64 / 173 / 353 / 35 7, 64 / 173 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 202 / 272 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 353, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / and / or 5 / 173 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202, or 5 / 173 / 272, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888.

[0174] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence that includes a variant substitution or set of substitutions provided in Tables 12-1 and 12-2, where the substitution or set of substitutions is relative to a reference sequence corresponding to SEQ ID NO:12.

[0175] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising a substitution at at least one amino acid position provided in Tables 12-1 and 12-2.

[0176] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence containing at least one substitution provided in Tables 12-1 and 12-2.

[0177] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or set of substitutions provided in Tables 12-1 and 12-2, wherein the substitution or set of substitutions is relative to SEQ ID NO: 828 or 888.

[0178] In some embodiments, the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide that comprises an amino acid sequence that includes the sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.

[0179] In some embodiments, the recombinant polynucleotide comprises an even-numbered SEQ ID NO: 854 to 1064 (e.g., 854, 856, 858, 860, 862, 864, 868, 870, 872, 874, 876, 878, 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 91 0, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992 , 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022, 1024, 1026, 1028, 1030, 1032, 1034, 1036, 1038, 1040, 1042, 1044, 1046, 1048, 1050, 1052, 1054, 1056, 1058, 10 In one embodiment, the present invention encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to any one of the sequences set forth in any of the following reference sequences:

[0180] In some embodiments, a recombinant polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference polypeptide sequence corresponding to SEQ ID NO: 827 or 887, wherein the recombinant polynucleotide encodes a polypeptide having leucine decarboxylase activity.

[0181] In some embodiments, the recombinant polynucleotide comprises an odd-numbered sequence among SEQ ID NOs: 853 to 1063 (e.g., 853, 855, 857, 859, 861, 863, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 957, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997 , 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 1017, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, or 1063) In one embodiment, the present invention relates to a nucleic acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence, wherein the polynucleotide encodes a polypeptide having leucine decarboxylase activity.

[0182] In some embodiments, the recombinant polynucleotide is selected from the odd SEQ ID NOs: 853 to 1063 (e.g., 853, 855, 857, 859, 861, 863, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911, 913, 915, 917, 919, 921, 923, 925, 927, 929, 931, 933, 935, 937, 939, 941, 943, 945, 947, 949, 951, 953, 955, 956, 957, 958, 959, 960, 961, 962, 963, 964, 965, 966, 967, 968, 969, 970, 971, 972, 973, 974, 975, 976, 977, 978, 979, 980, 981, 982, 983, 984, 985, 986, 987, 988, 989, 990, 991, 992, 993, 994, 995, 996, 997, 998, 999, 57, 959, 961, 963, 965, 967, 969, 971, 973, 975, 977, 979, 981, 983, 985, 987, 989, 991, 993, 995, 997, 999, 1001, 1003, 1005, 1007, 1009, 1011, 1013, 1015, 101 7, 1019, 1021, 1023, 1025, 1027, 1029, 1031, 1033, 1035, 1037, 1039, 1041, 1043, 1045, 1047, 1049, 1051, 1053, 1055, 1057, 1059, 1061, or 1063).

[0183] In some embodiments, the recombinant polynucleotide is capable of hybridizing under high stringency conditions to a reference polypeptide sequence encoding an engineered leucine decarboxylase polypeptide. In some embodiments, the reference sequence is selected from SEQ ID NO: 827 or 887, or a complement thereof, or a polynucleotide sequence encoding any of the engineered leucine decarboxylase polypeptides provided herein. In some embodiments, the recombinant polynucleotide capable of hybridizing under high stringency conditions encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NO: 828 or 888 at a residue position selected from any of the positions set forth in Tables 12-1 and 12-2. In some further embodiments, the recombinant polynucleotide comprises a polynucleotide that can hybridize under high stringency conditions to a reference recombinant polynucleotide selected from those provided in Tables 12-1 and 12-2, or has at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence corresponding to SEQ ID NO: 827 or 887. In some additional embodiments, a recombinant polynucleotide that hybridizes under high stringency conditions comprises a sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one polynucleotide reference sequence provided in Table 12-1 and Table 12-2 (e.g., a nucleic acid sequence comprising an odd-numbered SEQ ID NO: 853-1063), wherein the recombinant polynucleotide encodes a polypeptide having leucine decarboxylase activity.

[0184] In some embodiments, an isolated polynucleotide encoding any of the engineered leucine decarboxylase polypeptides described herein is engineered in various ways to facilitate expression of the leucine decarboxylase polypeptide. In some embodiments, the polynucleotide encoding the leucine decarboxylase polypeptide comprises an expression vector in which one or more control sequences are present to regulate expression of the leucine decarboxylase polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to insertion into a vector may be desirable or necessary depending on the expression vector used. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, the control sequences include, inter alia, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators. In some embodiments, an appropriate promoter is selected based on the choice of the host cell.For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the disclosure include, but are not limited to, the lac operon of E. coli, the agarase gene (dagA) of Streptomyces coelicolor, the levansucrase gene (sacB) of Bacillus subtilis, the alpha-amylase gene (amyL) of Bacillus licheniformis, the maltose amylase gene (amyM) of Bacillus stearothermophilus, the maltose amylase gene (amyM) of Bacillus amyloliquefaciens, and the alpha-amylase gene (amyL) of Bacillus stearothermophilus. Examples of promoters that can be used include promoters derived from the alpha-amylase gene (amyQ) of Bacillus amyloliquefaciens, the penicillinase gene (penP) of Bacillus licheniformis, the xylA and xylB genes of Bacillus subtilis, and prokaryotic beta-lactamase genes (see, e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci. USA, 1978, 75:3727-3731), and the tac promoter (see, e.g., DeBoer et al., Proc. Natl Acad. Sci. USA, 1983, 80: 21-25).Exemplary promoters for filamentous fungal host cells include, but are not limited to, promoters encoding Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid-stable alpha-amylase, Aspergillus Awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum acetamidase. Examples of yeast cell promoters include the trypsin-like protease from Saccharomyces oxysporum (see, for example, WO 96 / 00787), and the NA2-tpi promoter (a hybrid of promoters from the genes for neutral alpha-amylase from Saccharomyces niger and triosephosphate isomerase from Aspergillus oryzae), as well as mutant, truncated, and hybrid promoters thereof. Examples of yeast cell promoters are obtained from the genes for enolase from Saccharomyces cerevisiae (ENO-1), galactokinase from Saccharomyces cerevisiae (GAL1), alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase from Saccharomyces cerevisiae (ADH2 / GAP), and 3-phosphoglycerate kinase from Saccharomyces cerevisiae. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast, 1992, 8:423-488).

[0185] In some embodiments, the control sequence is also 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' end of the nucleic acid sequence encoding the leucine decarboxylase polypeptide. Any suitable terminator that functions in the selected host cell can be used in the present invention. For bacterial expression, the transcription terminator can be a Rho-dependent terminator that relies on Rho transcription factors, or a Rho-independent or intrinsic terminator that does not require transcription factors. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813. Examples of transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus nidulans glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus nidulans alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Examples of terminators 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).

[0186] In some embodiments, the control sequence is also 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' end of the nucleic acid sequence encoding the leucine decarboxylase polypeptide. Any suitable leader sequence that functions in the selected host cell finds use in the present invention. Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase and 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).

[0187] In some embodiments, the control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' end of a nucleic acid sequence that, upon transcription, is recognized by a host cell as a signal for adding polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that functions in a selected host cell finds use in the present invention. Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to, the genes for Aspergillus oryzae TAKA amylase, Aspergillus nidulans glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha-glucosidase. Useful polyadenylation sequences for yeast host cells are known (see, e.g., Guo and Sherman, Mol. Cell. Bio., 1995, 15:5983-5990).

[0188] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region that encodes an amino acid sequence linked to the amino terminus of a polypeptide and that directs the encoded polypeptide into the secretory pathway of a cell). In some embodiments, the 5' end of the coding sequence of the nucleic acid sequence essentially 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 unrelated to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of the selected host cell is used in expressing the engineered polypeptide. Signal peptide coding regions useful in bacterial host cells include, but are not limited to, the signal peptide coding regions obtained from the genes for Bacillus NClB11837 maltogenic amylase, Bacillus stearothermophilus alpha amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral protease (nprT, nprS, nprM), and Bacillus subtilis prsA. Additional signal peptides are known in the art (see, e.g., Simonen and Palva, Microbiol. Rev., 1993, 57:109-137). In some embodiments, signal peptide coding regions useful for filamentous fungal host cells include, but are not limited to, signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei apartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Signal peptides useful for yeast host cells include, but are not limited to, signal peptides obtained from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0189] In some embodiments, the control sequence is also a propeptide coding region that codes for an amino acid sequence located at the amino terminus of a polypeptide. The resulting polypeptide is referred to as a "proenzyme," "propolypeptide," or "zymogen." A propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide. The propeptide coding region can be obtained from any suitable source, including, but not limited to, the genes for alkaline protease (aprE) of Bacillus subtilis, neutral protease (nprT) of Bacillus subtilis, alpha factor of Saccharomyces cerevisiae, apartic protease of Rhizomucor miehei, and lactase of Myceliophthora thermophila (see, e.g., WO 95 / 33836). When both a signal peptide region and a propeptide region are present at the amino terminus of a polypeptide, the propeptide region is located adjacent to the amino terminus of the polypeptide, and the signal peptide region is located adjacent to the amino terminus of the propeptide region.

[0190] In some embodiments, regulatory sequences are also utilized. These sequences facilitate regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems include those that turn the expression of a gene 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 sequences include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha amylase promoter, the Niger glucoamylase promoter, and the Aspergillus glucoamylase promoter.

[0191] In another aspect, the invention relates to a recombinant expression vector comprising a polynucleotide encoding an engineered leucine decarboxylase polypeptide and one or more expression control regions, such as a promoter and terminator, an origin of replication, depending on the type of host into which it will be introduced. In some embodiments, the various nucleic acid and control sequences described herein are joined together to produce a recombinant expression vector that contains one or more convenient restriction sites that allow for the insertion or substitution of a nucleic acid sequence encoding a leucine decarboxylase polypeptide into such site. Alternatively, in some embodiments, the nucleic acid sequences of the invention are expressed by inserting the nucleic acid sequence or a nucleic acid construct containing the sequence into an appropriate expression vector for expression. In some embodiments involving the creation of an expression vector, the coding sequence is placed into the vector so that the coding sequence is operably linked to the appropriate expression control sequences.

[0192] A recombinant expression vector is any suitable vector (e.g., a plasmid or virus) that can be conveniently applied to recombinant DNA manipulations and can result in the expression of a leucine decarboxylase polynucleotide sequence. The choice of vector usually depends on the compatibility of the vector with the host cell into which the vector is to be introduced. The vector can be a linear or closed circular plasmid.

[0193] In some embodiments, the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extrachromosomal entity and whose replication is independent of chromosomal replication, such as a plasmid, an extrachromosomal element, a minichromosome, or an engineered chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector is a vector that, upon introduction into a host cell, is integrated into the genome and replicates together with the chromosome into which it is integrated. Furthermore, in some embodiments, a single vector or plasmid, or two or more vectors or plasmids that contain the total DNA to be introduced into the genome of the host cell, and / or a transposon are used.

[0194] In some embodiments, the expression vector contains one or more selectable markers that allow easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, heavy metal resistance, prototrophy for auxotrophs, and the like. Examples of bacterial selectable markers include, but are not limited to, the dal genes of Bacillus subtilis or Bacillus licheniformis, or markers that 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. niger or A. aspergillus), argB (ornithine carbamoyltransferase), bar (phosphinothricin acetyltransferase, e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'-phosphate decarboxylase, e.g., from A. niger or A. aspergillus), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof.

[0195] In another aspect, the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide of the present invention, said polynucleotide being operably linked to one or more control sequences for expressing the engineered leucine decarboxylase in the host cell. Suitable host cells for use in expressing the polypeptides encoded by the expression vectors of the invention are well known in the art and include, but are not limited to, bacterial cells, such as E. coli, Vibrio fluvialis, Streptomyces, and Salmonella typhimurium cells; fungal cells, such as yeast cells (e.g., Saccharomyces cerevisiae or Pichia pastoris (ATCC Accession No. 201178)); insect cells, such as Drosophila S2 cells and Spodoptera Sf9 cells; animal cells, such as CHO cells, COS cells, BHK cells, 293 cells, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various strains of E. coli (e.g., W3110(ΔfhuA) and BL21).

[0196] Thus, in another aspect, the invention provides a method for producing an engineered leucine decarboxylase polypeptide, the method comprising culturing a host cell capable of expressing a polynucleotide encoding an engineered leucine decarboxylase polypeptide under conditions suitable for expression of said polypeptide. In some embodiments, the method further comprises recovering the engineered leucine decarboxylase polypeptide from the culture and / or the host cell. In some embodiments, the method further comprises isolating and / or purifying the leucine decarboxylase polypeptide described herein.

[0197] Suitable culture media and growth conditions for host cells are well known in the art. Any suitable method of introducing a polynucleotide into a cell for expression of a leucine decarboxylase polypeptide is contemplated for use in the present invention. Suitable techniques include, but are not limited to, electroporation, biolistics, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0198] An engineered leucine decarboxylase polypeptide having the properties disclosed herein can be obtained by subjecting a polynucleotide encoding a natural or engineered leucine decarboxylase polypeptide to any suitable mutagenesis and / or directed evolution method known in the art and / or described herein. Exemplary directed evolution techniques are mutagenesis and / or DNA shuffling (see, e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; and U.S. Patent No. 6,537,746). Other directed evolution procedures that can be used include, among others, the staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 1998, 16:258-261), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 1994, 3:S136-S140), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA, 1996, 93:3525-3529).

[0199] Mutagenesis and directed evolution techniques can be readily applied to polynucleotides encoding leucine decarboxylase to generate libraries of variants that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution method can be used in the present invention and are well known in the art (see, e.g., U.S. Pat. Nos. 5,605,793, 5,811,238, 5,830,721, 5,834,252, 5,837,458, 5,928,905, 6,096,548, 6,117,679, 6,132,970, 6,165,793, 6,180,406, 6,251,674, 6,265,201, 6,277,638, 6,287, No. 861, No. 6,287,862, No. 6,291,242, No. 6,297,053, No. 6,303,344, No. 6,309,883, No. 6,319,713, No. 6,319,714, No. 6,323,030, No. 6,326,204, No. No. 6,335,160, No. 6,335,198, No. 6,344,356, No. 6,352,859, No. 6,355,484, No. 6,358,740, No. 6,358,742, No. 6,365,377, No. 6,365,408, No. 6,368, No. 861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6,391,552, No. 6,391,640, No. 6,395,547, No. 6,406,855, No. No. 6,406,910, No. 6,413,745, No. 6,413,774, No. 6,420,175, No. 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479, No. 652, No. 6,482,647, No. 6,483,011, No. 6,484,105, No. 6,489,146, No. 6,500,617, No. 6,500,639, No. 6,506,602, No. 6,506,603, No. 6,518,065, No. No. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,No. 430, No. 6,613,514, No. 6,653,072, No. 6,686,515, No. 6,703,240, No. 6,716,631, No. 6,825,001, No. 6,902,922, No. 6,917,882, No. 6,946,296, No. 6,961,664, No. 6,995,017, No. 7,024,312, No. 7,058,515, No. 7,105,297, No. 7,148,05 No. 4, No. 7,220,566, No. 7,288,375, No. 7,384,387, No. 7,421,347, No. 7,430,477, No. 7,462,469, No. 7,534,564, No. 7,620,500, No. 7,620,502, No. 7,629,170, No. 7,702,464, No. 7,747,391, No. 7,747,393, No. 7,751,986, No. 7,776,598 , No. 7,783,428, No. 7,795,030, No. 7,853,410, No. 7,868,138, No. 7,783,428, No. 7,873,477, No. 7,873,499, No. 7,904,249, No. 7,957,912, No. 7,981,614, No. 8,014,961, No. 8,029,988, No. 8,048,674, No. 8,058,001, No. 8,076,138, No. No. 8,108,150, No. 8,170,806, No. 8,224,580, No. 8,377,681, No. 8,383,346, No. 8,457,903, No. 8,504,498, No. 8,589,0 No. 85, No. 8,762,066, No. 8,768,871, No. 9,593,326, No. 9,665,694, No. 9,684,771, and all other patents related to the United States and other countries; Ling et al., Anal. Biochem., 1997, 254(2):157-78; Dale et al., Meth. Mol. Biol., 1996, 57:369-74; Smith, Ann. Rev. Genet., 1985, 19:423-462; Botstein et al., Science, 1985, 229:1193-1201; Carter, Biochem. J., 1986, 237:1-7; Kramer et al., Cell, 1984, 38:879-887; Wells et al.,Gene, 1985, 34:315-323; Minshull et al., Curr. Op. Chem. Biol., 1999, 3:284-290; Christians et al., Nat. Biotechnol., 1999, 17:259-264; Crameri et al., Nature, 1998, 391:288-291; Crameri, et al., Nat. Biotechnol., 1997, 15:436-438; Zhang et al., Proc. Nat. Acad. Sci. USA, 1997, 94:4504-4509; Crameri et al., Nat. Biotechnol., 1996, 14:315-319; Stemmer, Nature, 1994, 370:389-391; Stemmer, Proc. Nat. Acad. Sci. USA, 1994, 91:10747-10751; WO 95 / 22625; WO 97 / 0078; WO 97 / 35966; WO 98 / 27230; WO 00 / 42651; WO 01 / 75767; WO 2009 / 152336; and U.S. Patent Application Publication Nos. 2011 / 0082055, 2014 / 0005057, 2014 / 0214391, 2014 / 0221216, 2015 / 0133307, 2015 / 0134315, and 2015 / 0050658; all of which are incorporated herein by reference).

[0200] In some embodiments, the enzyme clones obtained after the mutagenesis treatment are screened by subjecting the enzyme preparation to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatment or other suitable assay conditions. Clones containing polynucleotides encoding leucine decarboxylase polypeptides are then isolated from the gene and sequenced to identify nucleotide sequence changes (if any) and used to express the enzyme in a host cell. Measurement of enzyme activity from an expression library can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0201] In the case of engineered polypeptides of known sequence, the polynucleotides encoding the enzymes can be prepared by standard solid-phase methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized separately and then linked (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods) to form any desired contiguous sequence. For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by classical phosphoramidite method automated synthesis (see, e.g., Beaucage et al., Tet. Lett., 1981, 22:1859-69; and Matthes et al., EMBO J., 1984, 3:801-05), which is typically performed by automated synthesis. Oligonucleotides are synthesized according to the phosphoramidite method (e.g., automatic DNA synthesizer, purification, annealing, ligation, and cloning in a suitable vector).

[0202] Thus, in some embodiments, a method of preparing an engineered leucine decarboxylase polypeptide may comprise (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any of the variant amino acid sequences described herein, and (b) expressing the leucine decarboxylase polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide may optionally have one or more (e.g., up to 3, 4, 5, or up to 10) deletions, insertions, and / or substitutions of amino acid residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residues. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the substitutions are conservative or non-conservative.

[0203] In some embodiments, the expressed engineered leucine decarboxylase polypeptides can be evaluated for any desired improved property or combination of properties (e.g., activity, selectivity, stability, etc.) using any suitable assay known in the art, including but not limited to the assays and conditions described herein.

[0204] In some embodiments, any engineered leucine decarboxylase polypeptide expressed in a host cell is recovered from the cells and / or culture medium using one or more of the well-known techniques for protein purification, such as lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, chromatography, among others.

[0205] Chromatographic techniques for the isolation of leucine decarboxylase polypeptides include, among others, reverse phase chromatography, high performance liquid chromatography, ion exchange chromatography, hydrophobic interaction chromatography, size exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depend, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, and the like, and will be apparent to one of skill in the art. In some embodiments, affinity techniques can be used to isolate improved leucine decarboxylases. Any antibody that specifically binds to the leucine decarboxylase polypeptide of interest can be used for affinity chromatography purification. To produce antibodies, various host animals, including, but not limited to, rabbits, mice, rats, and the like, are immunized by injection with a leucine decarboxylase polypeptide or a fragment thereof. In some embodiments, the leucine decarboxylase polypeptide or fragment is attached to a suitable carrier, such as BSA, by a side chain functional group or a linker attached to the side chain functional group.

[0206] In some embodiments, an engineered leucine decarboxylase polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., an E. coli strain) comprising a polynucleotide sequence encoding an engineered leucine decarboxylase polypeptide as described herein under conditions conducive to the production of an engineered leucine decarboxylase polypeptide, and recovering the engineered leucine decarboxylase polypeptide from the cell and / or culture medium. In some embodiments, the host cell produces two or more engineered leucine decarboxylase polypeptides.

[0207] composition In another aspect, the engineered leucine decarboxylase polypeptides of the present disclosure are prepared as compositions for various applications. These compositions are used in many fields, including, but not limited to, pharmaceuticals, dietary / nutraceuticals, food, feed, and fine chemical manufacturing. For example, in some embodiments, the present invention provides a food and / or feed comprising at least one engineered leucine decarboxylase variant and / or at least one polynucleotide sequence encoding at least one leucine decarboxylase variant. In some embodiments, the present invention provides a beverage comprising at least one engineered leucine decarboxylase variant. In some embodiments, the composition further comprises an effective amount of a coenzyme, such as pyridoxal 5' phosphate.

[0208] In some embodiments, the engineered leucine decarboxylase variant in the food, feed, and / or nutritional / dietary supplement is glycosylated.Furthermore, the engineered leucine decarboxylase variant can be used in any suitable edible enzyme delivery matrix.In some embodiments, the engineered leucine decarboxylase variant is present in an edible enzyme delivery matrix designed to rapidly distribute the leucine decarboxylase variant in the animal's digestive tract upon ingestion of the variant.

[0209] In some embodiments, engineered leucine decarboxylase polypeptides are used in the production of fine chemicals and other industrially important compounds (see, e.g., U.S. Patent Application Nos. 2013 / 0340119, 2013 / 0005012, and 2005 / 0260724, and WO 2012 / 122333).

[0210] Pharmaceuticals and Other Compositions The present invention also provides engineered leucine decarboxylase polypeptides suitable for use in pharmaceutical and other compositions such as dietary / nutritional supplements.

[0211] Depending on the method of administration, these compositions containing a therapeutically effective amount of engineered leucine decarboxylase according to the present invention are solid, semi-solid, or liquid. In some embodiments, the compositions contain other pharma- ceutically acceptable ingredients, such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, bulking agents, and other ingredients. Details of formulation and administration techniques are well known in the art and described in the literature.

[0212] In some embodiments, the engineered leucine decarboxylase polypeptide is formulated for use in an oral pharmaceutical composition. Any suitable form used to deliver the engineered leucine decarboxylase polypeptide is used in the present invention, including, but not limited to, pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol gels, gels, emulsions, implants, patches, sprays, ointments, salves, creams, pastes, jellies, paints, aerosols, chewing gums, demulcents, sticks, suspensions (including, but not limited to, oil suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled release formulations, suppositories, etc. In some embodiments, the engineered leucine decarboxylase polypeptide is provided in a form suitable for injection (i.e., an injectable formulation). In some embodiments, the engineered leucine decarboxylase polypeptide is provided in a biocompatible matrix, such as a sol gel, including silica-based (e.g., oxysilane) sol gels. In some embodiments, the engineered leucine decarboxylase polypeptide is encapsulated. In some alternative embodiments, the engineered leucine decarboxylase polypeptides are encapsulated in nanostructures (e.g., nanotubes, small nanotubes, nanocapsules or microcapsules, microspheres, liposomes, etc.). Indeed, the present invention is not intended to be limited to a particular delivery formulation and / or delivery means. The engineered leucine decarboxylase polypeptides are intended to be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, implants, etc.

[0213] In some further embodiments, the pharmaceutical composition further comprises the coenzyme pyridoxal-5-phosphate, hi some embodiments, the pyridoxal-5-phosphate can be formulated with the engineered leucine decarboxylase as a stable composition or prepared immediately prior to administration.

[0214] In some embodiments, the engineered leucine decarboxylase polypeptides are chemically modified by glycosylation, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.), or other compounds (see, e.g., Ikeda, Amino Acids, 2005, 29:283-287; U.S. Patent Nos. 7,531,341, 7,534,595, 7,560,263, and 7,553,653; U.S. Patent Application Publication Nos. 2013 / 0039898, 2012 / 0177722, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery method and / or mechanism.

[0215] In some additional embodiments, the engineered leucine decarboxylase polypeptide is provided in a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises a crosslinked crystalline engineered leucine decarboxylase and a polymer having a reactive moiety that attaches to the enzyme crystal. The present invention also provides engineered leucine decarboxylase polypeptides in a polymer.

[0216] In some embodiments, compositions comprising engineered leucine decarboxylase polypeptides of the invention include one or more commonly used carrier compounds, including, but not limited to, sugars (e.g., lactose, sucrose, mannitol, and / or sorbitol), starches (e.g., corn, wheat, rice, potato, or other vegetable starches), celluloses (e.g., methylcellulose, hydroxypropylmethylcellulose, sodium carboxymethylcellulose), gums (e.g., gum arabic, gum tragacanth, gum guar, etc.), and / or proteins (e.g., gelatin, collagen, etc.). Additional components in oral formulations may include colorants and / or sweeteners (e.g., glucose, sucrose, and mannitol), and lubricants (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxypropylmethylcellulose phthalate, and / or any other suitable enteric coatings known in the art). In some embodiments, disintegrating or solubilizing agents are included (eg, cross-linked polyvinyl pyrrolidone, agar, alginic acid or a salt thereof such as sodium alginate). In some embodiments, the engineered leucine decarboxylase polypeptides are combined with various additional ingredients, including but not limited to, preservatives, suspending agents, thickening agents, humectants, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations.

[0217] In some embodiments, the engineered leucine decarboxylase polypeptide is combined with various additional ingredients, including but not limited to, preservatives, suspending agents, thickening agents, humectants, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations. In some embodiments, the engineered leucine decarboxylase polypeptide is administered to a subject in combination with other compounds used to treat MSUD, as well as any other suitable compounds.

[0218] In some embodiments, the present invention provides engineered leucine decarboxylase polypeptides suitable for use in reducing, ameliorating, or eliminating the signs and / or symptoms of MSUD, as described herein. The dose of engineered leucine decarboxylase polypeptide administered to a patient depends on the patient's genotype, the patient's general condition, and other factors known to those skilled in the art. In some embodiments, the composition is intended for single or repeated administration to the patient. In some embodiments, the concentration of engineered leucine decarboxylase polypeptide in the composition administered to the patient is intended to be sufficient to effectively treat, ameliorate, and / or prevent symptoms of the disease. In some embodiments, the engineered leucine decarboxylase polypeptide is administered in combination with other pharmaceutical and / or dietary compositions.

[0219] Industrial Compositions It is contemplated that the engineered leucine decarboxylase polypeptides of the present invention will find use in industrial compositions, including in areas such as food flavorings (such as cheese).

[0220] In some embodiments, the engineered leucine decarboxylase polypeptides are formulated for use in the food and / or feed industry. In some embodiments, the engineered leucine decarboxylase polypeptides are formulated as granule or pellet products, which are mixed with animal feed ingredients such as additional enzymes (e.g., cellulases, laccases, and amylases). In some alternative embodiments, the engineered leucine decarboxylase polypeptides are used in liquid animal feed compositions (e.g., aqueous or oil-based slurries). Thus, in some embodiments, the engineered leucine decarboxylase variants of the invention are sufficiently heat tolerant and thermostable to survive the processes used to produce pellets and other feeds / food products.

[0221] Therapeutic Uses and Methods In another embodiment, the engineered leucine decarboxylase polypeptide is used for the treatment and / or prevention of symptoms of conditions associated with dysfunction of leucine, isoleucine, and / or alloisoleucine metabolism. In some embodiments, the subject for treatment to reduce plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid has organic acidemia or an inborn error of amino acid metabolism. In some embodiments, the subject for treatment to reduce plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid has elevated leucine levels due to unknown etiology.

[0222] In some embodiments, engineered leucine decarboxylase polypeptides are used to treat and / or prevent symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels. In some embodiments, a method of treating and / or preventing symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels comprises administering to a subject in need thereof an engineered leucine decarboxylase in an amount effective to reduce the subject's plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels.

[0223] In some embodiments, the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels is maple syrup urine disease. In some embodiments, the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels is isovaleric acidemia. In some embodiments, the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels is 3-methylcrotonyl-CoA carboxylase deficiency.

[0224] In some embodiments, engineered leucine decarboxylase polypeptides are used to reduce the levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and / or ketoisocaproic acid in the plasma of a subject. In some embodiments, the levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and / or ketoisocaproic acid can be used as indicators of diseases or conditions, e.g., associated with dysfunction of amino acid metabolism, and / or as markers of the effectiveness of treatment with engineered leucine decarboxylase polypeptides. In some embodiments, engineered leucine decarboxylase polypeptides are used to reduce the levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and / or ketoisocaproic acid in the plasma of a subject, the method comprising administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase.

[0225] In some embodiments, the engineered leucine decarboxylase polypeptide is used to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels in a subject, the method comprising administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase. In some embodiments, the subject for treatment to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels has maple syrup urine disease. In some embodiments, the subject for treatment to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels has isovaleric acidemia. In some embodiments, the subject for treatment to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels has 3-methylcrotonyl-CoA carboxylase deficiency.

[0226] In some embodiments, an engineered leucine decarboxylase polypeptide is administered in an effective dose for treatment and / or prevention of symptoms of a condition associated with dysfunction of leucine, isoleucine, and / or alloisoleucine metabolism in a subject or to reduce levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid in plasma.

[0227] In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg / kg to 500 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg / kg to 400 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 1 mg / kg to 200 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 2 mg / kg to about 300 mg / kg, or about 5 mg / kg to about 200 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 1 mg / kg, about 5 mg / kg, about 10 mg / kg, about 20 mg / kg, about 30 mg / kg, about 40 mg / kg, about 50 mg / kg, about 60 mg / kg, about 70 mg / kg, about 80 mg / kg, about 90 mg / kg, about 100 mg / kg, about 120 mg / kg, about 140 mg / kg, about 160 mg / kg, about 180 mg / kg, about 200 mg / kg, about 250 mg / kg, about 300 mg / kg, about 350 mg / kg, or about 400 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of between 1 mg / kg and less than 25 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of between 5 mg / kg and less than 25 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg / kg to about 12.5 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 12.5 mg / kg.

[0228] In some embodiments for the treatment of maple syrup urine disease, the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg / kg to less than 25 mg / kg. In some embodiments for the treatment of maple syrup urine disease, the engineered leucine decarboxylase polypeptide is administered at a dose of 5 mg / kg to less than 25 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg / kg to about 12.5 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg / kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 12.5 mg / kg.

[0229] In some embodiments, the engineered leucine decarboxylase polypeptide is used to reduce plasma levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and / or ketoisocaproic acid in a subject. In some embodiments, the engineered leucine decarboxylase polypeptide is administered in an amount effective to reduce plasma levels of leucine, ketoisocaproic acid, and methionine in a subject in need thereof. In some embodiments, the engineered leucine decarboxylase polypeptide is administered to the subject for at least two or more consecutive days. In some embodiments, the engineered leucine decarboxylase polypeptide is administered to the subject for at least three or more consecutive days. In some embodiments, the engineered leucine decarboxylase polypeptide is administered continuously, e.g., for weeks, months, years, and / or as needed, to treat a subject in need thereof.

[0230] In some embodiments, for treatment and / or prevention of symptoms of a condition associated with dysfunction of leucine, isoleucine, and / or alloisoleucine metabolism or to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels in a subject, an engineered leucine decarboxylase polypeptide is administered at a dose that reduces plasma or serum leucine levels by about 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, and / or 50% or more compared to baseline leucine levels in an untreated subject with a disease or disorder.

[0231] In some embodiments, an engineered leucine decarboxylase polypeptide is administered immediately prior to, simultaneously with, and / or immediately after ingestion of a protein-containing meal for treatment and / or prevention of symptoms of a condition associated with dysfunction of leucine, isoleucine, and / or alloisoleucine metabolism or to reduce plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels in a subject.

[0232] In some embodiments, in any of the methods described herein, the engineered leucine decarboxylase polypeptide can be administered in combination with the coenzyme pyridoxal-5-phosphate. In some embodiments, the engineered leucine decarboxylase polypeptide can be combined with pyridoxal-5-phosphate prior to administration. In some embodiments, pyridoxal-5-phosphate is administered simultaneously with administration of the engineered leucine decarboxylase polypeptide.

[0233] In some embodiments, the subject treated with the engineered leucine decarboxylase polypeptide suffers from maple syrup urine disease, and wherein the symptoms of maple syrup urine disease are alleviated. In some embodiments, the subject treated with the engineered leucine decarboxylase polypeptide suffers from isovaleric acidemia, and wherein the symptoms of isovaleric acidemia are alleviated. In some embodiments, the subject treated with the engineered leucine decarboxylase polypeptide suffers from 3-methylcrotonyl-CoA carboxylase deficiency, and wherein the symptoms of 3-methylcrotonyl-CoA carboxylase deficiency are alleviated. In some embodiments, the subject is able to consume a diet that is less restrictive in leucine, isoleucine, and / or valine content than the diet required by subjects suffering from the disease. In some embodiments, the subject is an infant, a child, a young adult, or an adult.

[0234] In some embodiments, engineered leucine decarboxylase polypeptides useful in the therapeutic methods and uses herein include the engineered leucine decarboxylase polypeptides described above, as well as those described in International Patent Publication WO2021158686 and its corresponding Sequence Listing, which are incorporated herein by reference in their entireties. These engineered leucine decarboxylase polypeptides are also shown in the Appendix (Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and 11-2) and in the incorporated Sequence Listings of this disclosure.

[0235] In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, and / or 888. In some embodiments, the engineered leucine decarboxylase polypeptide comprises one or more substitutions relative to the reference sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.

[0236] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, and / or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.

[0237] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:2.

[0238] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:4.

[0239] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:6.

[0240] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:8.

[0241] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:10.

[0242] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:14.

[0243] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.

[0244] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12.

[0245] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase comprises the amino acid sequence at amino acid positions 2, 3, 5, 12, 14, 16, 19, 33, 34, 38, 39, 41, 47, 48, 51, 55, 63, 64, 66, 69, 76, 77, 80, 87, 89, 91, 92, 102, 106, 109, 118, 123, 126, 127, 132, 134, 135, 139, 140, 141, 156, 161, 164, 168, 170, 173, 181, 187, 189, 193, 194, 196, 198, 200, 201, 202, 211, 223, 228, 245, 255, 256, 259, 262, 264, 266, 268, 270, 273, 275, 276, 277, 278, 279, 280, 281, 282, 283, 284, 285, 286, 287, 288, 289, 290, 291, 292, 293, 300, 304, 305, 306, 307, 308, 309, 310, 311, 312, 313, 314, 315, 316, 3 63, 265, 267, 270, 272, 275, 290, 296, 299, 300, 303, 304, 312, 317, 319, 324, 328, 331, 338, 339, 340, 343, 349, 350, 352, 353, 357, 364, 365, 365, 366, 379, 380, 381, 382, ​​383, 384, 386, 388, 389, 390, 391, 393, 394, 395, 397, 398, 401, 404, or 405, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12.

[0246] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase comprises at least the following substitutions: 2E, 3M, 5M / V, 12G, 14I / T, 16Q / V, 19I / L, 33L, 34L, 38V, 39N / S, 41D, 47F, 48L, 51Q / E, 55I, 63C, 64S / A / N / E, 66S / N, 69I, 76V, 77L, 80G / K, 87R, 89P, 91A / Q, 92K, 102S, 106M, 109G, 118T / D, 123F / M / V, 126A / T, 127S, 132F, 134A / S, 135V, 139G, 140V, 141P, 15 6A / S, 161V, 164A / C, 168K, 170A / P, 173A / I / T, 181K / R / V, 187L, 189A / D, 193I, 194C / L, 196D / R, 198G, 2 00S, 201D / R, 202H, 211S, 223M, 228D, 245M, 255G / N / P / H, 256W, 259K / L / Q, 262D / G / H / I / S / T, 263T / V, 265P, 267L / I, 270R / A / T / L, 272A, 275S, 290I, 296D / E, 299A, 300R / K, 303Q, 304R, 312A / T, 317Q, 319A, 324N / S / M / N / T, 328N, 331V, 338S, 339A / D, 340T / V, 343A / E, 349T, 350S / E, 352A, 353I / L / D / N / S / W / E, 357S / C / M / V, 364K / R, 365E, 365E, 366A / M / Q / T / V, 379D / N / P / E, 380E, 381D / E, 382G / S, 383S, 384W, 386 * , 388A, 389G / P / Q, 390A / S / E / * , 391E / * , 393T, 394E, 395A / D / G / K / S, 397A, 398 * , 401Y / * , 404I, or 405D / H / L / E, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12.

[0247] In some further embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 12, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 5, 14, 14 / 34 / 38 / 39 / 102 / 267 / 275 / 350 / 357, 14 / 39 / 102 / 127 / 245 / 267 / 275 / 349 / 35 0, 34 / 38 / 39 / 102 / 127 / 275 / 357, 34 / 38 / 39 / 102 / 275 / 357, 34 / 38 / 39 / 127 / 245 / 349 / 350 / 357, 34 / 38 / 39 / 127 / 245 / 350 / 357, 34 / 39 / 102 / 127 / 264 / 275 / 3 57, 34 / 39 / 102 / 127 / 275 / 349 / 357, 34 / 39 / 102 / 264 / 275 / 350 / 357, 34 / 39 / 275 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 264 / 267 / 350 / 357, 38 / 39 / 102 / 127 / 267 / 275 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 350, 38 / 39 / 102 / 127 / 350 / 357, 38 / 39 / 127 / 245 / 267 / 357, 38 / 39 / 127 / 264 / 275, 38 / 39 / 127 / 264 / 350 / 357, 38 / 39 / 127 / 350 / 357, 38 / 39 / 127 / 357, 38 / 39 / 245 / 275 / 357, 38 / 39 / 264 / 267 / 275 / 350, 38 / 39 / 264 / 275 / 357, 38 / 39 / 275, 38 / 39 / 275 / 350, 39, 39 / 102 / 127 / 264 / 275 / 357, 39 / 102 / 264 / 275 / 357, 39 / 102 / 267 / 275 / 357, 39 / 127 / 245 / 264 / 267 / 275 / 350, 39 / 127 / 245 / 264 / 275 / 350 / 357 , 39 / 127 / 245 / 357, 39 / 127 / 267 / 275 / 350 / 357, 39 / 127 / 267 / 350 / 357, 39 / 127 / 357, 39 / 245 / 264 / 267 / 275 / 357, 39 / 264 / 267 / 275 / 350, 39 / 275 / 350 / 357,48, 139, 164, 196, 255, 299, 318, 324, 339, 343, 350, 353, 357, 364, 365, 379, 381, 386, 389, 391, 393, 394, 395, 397, 398, or 405, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 5M, 14I, 14T / 34L / 38V / 39N / 102S / 267I / 275S / 350E / 357V, 14T / 39N / 102S / 127S / 245M / 267I / 275S / 349T / 350E, 34L / 38V / 39N / 102S / 127S / 275S / 357V, 34L / 38V / 39N / 102 ... 357V, 34L / 38V / 39N / 127S / 245M / 349T / 350E / 357V, 34L / 38V / 39N / 127S / 245M / 350E / 357V, 34L / 39N / 102S / 127S / 264V / 275S / 3 57V, 34L / 39N / 102S / 127S / 275S / 349T / 357V, 34L / 39N / 102S / 264V / 275S / 350E / 357V, 34L / 39N / 275S / 349T / 350E / 357V, 38V / 39 N / 102S / 127S / 264V / 267I / 350E / 357V, 38V / 39N / 102S / 127S / 267I / 275S / 349T / 350E / 357V, 38V / 39N / 102S / 127S / 349T / 350E / 357V, 38V / 39N / 102S / 127S / 350E, 38V / 39N / 102S / 127S / 350E / 357V, 38V / 39N / 127S / 245M / 267I / 357V, 38V / 39N / 127S / 264V / 2 75S, 38V / 39N / 127S / 264V / 350E / 357V, 38V / 39N / 127S / 350E / 357V, 38V / 39N / 127S / 357V, 38V / 39N / 245M / 275S / 357V, 38V / 39N / 264V / 267I / 275S / 350E, 38V / 39N / 264V / 275S / 357V, 38V / 39N / 275S, 38V / 39N / 275S / 350E, 39N / 102S / 127S / 264V / 275S / 357V,<h2 style=";text-align:left;direction:ltr">39N / 102S / 264V / 275S / 357V、39N / 102S / 267I / 275S / 357V、39N / 127S / 245M / 264V / 267I / 275S / 350E、39N / 127S / 245M / 264V / 275S / 350E / 357 V、39N / 127S / 245M / 357V、39N / 127S / 267I / 275S / 350E / 357V、39N / 127 S / 267I / 350E / 357V、39N / 127S / 357V、39N / 245M / 264V / 267I / 275S / 35 7V、39N / 264V / 267I / 275S / 350E、39N / 275S / 350E / 357V、39S、48F、139 G、164A、164C、196D、196R、255G、255N、255P、299A、299V、318K、324M、 324S, 324T, 339A, 339D, 343A, 343E, 350S, 353D, 353E, 353L, 353N, 353S, 353W, 357C, 357M, 364K, 364R, 365E, 379D, 379P, 381D, 381E, 386<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 、389E、389G、389P、389Q、391<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 391E, 393T, 394E, 395A, 395D, 395G, 395K, 395S, 397A, 398<h2 style=";text-align:left;direction:ltr"> *, 405D, 405E, 405H, or 405L, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 12. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions K5M, H14I, H14T / I34L / C38V / T39N / T102S / V267I / T275S / N350E / I357V, H14T / T39N / T102S / T127S / I245M / V267I / T275S / V349T / N350E, I34L / C38V / T39N / T102S / T127S / T275S / I357V, I34L / C38V / T39N / T102S / T127S / T275S / I357V, I34L / C38V / T39N / T10 2S / T275S / I357V, I34L / C38V / T39N / T127S / I245M / V349T / N350E / I357V, I34L / C38V / T39N / T127S / I245M / N350E / I357V, I34L / T39N / T102S / I34L / T39N / T 275S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / I264V / V267I / N350E / I357V, C38V / T39N / T102S / T127S / V267I / T275S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / N350E, C38V / T39N / T102S / T127S / N350E / I357V, C38V / T 39N / T127S / I245M / V267I / I357V, C38V / T39N / T127S / I264V / T275S, C38V / T39N / T127S / I264V / N350E / I357V, C38V / T39N / T127S / N350E / I35 7V, C38V / T39N / T127S / I357V, C38V / T39N / I245M / T275S / I357V, C38V / T39N / I264V / V267I / T275S / N350E, C38V / T39N / I264V / T275S / I357V,C38V / T39N / T275S, C38V / T39N / T275S / N350E, T39N / T102S / T127S / I264V / T275S / I357V, T39N / T102S / I264V / T275S / I357V, T39N / T102S / V267I / T275S / I357V, T39N / T127S / I245M / I264V / V 267I / T275S / N350E, T39N / T127S / I245M / I264V / T275S / N350E / I357V, T39N / T127S / I245M / I35 7V, T39N / T127S / V267I / T275S / N350E / I357V, T39N / T127S / V267I / N350E / I357V, T39N / T127S / I357V, T39N / I245M / I264V / V267I / T275S / I357V, T39N / I264V / V267I / T275S / N350E, T39N / T27 5S / N350E / I357V, T39S, L48F, N139G, I164A, I164C, K196D, K196R, H255G, H255N, H255P, K299A , K299V, R318K, R324M, R324S, R324T, Q339A, Q339D, H343A, H343E, N350S, R353D, R353E, R353L , R353N, R353S, R353W, I357C, I357M, L364K, L364R, Q365E, K379D, K379P, A381D, A381E, D386, * , K389E, K389G, K389P, K389Q, A391 * , A391E, K393T, K394E, R395A, R395D, R395G, R395K, R395S, T397A, P398 * , T405D, T405E, T405H, or T405L, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12.

[0248] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 38, wherein the amino acid sequence of the engineered leucine decarboxylase is at or near amino acid positions 48 / 64 / 164 / 324 / 343 / 353 / 357 / 364. , 48 / 64 / 164 / 324 / 343 / 364, 48 / 64 / 164 / 353 / 357 / 364, 48 / 64 / 357 / 364, 64 / 164 / 324 / 343 / 353 / 357 / 364, 64 / 164 / 324 / 343 / 357 / 364, 64 / 164 / 353 / 357, 64 / 318 / 324 / 357 / 364, 64 / 324 / 353 / 357 / 364, 132 / 255 / 339 / 379 / 395, 164 / 196 / 324 / 357 / 36 4, 164 / 318 / 324 / 343 / 353 / 357, 164 / 318 / 324 / 357 / 364, 164 / 324 / 343 / 353 / 357 / 364, 164 / 324 / 357 / 364, 164 / 353 / 357 / 364, 164 / 364, 196 / 318 / 324 / 353 / 357 / 364, 318 / 343 / 357, 324 / 343 / 357 / 364, 324 / 353 / 357 / 364, 324 / 357 / 364, 339 / 379 / 38 and 394 / 397, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:38.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 48F / 64E / 164A / 324M / 343E / 353E / 357C / 364K, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164C / 353N / 357V / 364R, 48F / 64E / 357M / 364K, 64E / 164A / 324M / 343E / 364R, M / 343E / 353D / 357V / 364K, 64E / 164A / 324M / 343E / 357C / 364R, 64E / 164C / 353D / 357V, 64E / 318K / 324S / 357V / 364R , 64E / 324M / 353N / 357C / 364R, 132F / 255P / 339A / 379D / 395D, 164A / 196D / 324M / 357C / 364K, 164A / 318K / 324M / 343 E / 353E / 357C, 164A / 324M / 343E / 353D / 357C / 364R, 164A / 324M / 357C / 364K, 164A / 353W / 357C / 364R, 164A / 364R, 164C / 318K / 324S / 357V / 364R, 164C / 324M / 343E / 353D / 357V / 364R, 164C / 353D / 357V / 364K, 164C / 353D / 357V / 364 R, 164C / 353W / 357C / 364R, 196D / 318K / 324M / 353N / 357C / 364K, 318K / 343E / 357C, 318K / 343E / 357M, 324M / 343E / 3 57V / 364K, 324M / 357M / 364R, 324N / 353W / 357C / 364K, 339A / 379D / 389G / 394E / 395D, 339A / 389G / 395K, 339A / 391. * , 339A / 394E / 395K / 405D, 357V / 364R, 379D / 386 *, 379D / 394E / 395D / 397A / 404I / 405H, 379D / 394E / 395K / 397A / 405D, 389G / 394E / 395D / 397A / 405D, or 394E / 397A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:38.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions L48F / A64E / I164A / R324M / H343E / R353E / I357C / L364K, L48F / A64E / I164A / R324M / H343E / L364R, L48F / A64E / I164C / R353N / I357V / L364R, L48F / A64E / I357M / L364K, A64E / I164A / R324M / H343E / R353E / I357C / L364R R353D / I357V / L364K, A64E / I164A / R324M / H343E / I357C / L364R, A64E / I164C / R353D / I357V, A64E / R318K / R324S / I357V / L364R, A64E / R324M / R353N / I357C / L364R, Y132F / H255P / Q339A / K379D / R395D, I164A / K196D / R324M / I357C / L364K, I164A / R318K / R324M / H343E / R3 53E / I357C, I164A / R324M / H343E / R353D / I357C / L364R, I164A / R324M / I357C / L364K, I164A / R353W / I357C / L364R, I164A / L364R, I164 C / R318K / R324S / I357V / L364R, I164C / R324M / H343E / R353D / I357V / L364R, I164C / R353D / I357V / L364K, I164C / R353D / I357V / L364R, I164C / R353W / I357C / L364R, K196D / R318K / R324M / R353N / I357C / L364K, R318K / H343E / I357C, R318K / H343E / I357M, R324M / H343E / I3 57V / L364K, R324M / I357M / L364R, R324N / R353W / I357C / L364K, Q339A / K379D / K389G / K394E / R395D, Q339A / K389G / R395K, Q339A / A391. * , Q339A / K394E / R395K / T405D, I357V / L364R, K379D / D386 *, K379D / K394E / R395D / T397A / R404I / T405H, K379D / K394E / R395K / T397A / T405D, K389G / K394E / R395D / T397A / T405D, or K394E / T397A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 38.

[0249] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:234, wherein the amino acid sequence of the engineered leucine decarboxylase is at or near amino acid positions 2, 3, 33, 48 / 64 / 255, 48 / 255 / 339, 48 / 255 / 379, 64, 64 / 255, 69, 161, 193, 255, 255 / 318 / 379, 259, 263, 318 / 339 / 379, 324, 324 / 389 / 3 94, 324 / 389 / 394 / 395, 324 / 389 / 394 / 397, 324 / 394, 324 / 394 / 395, 324 / 394 / 395, 324 / 394 / 395 / 397, 324 / 395, 339, 340, 380, 382, ​​389, 389 / 394, 389 / 394 / 395, 389 / 394 / 395 / 397, 389 / 394 / 397, 389 / 395, 389 / 397, 390, 394, 394 / 395, 394 / 395 / 397, 395, 395 / 397, 397, 401, or 405, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:234.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 2E, 3M, 33L, 48F / 64E / 255P, 48F / 255P / 339A, 48F / 255P / 379D, 64E, 64E / 255P, 64S, 69I, 161V, 193I, 255P, 255P / 318K / 379D, 259L, 263T, 263V, 318K / 339A / 379D, 324N, 324N / 394E / 395K / 397A, 324N / 395D, 324S / 389G / 394E ... 389G / 394E / 395D, 324S / 389G / 394E / 397A, 324S / 394E, 324S / 394E / 395K, 32 4S / 394E / 395K / 397A, 324S / 395K, 339A, 340T, 340V, 380E, 382S, 389G, 389G / 394E, 389G / 394E / 395D, 389G / 394E / 395D / 397A, 389G / 394E / 395K, 389G / 3 94E / 395K / 397A, 389G / 394E / 397A, 389G / 395D, 389G / 395K, 389G / 397A, 390. * , 390A, 390E, 390S, 394E, 394E / 395D, 394E / 395K / 397A, 395D / 397A, 395K, 397A, 401 *, 401Y, or 405H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 234. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions G2E, N3M, F33L, L48F / A64E / H255P, L48F / H255P / Q339A, L48F / H255P / K379D, A64E, A64E / H255P, A64S, V 69I, T161V, M193I, H255P, H255P / R318K / K379D, R259L, S263T, S263V, R318K / Q339A / K37 9D, M324N, M324N / K394E / R395K / T397A, M324N / R395D, M324S / K389G / K394E, M324S / K389G / K394E / R395D, M324S / K389G / K394E / T397A, M324S / K394E, M324S / K394E / R395K, M324S / K394E / R395K / T397A, M324S / R395K, Q339A, S340T, S340V, A380E, A382S, K389G, K389G / K 394E, K389G / K394E / R395D, K389G / K394E / R395D / T397A, K389G / K394E / R395K, K389G / K3 94E / R395K / T397A, K389G / K394E / T397A, K389G / R395D, K389G / R395K, K389G / T397A, P390 * , P390A, P390E, P390S, K394E, K394E / R395D, K394E / R395K / T397A, R395D / T397A, R395K, T397A, A401 * , A401Y, or T405H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 234.

[0250] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:284, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 2 / 64 / 69 / 324 / 380 / 382 / 388 / 389, 3 / 64 / 69 / 263 / 339 / 380 / 388, 3 / 64 / 69 ... 64 / 69 / 390, 3 / 64 / 379 / 380 / 390, 3 / 69 / 263 / 380, 3 / 69 / 324, 3 / 69 / 324 / 380 / 382 / 389 / 390, 12 / 135 / 259 / 263, 12 / 135 / 263 / 382, 12 / 259 / 263 / 304, 48 / 64 / 255, 64 / 69, 64 / 69 / 189 / 259 / 263 / 304, 64 / 69 / 189 / 259 / 263 / 304 / 339 / 340 / 379, 64 / 69 / 223 / 388, 64 / 69 / 223 / 388 / 389 / 390, 64 / 69 / 304 / 379 / 382 , 64 / 69 / 324, 64 / 69 / 324 / 339 / 380 / 389 / 390, 64 / 69 / 339, 64 / 69 / 339 / 382 / 388 / 389, 64 / 69 / 339 / 389 / 390, 64 / 69 / 379 / 380, 64 / 69 / 380 / 388 / 390, 64 / 69 / 389, 64 / 69 / 390, 64 / 255 / 263, 64 / 263, 64 / 324 / 339 / 389 / 390, 69 / 223 / 263 / 324 / 382 / 388 / 390, 69 / 223 / 324 / 379 / 380 / 382 / 388 / 390, 69 / 263, 69 / and / or 304 / 340 / 379 / 380 / 382, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:284.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 2E / 64S / 69I / 324S / 380E / 382S / 388A / 389G, 3M / 64S / 69I / 263T / 339A / 380E / 388A, 3M / 64S / 69I / 389G, 3M / 64S / 69I / 390. * , 3M / 64S / 379D / 380E / 390 * , 3M / 69I / 263T / 380E, 3M / 69I / 324S, 3M / 69I / 324S / 380E / 382S / 389G / 390 * , 12G / 135V / 259K / 263T, 12G / 135V / 263T / 382G, 12G / 259K / 263T / 304R, 48L / 64A / 255H, 64A / 255H / 263T, 64S / 69I, 64S / 69I / 189 A / 259Q / 263T / 304R / 339A / 340T / 379N, 64S / 69I / 189D / 259K / 263T / 304R, 64S / 69I / 223M / 388A, 64S / 69I / 223M / 388A / 389G / 390 * , 64S / 69I / 304R / 379E / 382G, 64S / 69I / 324S, 64S / 69I / 324S / 339A / 380E / 389G / 390 * , 64S / 69I / 339A, 64S / 69I / 339A / 382S / 388A / 389G, 64S / 69I / 339A / 389G / 390 * , 64S / 69I / 379D / 380E, 64S / 69I / 380E / 388A / 390 * , 64S / 69I / 389G, 64S / 69I / 390 * , 64S / 263T, 64S / 324S / 339A / 389G / 390 * , 69I / 223M / 263T / 324S / 382S / 388A / 390 * , 69I / 223M / 324S / 379D / 380E / 382S / 388A / 390 * , 69I / 263T, 69I / 263T / 324S, 69I / 263T / 339A, 69I / 263T / 388A, 69I / 263T / 389G / 390 *, 69I / 324S / 379D / 380E / 388A, 69I / 324S / 380E, 69I / 339A / 390 * , 69I / 382S / 390 * , 259K / 263T / 304R, 259K / 263T / 304R / 339A / 340T / 379N, 263T / 339A / 389G / 390 * , 263T / 390 * or 304R / 340T / 379D / 380E / 382G, where the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 284. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide has at least the substitution or set of substitutions G2E / E64S / V69I / M324S / A380E / A382S / Q388A / K389G, N3M / E64S / V69I / S263T / Q339A / A380E / Q388A, N3M / E64S / V69I / K389G, N3M / E64S / V69I / P390 * , N3M / E64S / K379D / A380E / P390 * , N3M / V69I / S263T / A380E, N3M / V69I / M324S, N3M / V69I / M324S / A380E / A382S / K389G / P390 * , S12G / L135V / R259K / S263T, S12G / L135V / S263T / A382G, S12G / R259K / S263T / A304R, F48L / E64A / P255H, E64A / P255H / S263T, E64S / V69I, E64S / V69I / T18 9A / R259Q / S263T / A304R / Q339A / S340T / K379N, E64S / V69I / T189D / R259K / S2 63T / A304R, E64S / V69I / A223M / Q388A, E64S / V69I / A223M / Q388A / K389G / P390 * , E64S / V69I / A304R / K379E / A382G, E64S / V69I / M324S, E64S / V69I / M324S / Q339A / A380E / K389G / P390 *, E64S / V69I / Q339A, E64S / V69I / Q339A / A382S / Q388A / K389G, E64S / V69I / Q339A / K389G / P390 * , E64S / V69I / K379D / A380E, E64S / V69I / A380E / Q388A / P390 * , E64S / V69I / K389G, E64S / V69I / P390 * , E64S / S263T, E64S / M324S / Q339A / K389G / P390 * ,V69I / A223M / S263T / M324S / A382S / Q388A / P390 * ,V69I / A223M / M324S / K379D / A380E / A382S / Q388A / P390 * , V69I / S263T, V69I / S263T / M324S, V69I / S263T / Q339A, V69I / S263T / Q388A, V69I / S263T / K389G / P390 * , V69I / M324S / K379D / A380E / Q388A, V69I / M324S / A380E, V69I / Q339A / P390 * , V69I / A382S / P390 * , R259K / S263T / A304R, R259K / S263T / A304R / Q339A / S340T / K379N, S263T / Q339A / K389G / P390 * , S263T / P390 * or A304R / S340T / K379D / A380E / A382G, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 284.

[0251] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:484, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is 3 / 304, 3 / 259 / 304, 3 / 259 / 304 / 324 / 339, 3 / 259 / 304 / 324 / 382, 3 / 259 / 304 / 382, 3 / 263 / 304 / 324, 3 / 263 / 304 / 324 / 339, 3 / 263 / 304 / 324 / 382, 3 / 304, 3 / 304 / 324, 16, 63, 77, 80, 87 / 270, 87 / 270 / 365, 87 / 328 / 365, 91, 92, 126, 140, 156, 168 / 27 0 / 328 / 338, 181, 194, 201, 256, 259, 259 / 263, 259 / 263 / 304, 259 / 263 / 304 / 324, 259 / 263 / 304 / 324 / 382, 259 / 263 / 304 / 379, 259 / 263 / 304 / 382, 259 / 304, 259 / 304 / 324, 259 / 304 / 324 / 339, 259 / 304 / 324 / 339 / 382, 259 / 304 / 382, 262, 263 / 30 4, 263 / 304 / 324, 263 / 304 / 324 / 339, 263 / 304 / 324 / 382, 263 / 324, 270, 270 / 319, 270 / 328 / 338, 270 / 328 / 338 / 365, 304, 304 / 324, 324, 328, 352, 365, 366, or 382, ​​wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 3M / 194L / 304R, 3M / 259K / 263T / 304R, 3M / 259K / 304R, 3M / 259K / 304R, 3M / 259K / 304R / 324S / 339A, 3M / 259K / 304R / 324S / 382S, 3M / 259K / 304R / 382S, 3M / 263T / 304R / 324S, 3M / 263T / 304R / 324S / 339A,3M / 263T / 304R / 324S / 382S, 3M / 304R, 3M / 304R / 324S, 16Q, 16V, 63C, 77L, 80G, 80K, 87R / 270R, 87R / 27 0R / 365E, 87R / 328N / 365E, 91A, 91Q, 92K, 126A, 126T, 140V, 156A, 156S, 168K / 270R / 328N / 338S, 181K, 181R, 181V, 194C, 194L, 201D, 256W, 259K, 259K / 263T, 259K / 263T / 304R, 259K / 263T / 304R / 324S, 259K / 263T / 304R / 324S / 382S, 259K / 263T / 304R / 379D, 259K / 263T / 304R / 382S, 259K / 304R, 259K / 304R / 324 S, 259K / 304R / 324S / 339A, 259K / 304R / 324S / 339A / 382S, 259K / 304R / 382S, 262D, 262G, 262H, 262I, 26 2S, 262T, 263T / 304R, 263T / 304R / 324S, 263T / 304R / 324S / 339A, 263T / 304R / 324S / 382S, 263T / 324S, 2 70R, 270R / 319A, 270R / 328N / 338S, 270R / 328N / 338S / 365E, 304R, 304R / 324S, 324S, 328N, 352A, 365E, 366A, 366L, 366M, 366Q, 366T, 366V, or 382S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions N3M / F194L / A304R, N3M / R259K / S263T / A304R, N3M / R259K / A304R, N3M / R259K / A304R / M324S / Q339A ... S / A382S, N3M / R259K / A304R / A382S, N3M / S263T / A304R / M324S, N3M / S263T / A304R / M324S / Q339A, N3 M / S263T / A304R / M324S / A382S, N3M / A304R, N3M / A304R / M324S, R16Q, R16V, A63C, E77L, A80G, A80K,H87R / L270R, H87R / L270R / Q365E, H87R / C328N / Q365E, E91A, E91Q, E92K, D126A, D126T, M140V, G156A, G156S , C168K / L270R / C328N / P338S, T181K, T181R, T181V, F194C, F194L, E201D, Y256W, R259K, R259K / S263T, R259 K / S263T / A304R, R259K / S263T / A304R / M324S, R259K / S263T / A304R / M324S / A382S, R259K / S263T / A304R / K37 9D, R259K / S263T / A304R / A382S, R259K / A304R, R259K / A304R / M324S, R259K / A304R / M324S / Q339A, R259K / A30 4R / M324S / Q339A / A382S, R259K / A304R / A382S, R262D, R262G, R262H, R262I, R262S, R262T, S263T / A304R, S2 63T / A304R / M324S, S263T / A304R / M324S / Q339A, S263T / A304R / M324S / A382S, S263T / M324S, L270R, L270R / I3 19A, L270R / C328N / P338S, L270R / C328N / P338S / Q365E, A304R, A304R / M324S, M324S, C328N, D352A, Q365E, H366A, H366L, H366M, H366Q, H366T, H366V, or A382S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484.

[0252] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:594, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is The amino acid sequence is at amino acid positions 16 / 63 / 80 / 126 / 168 / 366, 16 / 63 / 80 / 126 / 181 / 194 / 259 / 324 / 328 / 366, 16 / 63 / 126 / 168 / 270 / 328 / 366, 16 / 80 / 126 / 324 / 366, 16 / 80 / 126 / 366, 16 / 80 / 168, 16 / 80 / 168 / 270 / 366, 16 / 80 / 168 / 324, 16 / 80 / 168 / 366, 16 / 80 / 324, 16 / 91 / 126 / 168 / 324 / 366, 16 / 126 / 168 / 366, 16 / 168 / 259 / 366, 16 / 168 / 270 / 324 / 366, 16 / 168 / 324 / 328 / 366, 16 / 168 / 324 / 366, 16 / 168 / 366, 16 / 259 / 263 / 328, 16 / 32 and / or 168 / 366, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 16Q / 63C / 80K / 126T / 168K / 366M, 16Q / 63C / 80K / 126T / 181R / 194C / 259K / 324S / 328N / 366M, 16Q / 63C / 126T / 168K / 270R / 32 8N / 366M, 16Q / 80K / 126T / 324S / 366M, 16Q / 80K / 126T / 366M, 16Q / 80K / 168K, 16Q / 80K / 168K / 270R / 366M, 16Q / 80K / 168K / 324S, 16Q / 80K / 168K / 366M, 16Q / 80K / 324S, 16Q / 91A / 126T / 168K / 324S / 366M , 16Q / 126T / 168K / 366M, 16Q / 168K / 259K / 366M, 16Q / 168K / 270R / 324S / 366M, 16Q / 168K / 324S / 328 N / 366M, 16Q / 168K / 324S / 366M, 16Q / 168K / 366M, 16Q / 259K / 263T / 328N, 16Q / 324S / 328N / 366M, 16Q 594. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitution or set of substitutions R16Q / A63C / A80K / D126T / C168K / H366M, 80K / 126T / 168K / 270R / 366M, 80K / 126T / 168K / 366M, 80K / 126T / 181R / 270R / 324S / 366M, 80K / 168K / 270R / 366M, or 168K / 366M, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 594. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitution or set of substitutions R16Q / A63C / A80K / D126T / C168K / H366M, 80K / 126T / 168K / 270R / 366M, 80K / 126T / 168K / 366M, 80K / 126T / 181R / 270R / 324S / 366M, 80K / 168K / 270R / 366M, or 168K / 366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A80K / D126T / M324S / H366M, R16Q / A80K / D126T / H366M, R16Q / A80K / C168K, R16Q / A80K / C168K / L270R / H366M, R16 Q / A80K / C168K / M324S, R16Q / A80K / C168K / H366M, R16Q / A80K / M324S, R16Q / E91A / D126T / C168K / M324S / H366M, R16Q / D 126T / C168K / H366M, R16Q / C168K / R259K / H366M, R16Q / C168K / L270R / M324S / H366M, R16Q / C168K / M324S / C328N / H366M, R16Q / C168K / M324S / H366M, R16Q / C168K / H366M, R16Q / R259K / S263T / C328N, R16Q / M324S / C328N / H366M, R16Q / C328N / H366M, A80K / D126T / C168K / L270R / H366M, A80K / D126T / C168K / H366M, A80K / D126T / T181R / L270R / M324S / H366M, A80K / C168K / L270R / H366M, or C168K / H366M, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.

[0253] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 686, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at amino acid positions 66 / 76 / 118 / 141 / 201 / 300, 66 / 76 / 198 / 200 / 296 / 303, 66 / 76 / 198 / 200 / 300, 66 / 118 / 200 / 296 / 303 / 317, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 200, 76 / 118 / 141 / 200 / 296, 76 / 141 / 198 / 200 / 201 / 300, 80 / 201 / 270, 80 / 270, 80 / 270 / 324, 89 / 118 / 200, 106 / 270 / 324 / 352, 118 / 141 / 200, 126, 126 / 201 / 270 / 324, 12 6 / 270, 141 / 144 / 198 / 200 / 300, 156 / 270, 156 / 270 / 324, 201 / 270, 201 / 270 / 352, 270, or 270 / 324, where the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:686.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 66N / 76V / 118D / 141P / 201R / 300K, 66N / 76V / 198G / 200S / 296E / 303Q, 66N / 76V / 198G / 200S / 300K, 66N / 118D / 200S / 296E / 303Q / 317Q, 66N / 118D / 296E, 66N / 118D / 296E / 300K, 66N / 200S, 76V / 118D / 141P / 200S / 296E, 76V / 141P / 198G / 200S / 201R / 300 K, 80K / 201D / 270R, 80K / 270R, 80K / 270R / 324S, 89P / 118D / 200S, 106M / 270R / 324S / 352A, 118D / 141P / 200S, 126T, 126T / 201D / 270R / 324S, 126T / 270R, 141P / 144V / 198G / 200S / 300K, 156A / 270R, 156A / 270R / 324S, 201D / 270R, 201D / 270R / 352A, 270R, or 270R / 324S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions S66N / R76V / T118D / R141P / E201R / R300K, S66N / R76V / A198G / H200S / D296E / A303Q, S66N / R76V / A198G / H200S / R 300K, S66N / T118D / H200S / D296E / A303Q / K317Q, S66N / T118D / D296E, S66N / T118D / D296E / R3 00K, S66N / H200S, R76V / T118D / R141P / H200S / D296E, R76V / R141P / A198G / H200S / E201R / R300 K, A80K / E201D / L270R, A80K / L270R, A80K / L270R / M324S, A89P / T118D / H200S, L106M / L270R / M324S / D352A, T118D / R141P / H200S, D126T, D126T / E201D / L270R / M324S, D126T / L270R, R141 P / M144V / A198G / H200S / R300K, G156A / L270R, G156A / L270R / M324S, E201D / L270R, E201D / L270R / D352A, L270R, or L270R / M324S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.

[0254] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:686, wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:686. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitution or set of substitutions 19I, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 173I, 173T, 187L, 211S, or 312A, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least the substitution or set of substitutions L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A211S, or T312A, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.

[0255] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:688, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at or near amino acid positions 19 / 109 / 123 / 141 / 170 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 123 / 141 / 170 / 198 ... , 19 / 109 / 123 / 141 / 170 / 198 / 211 / 270 / 312, 19 / 109 / 123 / 170 / 211 / 270 / 312, 19 / 109 / 123 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 170 / 173 / 211 / 270 / 312, 19 / 109 / 211 / 270 / 312, 109 / 170 / 211 / 270 / 312, or 109 / 211 / 270 / 312, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:688. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 19I / 109G / 123F / 170A / 211S / 270R / 312A, 19I / 109G / 123F / 198G / 200S / 211S / 270R / 312A, 19I / 109G / 123V / 141P / 170A / 198G / 200S / 211S / 270R / 312A, 19I / 109G / 123V / 141P / 170A / 198G / 200S / 211S / 270R / 312A, 19I / 109G / 123V / 141P / 170A / 198G / 211S, 19I / 109G / 123V / 141P / 170A / 198G / 211S / 270R / 312A, 19I / 109G / 170A / 173I / 211S / 270R / 312A, 19I / 109G / 211S / 270R / 312A, 109G / 170A / 211S / 270R / 312A, or 109G / 211S / 270R / 312A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:688.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions L19I / L109G / Y123F / P170A / A211S / L270R / T312A, L19I / L109G / Y123F / A198G / H200S / A211S / L270R / T312A, L19I / L109G / Y123V / R141P / P170A / A198G / H200S / A211S / L270R / T312A, L19I / L109G / Y123V / R141P / P170A / A198G / H200S / A211S / L270R / T312A, A198G / A211S, L19I / L109G / Y123V / R141P / P170A / A198G / A211S / L270R / T312A, L19I / L109G / P170A / F173I / A211S / L270R / T312A, L19I / L109G / A211S / L270R / T312A, L109G / P170A / A211S / L270R / T312A, or L109G / A211S / L270R / T312A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 688.

[0256] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO:766, wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 5 / 41, 5 / 41 / 228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 5V / 41D, 5V / 41D / 228D, 33L, 41D, 47F, 51E, 51Q, 55I, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 353I, 353L, 357S, or 384W, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions K5V / H41D, K5V / H41D / T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, or P384W, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 766.

[0257] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 766, wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide is at amino acid positions 66, 67, 68, 69, 70, 71, 72, 73, 74, 75, 76, 77, 78, 79, 80, 81, 82, 83, 84, 85, 86, 87, 88, 89, 90, 91, 92, 93, 94, 95, 96, 97, 98, 99, or more sequence identity to SEQ ID NO: 766, and / or a substitution or set of substitutions at 6 / 118, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 118 / 300, 66 / 296, 66 / 296 / 300, 66 / 300, 118, 118 / 296, 118 / 296 / 300, 118 / 300, 296, 296 / 300, or 300, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 66S, 66S / 118T, 66S / 118T / 296D, 66S / 118T / 296D / 300R, 66S / 118T / 300R, 66S / 296D, 66S / 296D / 300R, 66S / 300R, 118T, 118T / 296D, 118T / 296D / 300R, 118T / 300R, 296D, 296D / 300R, or 300R, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions N66S, N66S / D118T, N66S / D118T / E296D, N66S / D118T / E296D / K300R, N66S / D118T / K300R, N66S / E296D, N66S / E296D / K300R, N66S / K300R, D118T, D118T / E296D, D118T / E296D / K300R, D118T / K300R, E296D, E296D / K300R, or K300R, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766.

[0258] In some embodiments, the engineered leucine decarboxylase polypeptide has an amino acid sequence at amino acid positions 5, 14, 14 / 34 / 38 / 39 / 102 / 267 / 275 / 350 / 357, 14 / 39 / 102 / 127 / 245 / 267 / 275 / 349 / 350, 34 / 38 / 39 / 102 / 127 / 275 / 357, 34 / 38 / 39 / 102 / 275 / 357, 34 / 38 / 39 / 127 / 245 / 349 / 350 / 357, 34 / 38 / 39 / 127 / 245 / 350 / 357, 34 / 39 / 102 / 127 / 264 / 275 ... 27 / 275 / 349 / 357, 34 / 39 / 102 / 264 / 275 / 350 / 357, 34 / 39 / 275 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 264 / 267 / 350 / 357, 38 / 39 / 102 / 127 / 267 / 275 / 349 / 350 / 35 7, 38 / 39 / 102 / 127 / 349 / 350 / 357, 38 / 39 / 102 / 127 / 350, 38 / 39 / 102 / 127 / 350 / 357, 38 / 39 / 127 / 245 / 267 / 357, 38 / 39 / 127 / 264 / 275, 38 / 39 / 127 / 264 / 350 / 357, 38 / 39 / 127 / 350 / 357, 38 / 39 / 127 / 357, 38 / 39 / 245 / 275 / 357, 38 / 39 / 264 / 267 / 275 / 350, 38 / 39 / 264 / 275 / 357, 38 / 39 / 275, 38 / 39 / 275 / 350, 39, 39 / 102 / 127 / 264 / 275 / 357, 39 / 102 / 264 / 275 / 357, 39 / 102 / 267 / 275 / 357, 39 / 127 / 245 / 264 / 267 / 275 / 350, 39 / 127 / 245 / 264 / 275 / 350 / 357, 39 / 127 / 245 / 3 57, 39 / 127 / 267 / 275 / 350 / 357, 39 / 127 / 267 / 350 / 357, 39 / 127 / 357, 39 / 245 / 264 / 267 / 275 / 357, 39 / 264 / 267 / 275 / 350, 39 / 275 / 350 / 357, 48, 139, 164, 196, 255, 299, 318, 324, 339, 343, 350, 353, 357, 364, 365, 379, 381, 386, 389, 391, 393, 394, 395, 397, 398, or 405;wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 12. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 5M, 14I, 14T / 34L / 38V / 39N / 102S / 267I / 275S / 350E / 357V, 14T / 39N / 102S / 127S / 245M / 267I / 275S / 349T / 350E, 34L / 38V / 39N / 102S / 127S / 275S / 357V, 34L / 38V / 39N / 102S / 275S / 357V, 34L / 38V / 39N / 102S / 245M / 34 9T / 350E / 357V, 34L / 38V / 39N / 127S / 245M / 350E / 357V, 34L / 39N / 102S / 1 27S / 264V / 275S / 357V, 34L / 39N / 102S / 127S / 275S / 349T / 357V, 34L / 39N / 102S / 264V / 275S / 350E / 357V, 34L / 39N / 275S / 349T / 350E / 357V, 38V / 3 9N / 102S / 127S / 264V / 267I / 350E / 357V, 38V / 39N / 102S / 127S / 267I / 275 S / 349T / 350E / 357V, 38V / 39N / 102S / 127S / 349T / 350E / 357V, 38V / 39N / 102S / 127S / 350E, 38V / 39N / 102S / 127S / 350E / 357V, 38V / 39N / 127S / 245 M / 267I / 357V, 38V / 39N / 127S / 264V / 275S, 38V / 39N / 127S / 264V / 350E / 3 57V, 38V / 39N / 127S / 350E / 357V, 38V / 39N / 127S / 357V, 38V / 39N / 245M / 2 75S / 357V, 38V / 39N / 264V / 267I / 275S / 350E, 38V / 39N / 264V / 275S / 357V , 38V / 39N / 275S, 38V / 39N / 275S / 350E, 39N / 102S / 127S / 264V / 275S / 357 V, 39N / 102S / 264V / 275S / 357V, 39N / 102S / 267I / 275S / 357V, 39N / 127S / 245M / 264V / 267I / 275S / 350E, 39N / 127S / 245M / 264V / 275S / 350E / 357V,<h2 style=";text-align:left;direction:ltr">39N / 127S / 245M / 357V, 39N / 127S / 267I / 275S / 350E / 357V, 39N / 127S / 267I / 350E / 357V, 39N / 127S / 357V, 39N / 245M / 264V / 267I / 275S / 357V, 39N / 264V / 267I / 275S / 350E, 39N / 275S / 350E / 357V, 39S, 48F, 139G 164A, 164C, 196D, 196R, 255G, 255N, 255P, 299A, 299V, 318K, 324M, 324S, 324T, 339A, 339D, 343A, 343E, 350S, 353D, 353E, 353L, 353N, 353S, 353W, 357C, 357M, 364K, 364R, 365E, 379D, 379P, 381D, 381E, 386<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 、389E、389G、389P、389Q、391<h2 style=";text-align:left;direction:ltr"> * <h2 style=";text-align:left;direction:ltr"> 391E, 393T, 394E, 395A, 395D, 395G, 395K, 395S, 397A, 398<h2 style=";text-align:left;direction:ltr"> *, 405D, 405E, 405H, or 405L, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 12. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions K5M, H14I, H14T / I34L / C38V / T39N / T102S / V267I / T275S / N350E / I357V, H14T / T39N / T102S / T127S / I245M / V267I / T275S / V349T / N350E, I34L / C38V / T39N / T102S / T127S / T275S / I357V, I34L / C38V / T39N / T102S / T127S / T275S / I357V, I34L / C38V / T39N / T10 2S / T275S / I357V, I34L / C38V / T39N / T127S / I245M / V349T / N350E / I357V, I34L / C38V / T39N / T127S / I245M / N350E / I357V, I34L / T39N / T102S / I34L / T39N / T 275S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / I264V / V267I / N350E / I357V, C38V / T39N / T102S / T127S / V267I / T275S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / V349T / N350E / I357V, C38V / T39N / T102S / T127S / N350E, C38V / T39N / T102S / T127S / N350E / I357V, C38V / T 39N / T127S / I245M / V267I / I357V, C38V / T39N / T127S / I264V / T275S, C38V / T39N / T127S / I264V / N350E / I357V, C38V / T39N / T127S / N350E / I35 7V, C38V / T39N / T127S / I357V, C38V / T39N / I245M / T275S / I357V, C38V / T39N / I264V / V267I / T275S / N350E, C38V / T39N / I264V / T275S / I357V,C38V / T39N / T275S, C38V / T39N / T275S / N350E, T39N / T102S / T127S / I264V / T275S / I357V, T39N / T102S / I264V / T275S / I357V, T39N / T102S / V267I / T275S / I357V, T39N / T127S / I245M / I264V / V 267I / T275S / N350E, T39N / T127S / I245M / I264V / T275S / N350E / I357V, T39N / T127S / I245M / I35 7V, T39N / T127S / V267I / T275S / N350E / I357V, T39N / T127S / V267I / N350E / I357V, T39N / T127S / I357V, T39N / I245M / I264V / V267I / T275S / I357V, T39N / I264V / V267I / T275S / N350E, T39N / T27 5S / N350E / I357V, T39S, L48F, N139G, I164A, I164C, K196D, K196R, H255G, H255N, H255P, K299A , K299V, R318K, R324M, R324S, R324T, Q339A, Q339D, H343A, H343E, N350S, R353D, R353E, R353L , R353N, R353S, R353W, I357C, I357M, L364K, L364R, Q365E, K379D, K379P, A381D, A381E, D386, * , K389E, K389G, K389P, K389Q, A391 * , A391E, K393T, K394E, R395A, R395D, R395G, R395K, R395S, T397A, P398 * , T405D, T405E, T405H, or T405L, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:12.

[0259] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 48 / 64 / 164 / 324 / 343 / 353 / 357 / 364, 48 / 64 / 164 / 324 / 343 / 364, 48 / 64 / 164 / 353 / 357 / 364, 48 / 64 / 357 / 364, 64 / 164 / 324 / 343 / 35 ... / 164 / 324 / 343 / 357 / 364, 64 / 164 / 353 / 357, 64 / 318 / 324 / 357 / 364, 64 / 324 / 353 / 357 / 364, 132 / 255 / 339 / 379 / 395, 164 / 196 / 324 / 357 / 364, 164 / 318 / 324 / 343 / 353 / 357, 164 / 318 / 324 / 357 / 364, 164 / 324 / 343 / 3 53 / 357 / 364, 164 / 324 / 357 / 364, 164 / 353 / 357 / 364, 164 / 364, 196 / 318 / 324 / 353 / 357 / 364, 318 / 343 / 357, 324 / 343 / 357 / 364, 324 / 353 / 357 / 364, 324 / 357 / 364, 339 / 379 / 389 / 394 / 395, 339 / 389 / 395, 339 / 391 , 339 / 394 / 395 / 405, 357 / 364, 379 / 386, 379 / 394 / 395 / 397 / 404 / 405, 379 / 394 / 395 / 397 / 405, 389 / 394 / 395 / 397 / 405, or 394 / 397, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:38.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 48F / 64E / 164A / 324M / 343E / 353E / 357C / 364K, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164C / 353N / 357V / 364R, 48F / 64E / 357M / 364K, 64E / 164A / 324M / 343E / 364R, M / 343E / 353D / 357V / 364K, 64E / 164A / 324M / 343E / 357C / 364R, 64E / 164C / 353D / 357V, 64E / 318K / 324S / 357V / 364R , 64E / 324M / 353N / 357C / 364R, 132F / 255P / 339A / 379D / 395D, 164A / 196D / 324M / 357C / 364K, 164A / 318K / 324M / 343 E / 353E / 357C, 164A / 324M / 343E / 353D / 357C / 364R, 164A / 324M / 357C / 364K, 164A / 353W / 357C / 364R, 164A / 364R, 164C / 318K / 324S / 357V / 364R, 164C / 324M / 343E / 353D / 357V / 364R, 164C / 353D / 357V / 364K, 164C / 353D / 357V / 364 R, 164C / 353W / 357C / 364R, 196D / 318K / 324M / 353N / 357C / 364K, 318K / 343E / 357C, 318K / 343E / 357M, 324M / 343E / 3 57V / 364K, 324M / 357M / 364R, 324N / 353W / 357C / 364K, 339A / 379D / 389G / 394E / 395D, 339A / 389G / 395K, 339A / 391. * , 339A / 394E / 395K / 405D, 357V / 364R, 379D / 386 *, 379D / 394E / 395D / 397A / 404I / 405H, 379D / 394E / 395K / 397A / 405D, 389G / 394E / 395D / 397A / 405D, or 394E / 397A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:38.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions L48F / A64E / I164A / R324M / H343E / R353E / I357C / L364K, L48F / A64E / I164A / R324M / H343E / L364R, L48F / A64E / I164C / R353N / I357V / L364R, L48F / A64E / I357M / L364K, A64E / I164A / R324M / H343E / R353E / I357C / L364R R353D / I357V / L364K, A64E / I164A / R324M / H343E / I357C / L364R, A64E / I164C / R353D / I357V, A64E / R318K / R324S / I357V / L364R, A64E / R324M / R353N / I357C / L364R, Y132F / H255P / Q339A / K379D / R395D, I164A / K196D / R324M / I357C / L364K, I164A / R318K / R324M / H343E / R3 53E / I357C, I164A / R324M / H343E / R353D / I357C / L364R, I164A / R324M / I357C / L364K, I164A / R353W / I357C / L364R, I164A / L364R, I164 C / R318K / R324S / I357V / L364R, I164C / R324M / H343E / R353D / I357V / L364R, I164C / R353D / I357V / L364K, I164C / R353D / I357V / L364R, I164C / R353W / I357C / L364R, K196D / R318K / R324M / R353N / I357C / L364K, R318K / H343E / I357C, R318K / H343E / I357M, R324M / H343E / I3 57V / L364K, R324M / I357M / L364R, R324N / R353W / I357C / L364K, Q339A / K379D / K389G / K394E / R395D, Q339A / K389G / R395K, Q339A / A391. * , Q339A / K394E / R395K / T405D, I357V / L364R, K379D / D386 *, K379D / K394E / R395D / T397A / R404I / T405H, K379D / K394E / R395K / T397A / T405D, K389G / K394E / R395D / T397A / T405D, or K394E / T397A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 38.

[0260] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises the amino acid sequence at amino acid positions 2, 3, 33, 48 / 64 / 255, 48 / 255 / 339, 48 / 255 / 379, 64, 64 / 255, 69, 161, 193, 255, 255 / 318 / 379, 259, 263, 318 / 339 / 379, 324, 324 / 389 / 394, 324 / 389 / 394 / 395, 324 / 389 / 394 / 397, 324 / 394, 324 / 394 / 395, 324 / 3 94 / 395 / 397, 324 / 395, 339, 340, 380, 382, ​​389, 389 / 394, 389 / 394 / 395, 389 / 394 / 395 / 397, 389 / 394 / 397, 389 / 395, 389 / 397, 390, 394, 394 / 395, 394 / 395 / 397, 395, 395 / 397, 397, 401, or 405, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:234. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 2E, 3M, 33L, 48F / 64E / 255P, 48F / 255P / 339A, 48F / 255P / 379D, 64E, 64E / 255P, 64S, 69I, 161V, 193I, 255P, 255P / 318K / 379D, 259L, 263T, 263V, 318K / 339A / 379D, 324N, 324N / 394E / 395K / 397A, 324N / 395D, 324S / 389G / 394E ... 389G / 394E / 395D, 324S / 389G / 394E / 397A, 324S / 394E, 324S / 394E / 395K, 32 4S / 394E / 395K / 397A, 324S / 395K, 339A, 340T, 340V, 380E, 382S, 389G, 389G / 394E, 389G / 394E / 395D, 389G / 394E / 395D / 397A, 389G / 394E / 395K, 389G / 3 94E / 395K / 397A, 389G / 394E / 397A, 389G / 395D, 389G / 395K, 389G / 397A, 390 *, 390A, 390E, 390S, 394E, 394E / 395D, 394E / 395K / 397A, 395D / 397A, 395K, 397A, 401 * , 401Y, or 405H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 234. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions G2E, N3M, F33L, L48F / A64E / H255P, L48F / H255P / Q339A, L48F / H255P / K379D, A64E, A64E / H255P, A64S, V 69I, T161V, M193I, H255P, H255P / R318K / K379D, R259L, S263T, S263V, R318K / Q339A / K37 9D, M324N, M324N / K394E / R395K / T397A, M324N / R395D, M324S / K389G / K394E, M324S / K389G / K394E / R395D, M324S / K389G / K394E / T397A, M324S / K394E, M324S / K394E / R395K, M324S / K394E / R395K / T397A, M324S / R395K, Q339A, S340T, S340V, A380E, A382S, K389G, K389G / K 394E, K389G / K394E / R395D, K389G / K394E / R395D / T397A, K389G / K394E / R395K, K389G / K3 94E / R395K / T397A, K389G / K394E / T397A, K389G / R395D, K389G / R395K, K389G / T397A, P390 * , P390A, P390E, P390S, K394E, K394E / R395D, K394E / R395K / T397A, R395D / T397A, R395K, T397A, A401 * , A401Y, or T405H, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 234.

[0261] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 2 / 64 / 69 / 324 / 380 / 382 / 388 / 389, 3 / 64 / 69 / 263 / 339 / 380 / 388, 3 / 64 / 69 / 389, 3 / 64 / 69 / 390, 3 / 64 / 379 / 380 / 390, 3 / 69 / 263 / 380, 3 / 69 / 324, 3 / 69 / 324 / 380 / 382 / 389 / 390, 12 / 135 / 259 / 263 ... 5 / 263 / 382, 12 / 259 / 263 / 304, 48 / 64 / 255, 64 / 69, 64 / 69 / 189 / 259 / 263 / 304, 64 / 69 / 189 / 259 / 263 / 304 / 339 / 340 / 379, 64 / 69 / 223 / 388, 64 / 69 / 223 / 388 / 389 / 390, 64 / 69 / 304 / 379 / 382, 64 / 69 / 324, 64 / 69 / 324 / 339 / 380 / 389 / 390, 64 / 69 / 339, 64 / 69 / 339 / 382 / 38 8 / 389, 64 / 69 / 339 / 389 / 390, 64 / 69 / 379 / 380, 64 / 69 / 380 / 388 / 390, 64 / 69 / 389, 64 / 69 / 390, 64 / 255 / 263, 64 / 263, 64 / 324 / 339 / 389 / 390, 69 / 223 / 263 / 324 / 382 / 388 / 390, 69 / 223 / 324 / 379 / 380 / 382 / 388 / 390, 69 / 263, 69 / 263 / 324, 69 / 263 / 339, 69 / 263 / 388, 69 263 / 390, 259 / 263 / 304, 259 / 263 / 304 / 339 / 340 / 379, 263 / 339 / 389 / 390, 263 / 390, or 304 / 340 / 379 / 380 / 382, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:284.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 2E / 64S / 69I / 324S / 380E / 382S / 388A / 389G, 3M / 64S / 69I / 263T / 339A / 380E / 388A, 3M / 64S / 69I / 389G, 3M / 64S / 69I / 390. * , 3M / 64S / 379D / 380E / 390 * , 3M / 69I / 263T / 380E, 3M / 69I / 324S, 3M / 69I / 324S / 380E / 382S / 389G / 390 * , 12G / 135V / 259K / 263T, 12G / 135V / 263T / 382G, 12G / 259K / 263T / 304R, 48L / 64A / 255H, 64A / 255H / 263T, 64S / 69I, 64S / 69I / 189 A / 259Q / 263T / 304R / 339A / 340T / 379N, 64S / 69I / 189D / 259K / 263T / 304R, 64S / 69I / 223M / 388A, 64S / 69I / 223M / 388A / 389G / 390 * , 64S / 69I / 304R / 379E / 382G, 64S / 69I / 324S, 64S / 69I / 324S / 339A / 380E / 389G / 390 * , 64S / 69I / 339A, 64S / 69I / 339A / 382S / 388A / 389G, 64S / 69I / 339A / 389G / 390 * , 64S / 69I / 379D / 380E, 64S / 69I / 380E / 388A / 390 * , 64S / 69I / 389G, 64S / 69I / 390 * , 64S / 263T, 64S / 324S / 339A / 389G / 390 * , 69I / 223M / 263T / 324S / 382S / 388A / 390 * , 69I / 223M / 324S / 379D / 380E / 382S / 388A / 390 * , 69I / 263T, 69I / 263T / 324S, 69I / 263T / 339A, 69I / 263T / 388A, 69I / 263T / 389G / 390 *, 69I / 324S / 379D / 380E / 388A, 69I / 324S / 380E, 69I / 339A / 390 * , 69I / 382S / 390 * , 259K / 263T / 304R, 259K / 263T / 304R / 339A / 340T / 379N, 263T / 339A / 389G / 390 * , 263T / 390 * or 304R / 340T / 379D / 380E / 382G, where the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 284. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions G2E / E64S / V69I / M324S / A380E / A382S / Q388A / K389G, N3M / E64S / V69I / S263T / Q339A / A380E / Q388A, N3M / E64S / V69I / K389G, N3M / E64S / V69I / P390 * , N3M / E64S / K379D / A380E / P390 * , N3M / V69I / S263T / A380E, N3M / V69I / M324S, N3M / V69I / M324S / A380E / A382S / K389G / P390 * , S12G / L135V / R259K / S263T, S12G / L135V / S263T / A382G, S12G / R259K / S263T / A304R, F48L / E64A / P255H, E64A / P255H / S263T, E64S / V69I, E64S / V69I / T18 9A / R259Q / S263T / A304R / Q339A / S340T / K379N, E64S / V69I / T189D / R259K / S2 63T / A304R, E64S / V69I / A223M / Q388A, E64S / V69I / A223M / Q388A / K389G / P390 * , E64S / V69I / A304R / K379E / A382G, E64S / V69I / M324S, E64S / V69I / M324S / Q339A / A380E / K389G / P390 *, E64S / V69I / Q339A, E64S / V69I / Q339A / A382S / Q388A / K389G, E64S / V69I / Q339A / K389G / P390 * , E64S / V69I / K379D / A380E, E64S / V69I / A380E / Q388A / P390 * , E64S / V69I / K389G, E64S / V69I / P390 * , E64S / S263T, E64S / M324S / Q339A / K389G / P390 * ,V69I / A223M / S263T / M324S / A382S / Q388A / P390 * ,V69I / A223M / M324S / K379D / A380E / A382S / Q388A / P390 * , V69I / S263T, V69I / S263T / M324S, V69I / S263T / Q339A, V69I / S263T / Q388A, V69I / S263T / K389G / P390 * , V69I / M324S / K379D / A380E / Q388A, V69I / M324S / A380E, V69I / Q339A / P390 * , V69I / A382S / P390 * , R259K / S263T / A304R, R259K / S263T / A304R / Q339A / S340T / K379N, S263T / Q339A / K389G / P390 * , S263T / P390 * or A304R / S340T / K379D / A380E / A382G, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 284.

[0262] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 3 / 194 / 304, 3 / 259 / 263 / 304, 3 / 259 / 304, 3 / 259 / 304 / 324 / 339, 3 / 259 / 304 / 324 / 382, 3 / 259 / 304 / 382, 3 / 263 / 304 / 324, 3 / 263 / 304 / 324 / 339, 3 / 263 / 304 / 324 / 382, 3 / 304, 3 / 304 / 324, 16, 63, 77, 80, 87 / 270, 87 / 270 / 365, 87 / 328 / 365, 91, 92, 126, 140, 156, 168 / 270 / 328 / 338, 181, 194, 201, 256, 259, 259 / 263, 259 / 263 / 304, 259 / 263 / 304 / 3 24, 259 / 263 / 304 / 324 / 382, 259 / 263 / 304 / 379, 259 / 263 / 304 / 382, 259 / 304, 259 / 304 / 324, 259 / 304 / 324 / 339, 259 / 304 / 324 / 339 / 382, 259 / 304 / 382, 262, 263 / 304, 263 / 304 / 324, 263 / 304 / 324 / 339, 26 3 / 304 / 324 / 382, 263 / 324, 270, 270 / 319, 270 / 328 / 338, 270 / 328 / 338 / 365, 304, 304 / 324, 324, 328, 352, 365, 366, or 382, ​​wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 3M / 194L / 304R, 3M / 259K / 263T / 304R, 3M / 259K / 304R, 3M / 259K / 304R / 324S / 339A, 3M / 259K / 304R / 324S / 382S, 3M / 259K / 304R / 382S , 3M / 263T / 304R / 324S, 3M / 263T / 304R / 324S / 339A, 3M / 263T / 304R / 324S / 382S, 3M / 304R, 3M / 304R / 324 S, 16Q, 16V, 63C, 77L, 80G, 80K, 87R / 270R, 87R / 270R / 365E, 87R / 328N / 365E, 91A, 91Q, 92K, 126A, 126T,140V, 156A, 156S, 168K / 270R / 328N / 338S, 181K, 181R, 181V, 194C, 194L, 201D, 256 W, 259K, 259K / 263T, 259K / 263T / 304R, 259K / 263T / 304R / 324S, 259K / 263T / 304R / 3 24S / 382S, 259K / 263T / 304R / 379D, 259K / 263T / 304R / 382S, 259K / 304R, 259K / 304R / 324S, 259K / 304R / 324S / 339A, 259K / 304R / 324S / 339A / 382S, 259K / 304R / 382S, 26 2D, 262G, 262H, 262I, 262S, 262T, 263T / 304R, 263T / 304R / 324S, 263T / 304R / 324S / 339A, 263T / 304R / 324S / 382S, 263T / 324S, 270R, 270R / 319A, 270R / 328N / 338S, 270R / 328N / 338S / 365E, 304R, 304R / 324S, 324S, 328N, 352A, 365E, 366A, 366L, 366M, 366Q, 366T, 366V, or 382S, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:484. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions N3M / F194L / A304R, N3M / R259K / S263T / A304R, N3M / R259K / A304R, N3M / R259K / A304R / M324S / Q339A, N3M / R259K / A304R / M324S / A382S, N3M / R259K / A304R / A382S, N3M / S263T / A304R / M324S, N3M / S263T / A 304R / M324S / Q339A, N3M / S263T / A304R / M324S / A382S, N3M / A304R, N3M / A304R / M324S, R16Q, R16V, A63C, E77L, A80G, A80K, H87R / L270R, H 87R / L270R / Q365E, H87R / C328N / Q365E, E91A, E91Q, E92K, D126A, D126T, M140V, G156A, G156S, C168K / L270R / C328N / P338S, T181K, T181R,T181V, F194C, F194L, E201D, Y256W, R259K, R259K / S263T, R259K / S263T / A304R, R259K / S 263T / A304R / M324S, R259K / S263T / A304R / M324S / A382S, R259K / S263T / A304R / K379D, R25 9K / S263T / A304R / A382S, R259K / A304R, R259K / A304R / M324S, R259K / A304R / M324S / Q339 A, R259K / A304R / M324S / Q339A / A382S, R259K / A304R / A382S, R262D, R262G, R262H, R262I, R262S, R262T, S263T / A304R, S263T / A304R / M324S, S263T / A304R / M324S / Q339A, S263T / A 304R / M324S / A382S, S263T / M324S, L270R, L270R / I319A, L270R / C328N / P338S, L270R / C32 8N / P338S / Q365E, A304R, A304R / M324S, M324S, C328N, D352A, Q365E, H366A, H366L, H366M, H366Q, H366T, H366V, or A382S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 484.

[0263] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of amino acid positions 16 / 63 / 80 / 126 / 168 / 366, 16 / 63 / 80 / 126 / 181 / 194 / 259 / 324 / 328 / 366, 16 / 63 / 126 / 168 / 270 / 328 / 366, 16 / 80 / 126 / 324 / 366, 16 / 80 / 126 / 366, 16 / 80 / 168, 16 / 80 / 168 / 270 / 366, 16 / 80 / 168 / 324, 16 / 80 / 168 / 366, 16 / 80 / 324, 16 / 91 / 126 / 168 / 324 / 366, 16 / 126 / 168 / 36 6, 16 / 168 / 259 / 366, 16 / 168 / 270 / 324 / 366, 16 / 168 / 324 / 328 / 366, 16 / 168 / 324 / 366, 16 / 168 / 366, 16 / 259 / 263 / 328, 16 / 324 / 328 / 366, 16 / 328 / 366, 80 / 126 / 168 / 270 / 366, 80 / 126 / 168 / 366, 80 / 126 / 181 / 270 / 324 / 366, 80 / 168 / 270 / 366, or 168 / 366, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 16Q / 63C / 80K / 126T / 168K / 366M, 16Q / 63C / 80K / 126T / 181R / 194C / 259K / 324S / 328N / 366M, 16Q / 63C / 126T / 168K / 270R / 32 8N / 366M, 16Q / 80K / 126T / 324S / 366M, 16Q / 80K / 126T / 366M, 16Q / 80K / 168K, 16Q / 80K / 168K / 270R / 366M, 16Q / 80K / 168K / 324S, 16Q / 80K / 168K / 366M, 16Q / 80K / 324S, 16Q / 91A / 126T / 168K / 324S / 366M , 16Q / 126T / 168K / 366M, 16Q / 168K / 259K / 366M, 16Q / 168K / 270R / 324S / 366M, 16Q / 168K / 324S / 328 N / 366M, 16Q / 168K / 324S / 366M, 16Q / 168K / 366M, 16Q / 259K / 263T / 328N, 16Q / 324S / 328N / 366M, 16Q / 328N / 366M, 80K / 126T / 168K / 270R / 366M, 80K / 126T / 168K / 366M, 80K / 126T / 181R / 270R / 324S / 366M, 80K / 168K / 270R / 366M, or 168K / 366M, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions R16Q / A63C / A80K / D126T / C168K / H366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366 M, R16Q / A80K / D126T / M324S / H366M, R16Q / A80K / D126T / H366M, R16Q / A80K / C168K, R16Q / A80K / C168K / L270R / H366 M, R16Q / A80K / C168K / M324S, R16Q / A80K / C168K / H366M, R16Q / A80K / M324S, R16Q / E91A / D126T / C168K / M324S / H366M , R16Q / D126T / C168K / H366M, R16Q / C168K / R259K / H366M, R16Q / C168K / L270R / M324S / H366M, R16Q / C168K / M324S / C 328N / H366M, R16Q / C168K / M324S / H366M, R16Q / C168K / H366M, R16Q / R259K / S263T / C328N, R16Q / M324S / C328N / H366 M, R16Q / C328N / H366M, A80K / D126T / C168K / L270R / H366M, A80K / D126T / C168K / H366M, A80K / D126T / T181R / L270R / M324S / H366M, A80K / C168K / L270R / H366M, or C168K / H366M, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:594.

[0264] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises the amino acid sequences at amino acid positions 66 / 76 / 118 / 141 / 201 / 300, 66 / 76 / 198 / 200 / 296 / 303, 66 / 76 / 198 / 200 / 300, 66 / 118 / 200 / 296 / 303 / 317, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 200, 76 / 118 / 141 / 200 / 296, 76 / 141 / 198 / 200 / 201 / 300, 80 / 201 / 270, and at least a substitution or set of substitutions in 80 / 270, 80 / 270 / 324, 89 / 118 / 200, 106 / 270 / 324 / 352, 118 / 141 / 200, 126, 126 / 201 / 270 / 324, 126 / 270, 141 / 144 / 198 / 200 / 300, 156 / 270, 156 / 270 / 324, 201 / 270, 201 / 270 / 352, 270, or 270 / 324, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:686. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 66N / 76V / 118D / 141P / 201R / 300K, 66N / 76V / 198G / 200S / 296E / 303Q, 66N / 76V / 198G / 200S / 300K, 66N / 118D / 200S / 296E / 303Q / 317Q, 66N / 118D / 296E, 66N / 118D / 296E / 300K, 66N / 200S, 76V / 118D / 141P / 200S / 296E, 76V / 141P / 198G / 200S / 201R / 300 K, 80K / 201D / 270R, 80K / 270R, 80K / 270R / 324S, 89P / 118D / 200S, 106M / 270R / 324S / 352A, 118D / 141P / 200S, 126T, 126T / 201D / 270R / 324S, 126T / 270R, 141P / 144V / 198G / 200S / 300K, 156A / 270R, 156A / 270R / 324S, 201D / 270R, 201D / 270R / 352A, 270R, or 270R / 324S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions S66N / R76V / T118D / R141P / E201R / R300K, S66N / R76V / A198G / H200S / D296E / A303Q, S66N / R76V / A198G / H200S / R 300K, S66N / T118D / H200S / D296E / A303Q / K317Q, S66N / T118D / D296E, S66N / T118D / D296E / R3 00K, S66N / H200S, R76V / T118D / R141P / H200S / D296E, R76V / R141P / A198G / H200S / E201R / R300 K, A80K / E201D / L270R, A80K / L270R, A80K / L270R / M324S, A89P / T118D / H200S, L106M / L270R / M324S / D352A, T118D / R141P / H200S, D126T, D126T / E201D / L270R / M324S, D126T / L270R, R141 P / M144V / A198G / H200S / R300K, G156A / L270R, G156A / L270R / M324S, E201D / L270R, E201D / L270R / D352A, L270R, or L270R / M324S, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.

[0265] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid position 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 19I, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 173I, 173T, 187L, 211S, or 312A, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A211S, and T312A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 686.

[0266] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises the amino acid positions 19 / 109 / 123 / 141 / 170 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 123 / 141 / 170 / 198 / 211, 19 / 109 / 123 / 141 / 170 / 198 / 211 / 270 / 312, 19 / 109 / 123 / 170 / 211 / 270 / 312, 19 / 109 / 123 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 170 / 173 / 211 / 270 / 312, 19 / 109 / 211 / 270 / 312, 109 / 170 / 211 / 270 / 312, or 109 / 211 / 270 / 312, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:688. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 19I / 109G / 123F / 170A / 211S / 270R / 312A, 19I / 109G / 123F / 198G / 200S / 211S / 270R / 312A, 19I / 109G / 123V / 141P / 170A ... / 109G / 123V / 141P / 170A / 198G / 211S, 19I / 109G / 123V / 141P / 170A / 198G / 211S / 270R / 312A, 19I / 109G / 170A / 173I / 211S / 270R / 312A, 19I / 109G / 211S / 270R / 312A, 109G / 170A / 211S / 270R / 312A, or 109G / 211S / 270R / 312A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:688.In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions L19I / L109G / Y123F / P170A / A211S / L270R / T312A, L19I / L109G / Y123F / A198G / H200S / A211S / L270R / T312A, L19I / L109G / Y123V / R141P / P170A / A198G / H200S / A211S / L270R / T312A, L19I / L109G / Y123V / R141P / P170A / A198G / H200S / A211S / L270R / T312A, A198G / A211S, L19I / L109G / Y123V / R141P / P170A / A198G / A211S / L270R / T312A, L19I / L109G / P170A / F173I / A211S / L270R / T312A, L19I / L109G / A211S / L270R / T312A, L109G / P170A / A211S / L270R / T312A, or L109G / A211S / L270R / T312A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 688.

[0267] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 5 / 41, 5 / 41 / 228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 5V / 41D, 5V / 41D / 228D, 33L, 41D, 47F, 51E, 51Q, 55I, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 353I, 353L, 357S, or 384W, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions K5V / H41D, K5V / H41D / T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, or P384W, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 766.

[0268] In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions at amino acid positions 66, 66 / 118, 66 / 118 / 296, 66 / 118 / 296 / 300, 66 / 118 / 300, 66 / 296, 66 / 296 / 300, 66 / 300, 118, 118 / 296, 118 / 296 / 300, 118 / 300, 296, 296 / 300, or 300, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions 66S, 66S / 118T, 66S / 118T / 296D, 66S / 118T / 296D / 300R, 66S / 118T / 300R, 66S / 296D, 66S / 296D / 300R, 66S / 300R, 118T, 118T / 296D, 118T / 296D / 300R, 118T / 300R, 296D, 296D / 300R, or 300R, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions N66S, N66S / D118T, N66S / D118T / E296D, N66S / D118T / E296D / K300R, N66S / D118T / K300R, N66S / E296D, N66S / E296D / K300R, N66S / K300R, D118T, D118T / E296D, D118T / E296D / K300R, D118T / K300R, E296D, E296D / K300R, or K300R, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:766.

[0269] In some embodiments, the engineered leucine decarboxylase polypeptide for the therapeutic uses and methods herein is an engineered leucine decarboxylase polypeptide as described above and disclosed herein.

[0270] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828 or 888.

[0271] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.

[0272] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO:828, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:828.

[0273] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0274] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828.

[0275] In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828, wherein the amino acid sequence is at or near amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 141 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 272 / 383, 41 / 141 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 126 / 267, 64 / 267 / 353 / 384, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 19 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 19 / 55 / 64 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:828. In some additional embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 170P / 270L / 383S, 270L, 41D / 173I, 272A, 5V / 141P / 272A / 383S, 41D / 383S, 41D / 141P / 187L / 272A / 290I,41D / 141P / 173I / 290I, 5V / 272A / 383S, 5V / 41D / 173I / 272A / 383S, 41D / 141P, 141P / 272A, 353E / 384W, 272A / 383S, 4 1D / 141P / 173I, 41D / 272A / 383S, 41D / 141P / 187L / 200S / 202H / 272A, 33L / 55I / 64N / 126A / 270L / 357S, 33L / 126A / 353 E / 357S, 55I / 64N / 267L / 353E / 384W, 33L / 64N / 357S, 126A / 267L, 64N / 267L / 353E / 384W, 33L / 55I / 64N / 357S, 19L / 64 N / 126A / 267L, 55I / 267L, 33L / 126A / 267L / 270T / 312T / 357S, 19L / 33L / 55I / 353E / 357S / 384W, 19L / 33L / 126A, 126A / 312T, 126A / 198G / 202H / 267L / 312T, 126A / 353E, 55I / 126A, 126A / 270T / 384W, 33L / 64N / 353E / 357S, 19L / 267L, 51E / 55I / 267L / 270T / 353E, 33L / 126A / 267L / 270T, 19L / 55I / 64N / 126A / 267L / 270T / 353E, 19L / 33L / 126A / 270T / 353E / 3 57S / 384W, 19L / 33L / 64N / 267L / 353E, 126A / 353E / 384W, 126A / 270T / 312T / 353E / 384W, 19L / 33L / 55I / 126A, 33L / 357S, 47F / 51E / 64N / 126A / 353E / 384W, or 126A, 126A / 270T, wherein the amino acid positions are relative to a reference sequence corresponding to sequence SEQ ID NO: 828. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions A170P / R270L / A383S, R270L, H41D / F173I, T272A, K5V / R141P / T272A / A383S, H41D / A383S, H41D / R141P / V187L / T272A / V290I, H41D / R141P / F173I / V290I, K5V / T272A / A383S, K5V / H41D / F173I / T272A / A383S, H41D / R141P,R141P / T272A, D353E / P384W, T272A / A383S, H41D / R141P / F173I, H41D / T272A / A383S, H41D / R141P / V187L / H200S / S202H / T272A , F33L / V55I / S64N / D126A / R270L / C357S, F33L / D126A / D353E / C357S, V55I / S64N / I267L / D353E / P384W, F33L / S64N / C357S, D12 6A / I267L, S64N / I267L / D353E / P384W, F33L / V55I / S64N / C357S, I19L / S64N / D126A / I267L, V55I / I267L, F33L / D126A / I267L / R 270T / A312T / C357S, I19L / F33L / V55I / D353E / C357S / P384W, I19L / F33L / D126A, D126A / A312T, D126A / A198G / S202H / I267L / A3 12T, D126A / D353E, V55I / D126A, D126A / R270T / P384W,, F33L / S64N / D353E / C357S, I19L / I267L, L51E / V55I / I267L / R270T / D35 3E, F33L / D126A / I267L / R270T, I19L / V55I / S64N / D126A / I267L / R270T / D353E, I19L / F33L / D126A / R270T / D353E / C357S / P384W , I19L / F33L / S64N / I267L / D353E, D126A / D353E / P384W, D126A / R270T / A312T / D353E / P384W, I19L / F33L / V55I / D126A, F33L / C357S, L47F / L51E / S64N / D126A / D353E / P384W, D126A, or D126A / R270T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828.

[0276] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least two or more substitutions at amino acid positions 33, 55, 64, 126, 270, and 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least three or more substitutions at amino acid positions 33, 55, 64, 126, 270, and 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 33 / 55, 33 / 64, 33 / 126, 33 / 270, 33 / 357, 55 / 64, 55 / 126, 55 / 270, 55 / 357, 64 / 126, 64 / 270, 64 / 357, 126 / 270, 126 / 357, or 270 / 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the engineered leucine decarboxylase polypeptide comprises at least a set of substitutions at amino acid positions 33 / 55 / 64, 33 / 55 / 126, 33 / 64 / 126, 55 / 64 / 270, 55 / 64 / 357, or 64 / 126 / 270, wherein said amino acids are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least substitutions at amino acid positions 33, 55, 64, 126, 270, and 357, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 828 or 888.In some embodiments, the substitutions at amino acid positions 33, 55, 64, 126, 270, and 357 are selected from 33L, 55I, 64N, 126A, 270L, and 357S. In some embodiments, the substitutions at amino acid positions 33, 55, 64, 126, 270, and 357 are selected from F33L, V55I, S64N, D126A, R270L, and C357S.

[0277] In some embodiments, the engineered leucine decarboxylase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence contains one or more substitutions relative to a reference sequence corresponding to SEQ ID NO: 888.

[0278] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein said amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0279] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase comprises amino acid residues 5V, 19L, 33L, 41D, 47F, 51E, 55I, 64S / N, 141P, 170P, 173I, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 290I, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0280] In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 202 / 357, 202 / 353, 5 / 51 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 353, 357, 64, 5 / 41 / 141, 41 / 141 / 173 / 202 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 64 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 353 / 357, 5 / 41 / 64 / 173 / 353 / 357, 64 / 173 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 202 / 272 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 353, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / 173 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202, or 5 / 173 / 272, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide contains at least one substitution or set of substitutions 64S / 173I / 202H / 353E / 384W,<h2 style=";text-align:left;direction:ltr">41D / 141P / 272A / 353E、141P / 202H / 272A / 353E / 357C、173I / 202H / 357C、202H / 353E、5V / 51E / 173I / 272A / 353E / 384W、51E / 202H / 272A / 357C、141P / 173I / 272A、272A、41D / 173I / 384W、41D / 64S / 141P / 353E / 357C / 383S、141P / 173I / 202H、5V / 51E / 64S / 202H / 353E、357C、64S、5V / 41D / 141P、41D / 141P / 173I / 2 02H / 353E, 353E, 202H / 357C, 51E / 141P, 202H / 272A / 353E, 202H, 51E / 141P, 173I / 353E / 384W, 41D / 141P / 173I / 202H / 272A / 353E / 383S / 384W, 64S / 202H / 357C, 5V / 64S / 353E / 383S / 384W, 41D / 272A / 353E / 383S, 41D / 173I / 272A / 353E / 357C, 51E / 141P / 272A / 353E / 357C / 383S / 384W, 41D / 353E / 357C, 173I / 2 72A / 353E / 357C, 5V / 41D / 64S / 173I / 353E / 357C, 64S / 173I / 357C, 51E / 272A, 51E / 64S / 357C / 384W, 51E / 141P / 173I / 272A / 353E, 64S / 202H / 272A / 353E / 357C / 384W, 51E / 272A / 357C, 51E / 173I / 272A / 353E / 384W, 353E / 384W, 202H / 272A / 357C, 64S / 141P / 173I / 202H / 353E / 357C, 5V / 41D / 51E / 202H / 357C / 38 3S, 5V / 51E / 173I / 272A / 383S, 41D / 141P / 272A, 51E / 173I, 5V / 353E, 41D / 64S / 173I / 272A / 353E / 383S, 5V / 64S / 173I / 272A / 353E, 51E / 64S, 41D / 357C / 38 3S, 41D / 173I / 353E / 357C, 202H / 272A / 383S, 202H / 272A, 353E / 357C, 41D / 173I / 202H / 272A / 357C, 141P / 173I / 202H / 272A / 353E / 357C, 64S / 141P / 202H,or 5V / 173I / 272A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888. In some embodiments, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or set of substitutions N64S / F173I / S202H / D353E / P384W, H41D / R141P / T272A / D353E, R141P / S202H / T272A / D353E / S357C, F173I / S202H / S357C, S202H / D353E, K5V / L51E / F173I / T272A / D353E / P384W, L51E / S202H / T272A / S357C, R141P / F 173I / T272A, T272A, H41D / F173I / P384W, H41D / N64S / R141P / D353E / S3 57C / A383S, R141P / F173I / S202H, K5V / L51E / N64S / S202H / D353E, S357 C, N64S, K5V / H41D / R141P, H41D / R141P / F173I / S202H / D353E, D353E, S202H / S357C, L51E / R141P / S202H / T272A / D353E, S202H, L51E / R141P / F 173I / D353E / P384W, H41D / R141P / F173I / S202H / T272A / D353E / A383S / P384W, N64S / S202H / S357C, K5V / N64S / D353E / A383S / P384W, H41D / T27 2A / D353E / A383S, H41D / F173I / T272A / D353E / S357C, L51E / R141P / T27 2A / D353E / S357C / A383S / P384W, H41D / D353E / S357C, F173I / T272A / D3 53E / S357C, K5V / H41D / N64S / F173I / D353E / S357C, N64S / F173I / S357C , L51E / T272A, L51E / N64S / S357C / P384W, L51E / R141P / F173I / T272A / D 353E, N64S / S202H / T272A / D353E / S357C / P384W, L51E / T272A / S357C, L 51E / F173I / T272A / D353E / P384W, D353E / P384W, S202H / T272A / S357C,N64S / R141P / F173I / S202H / D353E / S357C, K5V / H41D / L51E / S202H / S357C / A383S, K5V / L51E / F173I / T272A / A383S, H41D / R141P / T27 2A, L51E / F173I, K5V / D353E, H41D / N64S / F173I / T272A / D353E / A383S, K5V / N64S / F173I / T272A / D353E, L51E / N64S, H41D / S357C / A3 83S, H41D / F173I / D353E / S357C, S202H / T272A / A383S, S202H / T272A, D353E / S357C, H41D / F173I / S202H / T272A / S357C, R141P / F173I / S202H / T272A / D353E / S357C, N64S / R141P / S202H, or K5V / F173I / T272A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO: 888.

[0281] In some embodiments of the therapeutic uses and methods of treatment, the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises a substitution or set of substitutions of an engineered leucine decarboxylase provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and / or 12-2.

[0282] In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises the amino acid sequence of an engineered leucine decarboxylase provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and / or 12-2.

[0283] In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a sequence corresponding to an even-numbered SEQ ID NO:2-1064. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having an even-numbered SEQ ID NO:2-1064. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO:16-1064.

[0284] In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 16-852. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 16-204. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 206-278. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 280-390. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 392-484. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 486-636. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 638-686. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 688-736. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 738-762. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 764-780. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 782-822. In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO: 824-852.In some embodiments of the therapeutic uses and methods of treatment, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO:854-1064.

[0285] In some embodiments, the leucine decarboxylase for therapeutic uses and methods herein exhibits at least one improved property compared to wild-type leucine decarboxylase of Planctomycetaceae bacterial species. In some embodiments, the engineered leucine decarboxylase polypeptide exhibits higher activity on leucine than wild-type leucine decarboxylase of Planctomycetaceae bacterial species or leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is more stable than wild-type leucine decarboxylase of Planctomycetaceae bacterial species or leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is more resistant to proteolysis than wild-type leucine decarboxylase of Planctomycetaceae bacterial species or leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is more tolerant to low pH environments than wild-type leucine decarboxylase of Planctomycetaceae bacterial species or leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is less immunogenic than a wild-type leucine decarboxylase from a Planctomycetaceae bacterial species or a leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is more serum stable than a wild-type leucine decarboxylase from a Planctomycetaceae bacterial species or a leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO: 12.

[0286] In some additional embodiments, the polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used in gene therapy to treat and / or prevent symptoms of a disease or condition associated with dysfunction of leucine, isoleucine, and / or alloisoleucine metabolism. In some embodiments, the polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used in gene therapy to treat and / or prevent symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels. In some embodiments, the polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used in gene therapy to treat a subject with maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency. In some embodiments, the polynucleotide encoding an engineered leucine decarboxylase polypeptide is codon-optimized for expression in a human patient. In some embodiments, the polynucleotide for use in gene therapy is DNA or RNA. In some embodiments, the polynucleotide or composition thereof for use in gene therapy comprises mRNA.

[0287] These and other aspects of the present invention may be better understood in connection with the following non-limiting examples, which are provided for illustrative purposes only and are not intended to limit the scope of the invention in any way. Experimental Example

[0288] The following examples, including the experiments and results achieved, are provided for illustrative purposes only and are not to be construed as limiting the invention.

[0289] In the experimental disclosure that follows, the following abbreviations apply: ppm (parts per million); M (mol); mM (millimolar); μM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); μg and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); μM and μm (micrometers); sec. (seconds); min(s) (minutes); h(s) and hr(s) (hours); U (units); MW (molecular weight); rpm (revolutions per minute); psi and PSI (pounds per square inch); °C (degrees Celsius); RT and rt (room temperature); CDS (coding sequence); DNA (deoxyribonucleic acid); RNA (ribonucleic acid); AUC (area under the curve); limit of quantification (LOQ); E. coli W3110 (a commonly used laboratory strain of E. coli, Coli Genetic Stock Center [CGSC], New Haven, CT available from); iMSUD (Intermediate Maple Syrup Urine Disease); HTP (High-Throughput); HPLC (High-Pressure Liquid Chromatography); LC (Liquid Chromatography); MS (Mass Spectrometry); LC-MS / MS (Liquid Chromatography Coupled with Two Mass Spectrometers); SPE (Solid Phase Extraction); KIC (Ketoisocaproic Acid); IPTG (Isopropyl β-D-1-thiogalactopyranoside); PLP (Pyridoxal 5'-phosphate); BSA (Bovine Serum Albumin); BW (Body Weight); MSUD (Maple Syrup Urine Disease); FIOPC (Fold Improvement over Positive Control); LB (Luria Broth); TB (Terrific Broth); Innovative Research (Innovative Research, Novi, MI); Microfluidics (Microfluidics Corp., Newton, MA); Thermotron (Thermotron, Holland, MI); Waters (Waters Corp., Milford, MA); Infors (Infors AG, Bottmingen, Switzerland); Cambridge Isotope Laboratories (Cambridge Isotope Laboratories, Inc., Tewksbury, MA); Sigma-Aldrich (Sigma-Aldrich, St. Louis, MO); Applied Biosystems (Applied Biosystems, part of Life Technologies, Corp., Grand Island, NY), Agilent (Agilent Technologies, Inc., Santa Clara, CA); Thermo Scientific (part of Thermo Fisher Scientific, Waltham, MA); Gibco (Gibco, part of Thermo Fisher Scientific, Waltham, MA); (Khuner Shaker, Inc., Santa Clara, CA); BN Labs (British Nutritions, LLC, Irvine, CA); GraphPad Software (GraphPad Software, San Diego, CA); and Corning (Corning, Inc., Palo Alto, CA). .

[0290] Example 1 High-speed (HTP) growth of Escherichia coli expressing leucine decarboxylase (LDC) variants and LDC screening conditions HTP growth of Escherichia coli expressing LDC variants Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose. After overnight incubation at 37°C, the cells were selected in wells of 96-well shallow flat-bottom plates (Nunc™, Thermo-Scientific) filled with 180 μl / well of LB supplemented with 1% glucose. Cultures were grown overnight (18-20 h) on a shaker (200 rpm, 30°C, 85% relative humidity; Kulner). Samples (20 μL) of the overnight culture were transferred to Costar® 96-well deep-bottom plates (Corning) filled with 380 μL of Terrific Broth supplemented with selection compounds. Plates were incubated in a shaker (250 rpm, 30°C, 85% relative humidity; Kulner) for approximately 2 h. The cells were then induced with 40 μL of 10 mM IPTG and incubated overnight (20–24 h) in a shaker (250 rpm, 30°C, 85% relative humidity, Kuhner). The cells were pelleted (4000 rpm x 20 min), the supernatant was discarded, and the cells were frozen at -80°C and then lysed.

[0291] Dissolution of HTP pellets E. coli cell pellets were lysed in 400 μL of lysis buffer (20 mM sodium phosphate (pH 7), 0.04 mM pyridoxal 5'-phosphate (PLP), 1 mg / ml lysozyme, 0.5 g / L polymyxin B sulfate (PMBS)). The mixture was stirred for 1.5 h at room temperature and after pelleting (4000 rpm x 10 min), the clarified lysate was pre-incubated for 1 h at 60 °C in a Multitron plate shaker (250 or 400 rpm, Infors HT). The heat-treated lysate was pelleted (4000 rpm x 10 min) and the supernatant was used for the HTP assay.

[0292] HTP analysis of LDC activity in clarified lysates LDC activity was assessed by adding diluted heat-treated clarified lysate to a reaction mixture to a final concentration of 3 mM leucine in 20 mM sodium phosphate (pH 7.0) or a mixture of simulated intestinal fluid salts. In some experiments, a reaction mixture with a final concentration of 3 mM leucine, isoleucine-d10, valine, methionine, and cysteine ​​(5AA mix) was used. Reactions were incubated for 1 h at 37°C and 250 rpm on a Multitron plate shaker (Infors HT), then quenched with 3 volumes of acetonitrile containing 0.1% formic acid, centrifuged for 10 min at 4°C at 4000 rpm, and diluted 50-fold with water. The resulting samples were analyzed by RapidFire®-MS / MS (Agilent) for isopentylamine, a decarboxylation product of leucine, and relative activity was determined by dividing the sample isopentylamine peak area by the backbone isopentylamine peak area to calculate FIOP (fold improvement over parent compound) values.

[0293] HTP analysis of clarified lysates pretreated to mimic gastric and intestinal conditions Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin to mimic the gastric environment. Specifically, heat-treated clarified lysates were pre-incubated 1:1 with McIlvaine buffer (pH 2.8-3) and pepsin at a final concentration of 0.8 g / L in a Costar® 96-well round-bottom plate (Corning). The plate was sealed and incubated for 2 h at 37°C in a Multitron plate shaker (250 rpm, Infors HT). The simulated gastric fluid challenged lysates were then pre-incubated 1:1 with trypsin at a final concentration of 4 g / L and chymotrypsin at a final concentration of 1.5 g / L in a Multitron plate shaker (250 rpm, Infors HT) at 37°C for 2 h to mimic the intestinal environment. After this incubation, the samples were centrifuged and 40 μL of sample was added to 60 μL of reaction mixture to give a final concentration of 3 mM leucine in 20 mM sodium phosphate (pH 7) or a mixture of simulated intestinal fluid salts. In some experiments, a reaction mixture giving a final concentration of 3 mM leucine, isoleucine-d10, valine, methionine, and cysteine ​​(5AA mix) was used. The activity of the LDC variants was then measured as described in Example 1.3 above.

[0294] Example 2 Screening results for LDC variants Library variants were generated by recombining beneficial mutations into the LDC polypeptide of SEQ ID NO:828. HTP growth and lysis of E. coli cells expressing the LDC variants was performed as described in Example 1. The variants were screened for LDC activity in a 5AA mixture dissolved in simulated intestinal fluid salt solution after 2 hours of simulated gastric fluid challenge (50% McIlvaine's buffer pH 3, 0.8 g / L pepsin) and 2 hours of intestinal fluid challenge (200 mM sodium phosphate pH 8, 4 g / L trypsin, 1.5 g / L chymotrypsin). LDC activity was measured as described in Example 1, and analysis of the data for SEQ ID NO:828 is listed in Table 12-1. [Table 12.1-1] [Table 12.1-2] [Table 12.1-3] [Table 12.1-4] [Table 12.1-5] [Table 12.1-6] [Table 12.1-7]

[0295] Based on the results in Table 12-2, SEQ ID NO:888 was selected as the backbone. The beneficial mutations identified in Table 12-1 were incorporated into the backbone. The resulting variants were screened for activity in an unstimulated challenge against leucine by incubating 20 μL of 80-fold diluted heat-treated clarified lysate with 80 μL of reaction mixture to a final concentration of 3 mM leucine and 0.01 mM PLP in 20 mM sodium phosphate, pH 7. The variants were also screened for LDC activity in a 5AA mixture containing 0.01 mM PLP in 20 mM sodium phosphate, pH 7 after 2 hours of simulated gastric fluid stimulation (50% McIlvaine buffer, pH 2.8, 0.8 g / L pepsin) and 2 hours of simulated intestinal fluid stimulation (200 mM sodium phosphate, pH 8, 4 g / L trypsin, 1.5 g / L chymotrypsin). LDC activity was measured as described in Example 1 and analysis of the data for SEQ ID NO:888 is listed in Table 12-2. [Table 12.2-1] [Table 12.2-2] [Table 12.2-3] [Table 12.2-4] [Table 12.2-5] [Table 12.2-6] [Table 12.2-7] [Table 12.2-8] [Table 12.2-9] [Table 12.2-10]

[0296] Example 3 Pharmacodynamic (PD) testing of LDC variants in an intermediate MSUD mouse model Intermediate maple syrup urine disease (iMSUD; Dbt tm1GehiMSUD mice (Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh / J, JacksonLabs #006999) were subjected to leucine restriction (Research Diets #A05080202i, leucine-free diet supplemented with 5.75 g / L leucine, Sigma #L8912) at weaning to promote health and extend survival. When iMSUD mice reached approximately 20 g body weight (BW), 18 untreated mice were randomly assigned to the test group (mixed sex, 2-3.5 months old). During the test period, mice had free access to the leucine-free diet and leucine-supplemented water. Approximately 45 mg of whey (Grass Fed Whey Protein, BN Labs Lot No. U0637AL, whey protein powder containing 8.57% w / w leucine) suspended in 100 μL of water was orally gavaged to each mouse. After the whey protein meal, mice were orally gavaged (100 μL / mouse) with either vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or enzyme (LDC of SEQ ID NO: 484, SEQ ID NO: 686, or SEQ ID NO: 766, administered at 200 mg / kg diluted in vehicle). Blood was collected from the tail vein at scheduled time points (pre-dose and 15, 30, 60, 120, and 240 min post-dose). Plasma was extracted from the collected blood samples and analyzed by LC-MS to determine leucine concentration using a standard curve (range: 2-3050 mM, LOQ: 2 μM).

[0297] Plasma leucine in mice increased significantly after administration of the whey protein diet and vehicle. Treatment with engineered leucine decarboxylase immediately after the whey diet significantly inhibited the leucine spike, with inhibition enhanced in the more evolved variants, both in terms of the time course data (FIG. 1A) and the area under the curve (AUC, FIG. 1B; inhibition of 21% (p<0.05), 30% (p<0.01), or 39% (p<0.001) for LDCs of SEQ ID NO:484, SEQ ID NO:686, and SEQ ID NO:766, respectively, compared to vehicle).

[0298] Example 4 Pharmacodynamic (PD) dose-response study of the LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model Thirteen drug-experienced iMSUD mice from a previous study were randomly grouped (mixed sex, 2.5-4 months old, weighing approximately 20 g) after a minimum of 2 weeks washout. Mice had free access to a leucine-free diet and leucine-supplemented water (5.75 g / L leucine) for the duration of the study. Each mouse was gavaged with approximately 45 mg of whey (Grass Fed Whey Protein; BN Labs Lot No. U0637AL; whey protein powder containing 8.57% w / w leucine) suspended in 100 μL of water. After the whey protein diet, mice were gavaged (100 μL / mouse) with vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or LDC of SEQ ID NO: 766 (50, 100, or 200 mg / kg, diluted in vehicle). Blood was collected from the tail vein at scheduled time points (pre-dose and 15, 30, 60, 120, and 240 min post-dose). Plasma was extracted from collected blood samples and analyzed by LC-MS to determine the levels of leucine, isoleucine, valine, phenylalanine, and methionine using their respective standard curves (LOQ: 2 μM). Background (pre-dose) was subtracted to correct for high baseline variation between mice.

[0299] A significant increase in plasma leucine was observed after administration of the whey protein meal and vehicle. Treatment with all doses of LDC of SEQ ID NO: 766 immediately after whey significantly inhibited the leucine spike in a dose-dependent manner, both in the background-subtracted time course data (Figure 2A) and in the incremental AUC (iAUC), which is the AUC calculated using the background-subtracted time course data (Figure 2B; 40% (p<0.01), 57% (p<0.001), or 92% (p<0.0001) inhibition at 50, 100, and 200 mg / kg LDC of SEQ ID NO: 766, respectively, compared to vehicle). The whey protein meal caused spikes in other amino acids evaluated, but LDC did not induce inhibition at any dose. The mean iAUC±SEM data for several amino acids tested are shown in Table 13-1 below. [Table 13-1]

[0300] Example 5 Pharmacodynamic (PD) dose-response study of evolved LDC variants compared to the LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model Drug-naive iMSUD mice were subjected to leucine restriction (Research Diets #A05080202i, leucine-free diet supplemented with 5.75 g / L leucine; Sigma #L8912) at weaning to promote health and extend survival. Twenty-four iMSUD mice weighing ≥20 g were randomly assigned to test groups (mixed sex, 2.5–6 months of age). Leucine-supplemented water (5.75 g / L) was replaced with regular water approximately 40–60 min before the first blood draw and after the final blood draw. Mice had free access to leucine-free chow and water during the test period. Approximately 45 mg of whey (Grass Fed Whey Protein, BN Labs lot no. U0637AL, whey protein powder containing 8.57% w / w leucine) was suspended in 100 μL of water and orally gavaged to each mouse. After a whey protein meal, mice were gavaged (100 μL / mouse) with vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or engineered leucine decarboxylase (SEQ ID NO: 766 or SEQ ID NO: 828, 100 mg / kg dose diluted in vehicle). Blood was collected from the tail vein at scheduled time points (pre-dose and 15, 30, 60, 120, 240 min post-dose). Plasma was extracted from the collected blood samples and analyzed by LC-MS to determine the levels of leucine, isoleucine, valine, phenylalanine, and methionine using the respective standard curves (LOQ: 2 μM).

[0301] A significant increase in serum leucine was observed after administration of the whey protein meal and vehicle. Treatment with engineered leucine decarboxylase immediately after whey significantly inhibited the leucine spike, both in terms of time course data (Figure 3A) and AUC (Figure 3B; inhibition of 28% (SEQ ID NO: 766) and 45% (SEQ ID NO: 828, p<0.05) compared to vehicle). The whey protein meal caused spikes in other amino acids evaluated, but LDC did not induce inhibition with either leucine decarboxylase variant. The mean iAUC±SEM data for several amino acids tested are shown in Table 14-1 below. [Table 14-1]

[0302] Example 6 Pharmacodynamic (PD) dose-response study of LDC variants in healthy cynomolgus monkeys Thirteen male cynomolgus monkeys were fasted overnight prior to dosing. Each monkey was gavaged with approximately 10 g of whey (Grass Fed Whey Protein; BN Labs Lot No. U0637AL; whey protein powder containing 8.57% w / w leucine) suspended in 20 mL of water. Cynomolgus monkeys were gavaged with vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or engineered leucine decarboxylase (SEQ ID NO: 484, SEQ ID NO: 686, or SEQ ID NO: 766, dosed at 25, 50, or 100 mg / kg diluted in vehicle) as a suspension (2.5 mL / kg, 7.5-10 mL / monkey). A standard diet (Certified Primate Chow 2055C; Envigo) was given to all monkeys approximately 8 hours after treatment. Blood was collected from the femoral vein twice before dosing (-1.5 h and -30 min) and at 5, 15, 30 min, and 1, 2, 4, 8, 12, and 24 h after dosing. Plasma was extracted from the collected blood samples and analyzed by LC-MS for leucine, isoleucine, valine, phenylalanine, methionine, and tyrosine levels against their respective standard curves (LOQ: 4, 8, 8.5, 6, 7, and 11 μM, respectively).

[0303] Oral gavage of a whey meal followed immediately by vehicle administration increased leucine concentrations, reaching a maximum of approximately 1165 μM at 4 hours. Treatment with LDC immediately after the whey protein meal inhibited the plasma leucine spike in all variants, with both the time course data (Figures 4A-4C) and AUC (Figure 4D) indicating enhanced inhibition in the more evolved variants. The reduction in plasma leucine in the LDC variants is also shown in Table 15-1. [Table 15-1]

[0304] The whey protein diet caused a sharp increase in the other amino acids evaluated, but LDC caused no suppression at any dose. Mean AUC±SEM data for several additional amino acids tested are presented in Tables 15-2, 15-3, and 15-4. [Table 15-2] [Table 15-3] [Table 15-4]

[0305] Example 7 Pharmacodynamic (PD) dose-response and 3-day repeat dose study with the LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys Ten male cynomolgus monkeys were fasted overnight prior to dosing. Each monkey was gavaged with approximately 10 g of whey (Grass Fed Whey Protein; BN Labs Lot No. U0637AL; whey protein powder containing 8.57% w / w leucine) suspended in 20 mL of water. Cynomolgus monkeys were gavaged with vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or LDC of SEQ ID NO: 766 (6.25, 12.5, and 25 mg / kg, diluted in vehicle) as a suspension (2.5 mL / kg, 7.5-10 mL / monkey). A standard diet (Certified Primate Diet 5048; PMI) was given to all monkeys approximately 8 hours after dosing. Blood was collected from the femoral vein pre-dose and then 5, 15, 30 min, 1, 2, 4, 8, 12, and 24 h post-dose. Plasma was extracted from the collected blood samples and analyzed by LC-MS for leucine, ketoisocaproic acid (KIC), isoleucine, valine, methionine, and phenylalanine levels against their respective standard curves (LOQ: 15, 10, 10, 15, 5, and 10 μM, respectively).

[0306] Oral gavage of a whey meal followed immediately by vehicle administration increased leucine and KIC levels to a maximum of approximately 877 and 77 μM, respectively, at 2 hours. Treatment with LDC of SEQ ID NO: 766 immediately after a whey protein meal significantly suppressed the spike in plasma leucine and KIC at all doses, both in the background-subtracted time course data (Figures 5A and 5C) and in iAUC (Figures 5B and 5D and Table 16-1). [Table 16-1]

[0307] The protein diet led to increases in the other amino acids evaluated, but LDC did not induce suppression at any dose. The mean iAUCs for several of the amino acids tested are shown in Table 16-2. [Table 16-2]

[0308] After a 3-week washout, all 10 cynomolgus monkeys were again randomly divided into two groups and fasted overnight before daily dosing. Each monkey was gavaged with approximately 10 g of whey protein meal suspended in 20 mL of water. Cynomolgus monkeys were gavaged with vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or LDC of SEQ ID NO: 766 (25 mg / kg, diluted in vehicle) as a suspension (2.5 mL / kg, 7.5-10 mL / monkey). Approximately 8 hours after treatment, all monkeys were given a standard diet (Certified Primate Diet 5048; PMI). This protocol was repeated for three consecutive days, with cynomolgus monkeys maintaining the same group designation throughout the study. On each dosing day, blood was collected from the femoral vein pre-dose, 1, 2, 4, 8, 12, and 24 hours post-dose (day 3 only). Plasma was extracted from the collected blood samples and analyzed by LC-MS, and the levels of leucine, isoleucine, valine, methionine, and phenylalanine were analyzed against their respective standard curves (LOQ: 15, 10, 15, 5, and 10 μM, respectively).

[0309] Oral gavage of a whey meal followed immediately by vehicle administration resulted in a similar increase in leucine 2 hours after dosing on each day (Figure 6A). Treatment with LDC of sequence number 766 immediately after a whey protein meal significantly suppressed plasma leucine on all days. Repeated dosing was observed to increase the separation between vehicle and treated baselines (pre-dose values ​​on days 2 and 3, Figure 6A), suggesting an additive effect. The AUC data (Figure 6B) also supported the additive effect of treatment, with the greatest reduction observed on day 3 (47%) compared to 32% and 34% on days 1 and 2, respectively. To better assess baseline differences due to repeated meal stimulus administration, daily data were reanalyzed by subtracting the pre-meal value on day 1 as baseline (Figures 6C and 6D). Normalization made it more clear that repeated meal stimulus administration increases baseline in the vehicle group, while baseline in the treated group continues to decrease toward the pre-meal baseline on day 1 (Figure 6C). The maximal effect of treatment was again seen on day 3 of meal challenge (FIG. 6D). Table 16-3 also shows the % reduction in plasma leucine compared to vehicle. [Table 16-3]

[0310] The protein diet produced increases in the other amino acids evaluated on all three days, whereas LDC caused a significant suppression only on the third day of continuous administration (Figures 7A and 7B). The mean iAUC±SEM of the additional amino acids tested are shown in Table 16-4. [Table 16-4]

[0311] Example 8 Pharmacodynamic (PD) dose-response study of LDC of SEQ ID NO:828 in an intermediate MSUD mouse model Intermediate maple syrup urine disease (iMSUD; Dbt tm1GehTg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh / J, Jackson Labs #006999) mice were subjected to leucine restriction (Research Diets #A05080202i, leucine-free diet supplemented with 5.75 g / L leucine, Sigma #L8912) at weaning to promote health and extend survival. When iMSUD mice reached approximately 20 g body weight (BW), 14 mice were randomly assigned to test groups (mixed sex, 2-4.7 months of age). During the test period, mice had free access to the leucine-free diet and leucine-supplemented water. Each mouse was gavaged with approximately 45 mg of whey (Grass Fed Whey Protein; BN Labs Lot No. X01010AL0R; containing 8.613% w / w leucine, 0% free amino acids) suspended in 100 μL of water. After the whey protein meal, the mice were gavaged with either vehicle (1 mM sodium phosphate + 0.4 mM PLP, pH 7.5) or enzyme (SEQ ID NO: 828, dosed at 25, 50, 100 mg / kg, diluted in vehicle without PLP). The study design followed a two-phase crossover with a two-week washout in between. Blood was collected from the tail vein at scheduled time points (30 min pre-dose, 30, 60, 120, and 240 min post-dose). Plasma was extracted from collected blood samples and analyzed by LC-MS / MS to measure leucine levels using a standard curve (range: 2-3050 mM; LOQ: 2 µM) to assess efficacy.

[0312] A significant increase in plasma leucine levels was observed after administration of the whey protein meal and vehicle. Treatment with LDC immediately after the whey meal significantly inhibited the leucine surge in a dose-dependent manner, both in the time course data and the area under the curve. Specifically, there was a statistically significant inhibition at doses of 100 mg / kg and 50 mg / kg compared to vehicle at all time points after administration (Figure 8A). Similarly, incremental area under the curve (iAUC) data showed a significant inhibition of plasma leucine of 40% and 54% with treatment of 50 and 100 mg / kg of SEQ ID NO: 828, respectively (Figure 8B, Table 17). No statistically significant effect of treatment (iAUC) was observed on plasma levels of isoleucine, valine, methionine, or phenylalanine (Table 17). Statistical calculations and significance were determined using GraphPad Prism9 (GraphPad software). [Table 17]

[0313] Example 9 Pharmacodynamic (PD) dose-response study of SEQ ID NO:828 in healthy cynomolgus monkeys Twelve male cynomolgus monkeys were fasted overnight prior to dosing. Each monkey was gavaged with approximately 10 g of whey protein powder (Grass Fed Whey Protein; BN Labs Lot No. U06376AL; containing 8.63% w / w leucine) suspended in 20 mL of water. Cynomolgus monkeys were gavaged with vehicle (1 mM sodium phosphate + 0.4 mM PLP, pH 7.5) or enzyme (SEQ ID NO: 828, dosed at 6.25, 12.5, and 25 mg / kg diluted in vehicle without PLP) as a suspension (2.5 mL / kg). The study design was a two-stage crossover with a one-week washout in between. A standard diet (Certified Primate Diet 5048, PMI Inc.) was given to all cynomolgus monkeys approximately 8 hours after treatment. Blood was collected from the femoral vein twice (1 and 2 h) before dosing and then 5, 15, and 30 min and 1, 2, 4, 8, and 24 h after dosing. Plasma was extracted from the collected blood samples and analyzed by LC-MS / MS for leucine, isoleucine, valine, phenylalanine, and methionine levels against their respective standard curves (LOQ: 4, 8, 8.5, 6, and 7 μM, respectively).

[0314] Oral gavage of a whey meal followed immediately by vehicle administration increased leucine concentrations. Treatment with LDC immediately after a whey protein meal dose-dependently inhibited the spike in plasma leucine, with time-cour...

Claims

1. 1. An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80% sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein said amino acid sequence comprises one or more substitutions at amino acid positions 202, 272, 290 or 383, or a combination thereof, relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.

2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid sequence comprises at least one substitution from 202H, 272A, 290I, or 383S, or a combination thereof, and the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

3. 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to a reference sequence corresponding to SEQ ID NO: 828, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:

828.

4. 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to a reference sequence corresponding to SEQ ID NO: 888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:

828.

5. 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid sequence comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 126, 141, 170, 173, 187, 198, 200, 267, 270, 312, 353, 357, or 384, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

6. 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid sequence comprises at least the substitutions 5V, 19L, 33L, 41D, 47F, 51E, 551, 64S / N, 126A, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 2901, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

7. 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid sequence comprises at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

8. 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid sequence comprises at least the substitutions 33L, 55I, 64N, 126A, 270L, or 357S, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

9. The amino acid sequence is selected from the group consisting of amino acid positions 170 / 270 / 383, 270, 41 / 173, 272, 5 / 141 / 272 / 383, 41 / 383, 41 / 141 / 187 / 272 / 290, 41 / 141 / 173 / 290, 5 / 272 / 383, 5 / 41 / 173 / 272 / 383, 41 / 141, 141 / 272, 353 / 384, 272 / 383, 41 / 141 / 173, 41 / 272 / 383, 41 / 141 / 1 / 187 / 200 / 202 / 272, 33 / 55 / 64 / 126 / 270 / 357, 33 / 126 / 353 / 357, 55 / 64 / 267 / 35 / 384, 33 / 64 / 357, 126 / 267, 64 / 267 / 353 / 384, 33 / 55 / 64 / 357, 19 / 64 / 126 / 267, 55 / 267, 33 / 126 / 267 / 270 / 312 / 357, 19 / 33 / 55 / 353 / 357 / 384, 1 9 / 33 / 126, 126 / 312, 126 / 198 / 202 / 267 / 312, 126 / 353, 55 / 126, 126 / 270 / 384, 33 / 64 / 353 / 357, 19 / 267, 51 / 55 / 267 / 270 / 353, 33 / 126 / 267 / 270, 19 / 55 / 64 / 126 / 267 / 270 / 353, 19 / 33 / 126 / 270 / 353 / 357 / 384, 19 / 33 / 64 / 267 / 3 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least a substitution or set of substitutions at positions 53, 126 / 353 / 384, 126 / 270 / 312 / 353 / 384, 19 / 33 / 55 / 126, 33 / 357, 47 / 51 / 64 / 126 / 353 / 384, 126, or 126 / 270, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

10. The amino acid sequence may comprise at least one substitution or set of substitutions 170P / 270L / 383S, 270L, 41D / 173I, 272A, 5V / 141P / 272A / 383S, 41D / 383S, 41D / 141P / 187L / 272A / 290I, 41D / 141P / 173I / 290I, 5V / 272A / 383S, 5V / 41D / 173I / 272A / 383S, 41D / 141P, 141P / 272A, 353E / 384W, 272A / 383S, 41D / 141P / 173I, 41D / 272A / 383S A / 383S, 41D / 141P / 187L / 200S / 202H / 272A, 33L / 55I / 64N / 126A / 270L / 357S, 33L / 126A / 353E / 357S, 55I / 64N / 267L / 353E / 384W, 33L / 64 N / 357S, 126A / 267L, 64N / 267L / 353E / 384W, 33L / 55I / 64N / 357S, 19L / 64N / 126A / 267L, 55I / 267L, 33L / 126A / 267L / 270T / 312T / 357S, 19L / 33L / 55I / 353E / 357S / 384W, 19L / 33L / 126A, 126A / 312T, 126A / 198G / 202H / 267L / 312T, 126A / 353E, 55I / 126A, 126A / 270T / 384W, 33L / 6 4N / 353E / 357S, 19L / 267L, 51E / 55I / 267L / 270T / 353E, 33L / 126A / 267L / 270T, 19L / 55I / 64N / 126A / 267L / 270T / 353E, 19L / 33L / 126A / 270 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising the amino acid sequences: 19L / 33L / 64N / 267L / 353E, 126A / 353E / 384W, 126A / 270T / 312T / 353E / 384W, 19L / 33L / 55I / 126A, 33L / 357S, 47F / 51E / 64N / 126A / 353E / 384W, 126A, or 126A / 270T, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

11. The amino acid sequence comprises at least one substitution or set of substitutions A170P / R270L / A383S, R270L, H41D / F173I, T272A, K5V / R141P / T272A / A383S, H41D / A383S, H41D / R141P / V187L / T272A / V290I, H41D / R141P / F173I / V290I, K5V / T272A / A383S, K5V / H41D / F173I / T272A / A383S, H41D / R141P, R141P / T272A, D353E / P384W, T272A / A383S, H41D / R141P 1P / F173I, H41D / T272A / A383S, H41D / R141P / V187L / H200S / S202H / T272A, F 33L / V55I / S64N / D126A / R270L / C357S, F33L / D126A / D353E / C357S, V55I / S6 4N / I267L / D353E / P384W, F33L / S64N / C357S, D126A / I267L, S64N / I267L / D3 53E / P384W, F33L / V55I / S64N / C357S, I19L / S64N / D126A / I267L, V55I / I267L , F33L / D126A / I267L / R270T / A312T / C357S, I19L / F33L / V55I / D353E / C357S / P384W, I19L / F33L / D126A, D126A / A312T, D126A / A198G / S202H / I267L / A31 2T, D126A / D353E, V55I / D126A, D126A / R270T / P384W, F33L / S64N / D353E / C3 57S, I19L / I267L, L51E / V55I / I267L / R270T / D353E, F33L / D126A / I267L / R27 0T, I19L / V55I / S64N / D126A / I267L / R270T / D353E, I19L / F33L / D126A / R270T / D353E / C357S / P384W, I19L / F33L / S64N / I267L / D353E, D126A / D353E / P384W, D126A / R270T / A312T / D353E / P384W, I19L / F33L / V55I / D126A, F33L / C357S, L47F / L51E / S64N / D126A / D353E / P384W, D126A, or D126A / R270T, wherein2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

828.

12. 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising an amino acid sequence having at least 80% sequence identity to SEQ ID NO: 888, wherein the amino acid sequence contains one or more substitutions relative to the reference sequence corresponding to SEQ ID NO:

888.

13. 13. The engineered leucine decarboxylase polypeptide of claim 12, wherein the amino acid sequence comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

888.

14. 13. The engineered leucine decarboxylase polypeptide of claim 12, wherein the amino acid sequence comprises amino acid residues 5V, 19L, 33L, 41D, 47F, 51E, 551, 64S / N, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L / T, 272A, 2901, 312T, 353E, 357S / C, 383S, or 384W, or a combination thereof, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

888.

15. The amino acid sequence is selected from the group consisting of amino acid positions 64 / 173 / 202 / 353 / 384, 41 / 141 / 272 / 353, 141 / 202 / 272 / 353 / 357, 173 / 202 / 357, 202 / 353, 5 / 51 / 173 / 272 / 353 / 384, 51 / 202 / 272 / 357, 141 / 173 / 272, 272, 41 / 173 / 384, 41 / 64 / 141 / 353 / 357 / 383, 141 / 173 / 202, 5 / 51 / 64 / 202 / 353, 357, 64, 5 / 41 / 141, 41 / 141 / 173 / 2 02 / 353, 353, 202 / 357, 51 / 141 / 202 / 272 / 353, 202, 51 / 141 / 173 / 353 / 384, 41 / 141 / 173 / 202 / 272 / 353 / 383 / 384, 64 / 202 / 357, 5 / 64 / 353 / 383 / 384, 41 / 272 / 353 / 383, 41 / 173 / 272 / 353 / 357, 51 / 141 / 272 / 353 / 357 / 383 / 384, 41 / 353 / 357, 173 / 272 / 353 / 357, 5 / 41 / 64 / 173 / 353 / 357, 64 / 1 73 / 357, 51 / 272, 51 / 64 / 357 / 384, 51 / 141 / 173 / 272 / 353, 64 / 202 / 272 / 353 / 357 / 384, 51 / 272 / 357, 51 / 173 / 272 / 353 / 384, 353 / 384, 202 / 272 / 357, 64 / 141 / 173 / 202 / 353 / 357, 5 / 41 / 51 / 202 / 357 / 383, 5 / 51 / 173 / 272 / 383, 41 / 141 / 272, 51 / 173, 5 / 353, 41 / 64 / 173 / 272 / 353 / 383, 5 / 64 / 13. The engineered leucine decarboxylase polypeptide of claim 12, comprising at least a substitution or set of substitutions at: 173 / 272 / 353, 51 / 64, 41 / 357 / 383, 41 / 173 / 353 / 357, 202 / 272 / 383, 202 / 272, 353 / 357, 41 / 173 / 202 / 272 / 357, 141 / 173 / 202 / 272 / 353 / 357, 64 / 141 / 202, or 5 / 173 / 272, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

888.

16. The amino acid sequence comprises at least one substitution or set of substitutions 64S / 173I / 202H / 353E / 384W, 41D / 141P / 272A / 353E, 141P / 202H / 272A / 353E / 357C, 173I / 202H / 357C, 202H / 353E, 5V / 51E / 173I / 272A / 353E / 384W, 51E / 202H / 272A / 357C, 141P / 173I / 272A, 272A, 41D / 173I / 384W, 41D / 64S / 141P / 353E / 357C / 383S, 141P / 173I / 202H, 5V / 51E / 64S / 202H / 353E, 357C, 64S, 5V / 41D / 141P, 41D / 141P / 173I / 202H / 353E, 353E, 20 2H / 357C, 51E / 141P / 202H / 272A / 353E, 202H, 51E / 141P / 173I / 353E / 384W, 41 D / 141P / 173I / 202H / 272A / 353E / 383S / 384W, 64S / 202H / 357C, 5V / 64S / 353E / 383S / 384W, 41D / 272A / 353E / 383S, 41D / 173I / 272A / 353E / 357C, 51E / 141P / 272A / 353E / 357C / 383S / 384W, 41D / 353E / 357C, 173I / 272A / 353E / 357C, 5V / 41D / 64S / 173I / 353E / 357C, 64S / 173I / 357C, 51E / 272A, 51E / 64S / 357C / 384W , 51E / 141P / 173I / 272A / 353E, 64S / 202H / 272A / 353E / 357C / 384W, 51E / 272A / 357C, 51E / 173I / 272A / 353E / 384W, 353E / 384W, 202H / 272A / 357C, 64S / 141P / 173I / 202H / 353E / 357C, 5V / 41D / 51E / 202H / 357C / 383S, 5V / 51E / 173I / 272 A / 383S, 41D / 141P / 272A, 51E / 173I, 5V / 353E, 41D / 64S / 173I / 272A / 353E / 38 3S, 5V / 64S / 173I / 272A / 353E, 51E / 64S, 41D / 357C / 383S, 41D / 173I / 353E / 3 57C, 202H / 272A / 383S, 202H / 272A, 353E / 357C, 41D / 173I / 202H / 272A / 357C,13. The engineered leucine decarboxylase polypeptide of claim 12, comprising 141P / 173I / 202H / 272A / 353E / 357C, 64S / 141P / 202H, or 5V / 173I / 272A, wherein the amino acid positions are relative to a reference sequence corresponding to SEQ ID NO:

888.

17. The amino acid sequence comprises at least one substitution or set of substitutions N64S / F173I / S202H / D353E / P384W, H41D / R141P / T272A / D353E, R141P / S202H / T272A / D353E / S357C, F173I / S202H / S357C, S202H / D353E, K5V / L51E / F173I / T272A / D353E / P384W, L51E / S202H / T272A / S357C, R141P / F173I / T272A, T272A, H41D / F173I / P384W, H41D / N64S / R141P / D353E / S357C / A383S, R141P / F173I / S202H, K5V / L51E / N64S / S202H / D353 E, S357C, N64S, K5V / H41D / R141P, H41D / R141P / F173I / S202H / D353E, D353E , S202H / S357C, L51E / R141P / S202H / T272A / D353E, S202H, L51E / R141P / F17 3I / D353E / P384W, H41D / R141P / F173I / S202H / T272A / D353E / A383S / P384W, N64S / S202H / S357C, K5V / N64S / D353E / A383S / P384W, H41D / T272A / D353E / A383S, H41D / F173I / T272A / D353E / S357C, L51E / R141P / T272A / D353E / S357 C / A383S / P384W, H41D / D353E / S357C, F173I / T272A / D353E / S357C, K5V / H41 D / N64S / F173I / D353E / S357C, N64S / F173I / S357C, L51E / T272A, L51E / N64S / S357C / P384W, L51E / R141P / F173I / T272A / D353E, N64S / S202H / T272A / D3 53E / S357C / P384W, L51E / T272A / S357C, L51E / F173I / T272A / D353E / P384W, D353E / P384W, S202H / T272A / S357C, N64S / R141P / F173I / S202H / D353E / S35 7C, K5V / H41D / L51E / S202H / S357C / A383S, K5V / L51E / F173I / T272A / A383S,H41D / R141P / T272A, L51E / F173I, K5V / D353E, H41D / N64S / F173I / T272A / D353E / A383S, K5V / N64S / F173I / T272 A / D353E, L51E / N64S, H41D / S357C / A383S, H41D / F173I / D353E / S357C, S202H / T272A / A383S, S202H / T272A, D353 13. The engineered leucine decarboxylase polypeptide of claim 12, comprising the amino acid sequences: E / S357C, H41D / F173I / S202H / T272A / S357C, R141P / F173I / S202H / T272A / D353E / S357C, N64S / R141P / S202H, or K5V / F173I / T272A, wherein the amino acid positions are relative to a reference sequence for SEQ ID NO:

888.

18. The amino acid sequences of SEQ ID NOs: 854, 860, 862, 864, 866, 868, 870, 872, 876, 880, 884, 886, 914, 950, 952, 954, 956, 958, 960, 962, 964, 966, 970, 972, 974, 982, 986, 988, 990, 994, 996, 998, 1000, 1002 , 1004, 1008, 1014, 1018, 1020, 1022, 1024, 1028, 1030, 1032, 1034, 1036, 1042, 1044, 1048, 1052, 1054, 1058, 1060, 1062, and 1064.

19. 19. The engineered leucine decarboxylase polypeptide of any one of claims 1 to 18, wherein the engineered leucine decarboxylase polypeptide exhibits one or more improved properties compared to a wild-type leucine decarboxylase of a Planctomycetaceae species, or a leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO:

12.

20. 20. The engineered leucine decarboxylase polypeptide of claim 19, wherein the improved property is selected from: (i) increased activity towards leucine; (ii) increased resistance to proteolysis; (iii) increased tolerance to low pH environments; or (iv) increased thermostability, or any combination thereof, compared to a wild-type leucine decarboxylase of a Planctomycete species or a leucine decarboxylase having an amino acid sequence corresponding to SEQ ID NO:

12.

21. 19. The leucine decarboxylase polypeptide of any one of claims 1 to 18, wherein the engineered leucine decarboxylase polypeptide is purified.

22. A recombinant polynucleotide encoding at least one engineered leucine decarboxylase polypeptide according to any one of claims 1 to 18.

23. SEQ ID NOs: 853, 859, 861, 863, 865, 867, 869, 871, 875, 879, 883, 885, 913, 949, 951, 953, 955, 957, 959, 961, 963, 965, 969, 971, 981, 985, 987, 989, 993, 995, 997, 999, 1001, 1003, 1007, 1013, 1017, 101 23. The recombinant polynucleotide of claim 22, comprising a polynucleotide sequence having at least 70% sequence identity to any one of the reference polypeptide sequences of 9, 1021, 1023, 1027, 1029, 1031, 1033, 1035, 1041, 1043, 1047, 1051, 1053, 1057, 1059, 1061, and 1063.

24. 23. The recombinant polynucleotide of claim 22, wherein the polynucleotide sequence is codon-optimized.

25. The polynucleotide sequences are SEQ ID NOs: 853, 859, 861, 863, 865, 867, 869, 871, 875, 879, 883, 885, 913, 949, 951, 953, 955, 957, 959, 961, 963, 965, 969, 971, 981, 985, 987, 989, 993, 995, 997, 999, 1 23. The recombinant polynucleotide of claim 22, comprising any one of the sequences: 001, 1003, 1007, 1013, 1017, 1019, 1021, 1023, 1027, 1029, 1031, 1033, 1035, 1041, 1043, 1047, 1051, 1053, 1057, 1059, 1061, and 1063.

26. An expression vector comprising at least one recombinant polynucleotide according to claim 22.

27. 27. The expression vector of claim 26, wherein the recombinant polynucleotide is operably linked to a control sequence.

28. A host cell transformed with at least one polynucleotide according to claim 22.

29. 30. A method for producing an engineered leucine decarboxylase polypeptide in a host cell, comprising culturing the host cell of claim 28 under conditions such that at least one engineered leucine decarboxylase polypeptide is expressed.

30. 30. The method of claim 29, further comprising recovering the engineered leucine decarboxylase polypeptide from the culture and / or host cell.

31. 30. The method of claim 29, further comprising purifying the at least one engineered leucine decarboxylase polypeptide.

32. A pharmaceutical composition comprising at least one engineered leucine decarboxylase polypeptide according to any one of claims 1 to 18.

33. The pharmaceutical composition of claim 32, further comprising at least one pharmaceutically acceptable excipient and / or carrier.

34. The pharmaceutical composition of claim 32, wherein the composition is suitable for oral administration to humans.

35. The pharmaceutical composition of claim 32, wherein the composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion.

36. The pharmaceutical composition of claim 35, wherein the pill, tablet, capsule, or gelcap further comprises an enteric coating.

37. The pharmaceutical composition of claim 32, wherein the composition is suitable for parenteral injection into humans.

38. 33. The pharmaceutical composition of claim 32 for use in a method for treating and / or preventing symptoms of a disease or condition associated with elevated plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid, comprising: The method comprises administering the pharmaceutical composition to a subject in need of treatment and / or prevention, and said administration reduces plasma leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid levels.

39. 39. The pharmaceutical composition of claim 38, wherein the disease or condition associated with elevated plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid is maple syrup urine disease.

40. 39. The pharmaceutical composition of claim 38, wherein the disease or condition associated with elevated plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid is isovaleric acidemia.

41. 39. The pharmaceutical composition of claim 38, wherein the disease or condition associated with elevated plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid is 3-methylcrotonyl-CoA carboxylase deficiency.

42. 33. The pharmaceutical composition of claim 32 for reducing plasma levels of leucine, isoleucine, alloisoleucine, and / or ketoisocaproic acid in a subject.

43. 43. The pharmaceutical composition of claim 42, wherein the subject has maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.

44. 39. The pharmaceutical composition of claim 38, wherein the engineered leucine decarboxylase is administered in an amount effective to reduce plasma levels of leucine, ketoisocaproic acid, and methionine.

45. 39. The pharmaceutical composition of claim 38, wherein the composition is administered immediately before, simultaneously with, and / or immediately after ingestion of a protein-containing meal.

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