Engineered leucine decarboxylases

Abstract

To provide engineered leucine decarboxylases.SOLUTION: The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase (LDC) polypeptides. In some embodiments, the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis, and / or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability. The present invention also provides methods for use of the compositions comprising the engineered LDC polypeptides for therapeutic and industrial purposes.SELECTED DRAWING: None

Description

[Technical Field]

[0001] This application claims priority to U.S. Provisional Application No. 62 / 970,039, filed February 4, 2020, which is hereby incorporated by reference in its entirety for all purposes.

[0002] FIELD OF THE INVENTION The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase (LDC) polypeptides. In some embodiments, the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, reduced susceptibility to proteolysis, and / or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability. The present invention also provides methods for using compositions comprising engineered LDC polypeptides for therapeutic and industrial purposes. Reference to a "Sequence Listing," table, or computer program listing appendix submitted as an ASCII text file

[0003] The sequence listing written in file CX7-198WO2_ST25.TXT, having a size of 2.06 MB and created on February 3, 2021 in machine format IBM-PC, MS-Windows® operating system, is hereby incorporated by reference into this specification. [Background technology]

[0004] Background of the Invention "Leucineuria", "branched-chain alpha-leucine dehydrogenase Maple syrup urine disease (MSUD), also known as "BCKD deficiency" and "BCKD deficiency," is a rare inherited disorder of amino acid metabolism secondary to dysfunction in 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. (Menkes et al., Pediatrics 14:462-467

[1954] ) and characterized in the urine of affected newborns. It is named for its distinctive sweet odor. It is also characterized by loss of appetite, vomiting, fatigue, abnormal movements (e.g., hypertonia or hypotonia), and growth retardation. If untreated, the disease can progress to brain damage, convulsions, coma, permanent neurological damage, and death. Later in life, developmental delay, learning problems, seizures, and motor difficulties are common. There are four common forms classified based on the signs and symptoms of the disease. The most common and severe form is the "classic" form, which becomes apparent within two weeks of birth. Other forms are intermediate MSUD, intermittent MSUD, and thiamine-responsive MSUD. In the classic form, the disease becomes apparent after the newborn ingests protein-containing milk. This leads to an increase in isoleucine, leucine, and valine in the body, which becomes toxic to the brain. In the intermittent form, brain damage occurs during times of physical stress (e.g., infection, fever, or not eating for long periods of time), leading to metabolic decompensation.

[0005] Diagnostic testing for MSUD in newborns includes blood and urine amino acid testing to determine the concentrations of leucine, isoleucine, alloisoleucine, and valine in their body fluids. If MSUD is identified, signs of ketosis and acidosis will be present. At diagnosis and during symptomatic episodes, treatment involves a protein-free diet, with correction of metabolic consequences associated with increased amino acid levels. The use of special intravenous solutions reduces leucine levels (the most toxic) and corrects the energy deficit.

[0006] Current treatment involves dietary restriction of branched-chain amino acids (BCAAs). Insufficient levels of BCKDH complex enzymes result in the toxic deposition of BCAAs and their associated metabolites in the cerebrospinal fluid, blood, and tissues. Without treatment or consistent, intensive care, this results in numerous serious side effects (e.g., neurological dysfunction, seizures, and infant death). Although some BCAAs are reversed via renal clearance (resulting in the typical sweet maple syrup odor of affected patients' urine), this is not sufficient to provide relief from the accumulation of toxic amino acid levels in the body (Schadewalt and Wendel, Eur. J. Pediatr., 156(Suppl. 1): S62-66

[1997] ; and Skvorak, J. Inherit. Metab. Dis., 32(2):229-46

[2009] (See [Prior art documents] [Non-patent literature]

[0007] [Non-Patent Document 1] Menkes et al., Pediatrics 14:462-467

[1954] [Non-patent document 2] Schadewalt and Wendel, Eur. J. Pediatr., 156(Suppl. 1): S62-66

[1997] [Non-patent document 3] Skvorak, J. Inherit. Metab. Dis., 32(2):229-46

[2009] Summary of the Invention [Means for solving the problem]

[0008] Summary of the Invention The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase (LDC) polypeptides. In some embodiments, the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, reduced susceptibility to proteolysis, and / or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability. The present invention also provides methods for using compositions comprising engineered LDC polypeptides for therapeutic and industrial purposes.

[0009] The present invention is directed to engineered LDC polypeptides and biologically active fragments and analogs thereof that have improved properties when compared to wild-type LDC enzymes or reference LDC polypeptides under essentially the same conditions. The present invention is further directed to methods of using the engineered LDC polypeptides and biologically active fragments and analogs thereof in therapeutic and / or industrial compositions, and methods of using such compositions for therapeutic and / or industrial purposes.

[0010] The present invention provides engineered leucine decarboxylase polypeptides comprising 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, and / or 766, wherein the amino acid positions of the amino acid sequence are numbered with reference to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766.

[0011] The present invention also provides engineered leucine decarboxylase polypeptides, wherein the polypeptide sequence has 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. The present invention further provides engineered leucine decarboxylase polypeptides, wherein the polypeptide sequence has 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. The present invention further provides engineered leucine decarboxylase polypeptides, wherein the polypeptide sequence has 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. The present invention further provides engineered leucine decarboxylase polypeptides, wherein the polypeptide sequence has 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. The present invention further provides an engineered leucine decarboxylase polypeptide, wherein the polypeptide sequence has 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.The present invention further provides an engineered leucine decarboxylase polypeptide, wherein the polypeptide sequence has 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.

[0012] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is 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 / 357, 34 / 3 9 / 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,and at least one substitution or set of substitutions at one or more amino acid positions selected from 164, 196, 255, 299, 318, 324, 339, 343, 350, 353, 357, 364, 365, 379, 381, 386, 389, 391, 393, 394, 395, 397, 398, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO:12. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 ... / 38V / 39N / 127S / 245M / 349T / 350E / 357V, 34L / 38V / 39N / 127S / 245M / 350E / 357V, 34L / 39N / 102S / 127S / 264V / 275S / 357V, 34L / 39N / 102S / 127S / 275S / 349T / 357V, 34L / 39N / 102S / 264V / 275S / 350E / 357V, 34L / 39N / 275S / 349T / 350E / 357V, 38V / 39N / 102 S / 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 / 275S , 38V / 39N / 127S / 264V / 350E / 357V, 38V / 39N / 127S / 350E / 357V, 38V / 39N / 127S / 357V, 38V / 39N / 245M / 275S / 357V, 38V / 39N / 2 64V / 267I / 275S / 350E, 38V / 39N / 264V / 275S / 357V, 38V / 39N / 275S, 38V / 39N / 275S / 350E, 39N / 102S / 127S / 264V / 275S / 357V,39N / 102S / 264V / 275S / 357V, 39N / 102S / 267I / 275S / 357V, 39N / 127S / 245M / 264V / 267I / 275S / 3 50E, 39N / 127S / 245M / 264V / 275S / 350E / 357V, 39N / 127S / 245M / 357V, 39N / 127S / 267I / 275S / 35 0E / 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, 2 55N, 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*, 389E, 389G, 389 and at least one substitution or set of substitutions at one or more amino acid positions selected from P, 389Q, 391*, 391E, 393T, 394E, 395A, 395D, 395G, 395K, 395S, 397A, 398*, 405D, 405E, 405H, and 405L, wherein the amino acid positions are numbered with reference to SEQ ID NO:12. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of K5M, H14I, H14T / I34L / C38V / T39N / T102S / V267I / T275S / N350E / I357V, H14T / T39N / T102S / T127S / I245M / V267I / T275S / V349T / N350E, I34L / C38V / T39N / T1 02S / T127S / T275S / I357V, I34L / C38V / T39N / T102S / T275S / I357V, I34L / C38V / T39N / T127S / I245M / V349T / N 350E / I357V, I34L / C38V / T39N / T127S / I245M / N350E / I357V, I34L / T39N / T102S / T127S / I264V / T275S / I357V,<h2 style=";text-align:left;direction:ltr">I34L / T39N / T102S / T127S / T275S / V349T / I357V、I34L / T39N / T102S / I264V / T275S / N350E / I357V、I34L / T39N / T275S / V349T / N350E / I357V、C38V / T39N / T 102S / T127S / I264V / V267I / N350E / I357V、C38V / T39N / T102S / T127S / V267I / T275S / V349T / N350E / I357V、C38V / T39N / T102S / T127S / V349T / N350E / I357 V、C38V / T39N / T102S / T127S / N350E、C38V / T39N / T102S / T127S / N350E / I357 V、C38V / T39N / T127S / I245M / V267I / I357V、C38V / T39N / T127S / I264V / T275S C38V / T39N / T127S / I264V / N350E / I357V, C38V / T39N / T127S / N350E / I357V, C38V / T39N / T127S / I357V, C38V / T39N / I245M / T275S / I357V, C38V / T39N / I2 64V / V267I / T275S / N350E、C38V / T39N / I264V / T275S / I357V、C38V / T39N / T275S、C38V / T39N / T275S / N350E、T39N / T102S / T127S / I264V / T275S / I357V、T3 9N / T102S / I264V / T275S / I357V、T39N / T102S / V267I / T275S / I357V、T39N / T127S / I245M / I264V / V267I / T275S / N350E、T39N / T127S / I245M / I264V / T275S / N350E / I357V、T39N / T127S / I245M / I357V、T39N / T127S / V267I / T275S / N350E / I357V、T39N / T127S / V267I / N350E / I357V、T39N / T127S / I357V、T39N / I24 5M / I264V / V267I / T275S / I357V、T39N / I264V / V267I / T275S / N350E、T39N / T275S / 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, and T405L, wherein the amino acid positions are numbered with reference to SEQ ID NO: 12.

[0013] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is selected from the group consisting of 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 / 364, 1 64 / 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 / 389 / 394 / and at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 394 / 397, wherein the amino acid positions are numbered with reference to SEQ ID NO:38. In some further embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 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 / 324 M / 343E / 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 / 364R, 16 4C / 353W / 357C / 364R, 196D / 318K / 324M / 353N / 357C / 364K, 318K / 343E / 357C, 318K / 343E / 357M, 324M / 343E / 357V / 364K , 324M / 357M / 364R, 324N / 353W / 357C / 364K, 339A / 379D / 389G / 394E / 395D, 339A / 389G / 395K, 339A / 391*, 339A / 394E / 3 and at least one substitution or set of substitutions at one or more amino acid positions selected from 95K / 405D, 357V / 364R, 379D / 386*, 379D / 394E / 395D / 397A / 404I / 405H, 379D / 394E / 395K / 397A / 405D, 389G / 394E / 395D / 397A / 405D, and 394E / 397A, wherein the amino acid positions are numbered with reference to SEQ ID NO:38. In some further embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / R353D / I357V / L364K, A64E / I164A / R324M / H343E / I357C / L364R, A64E / I164C / R353D / I357V, A64E / R318K / R324 S / I357V / L364R, A64E / R324M / R353N / I357C / L364R, Y132F / H255P / Q339A / K379D / R395D, I164A / K196D / R324M / I357C / L364K, I164A / R318K / R324M / H343E / R353E / I357C, I164A / R324M / H343E / R353D / I357C / L364R, I164A / R324M / I357C / L364K, I164A / R353W / I357C / L364R, I164A / L364R, I164C / R318K / R324S / I357V / L364R, I164C / R324M / H343E / R353D / I357V / L364R, I164C / R353D / I357V / L364K, I164C / R353D / I357V / L 364R, I164C / R353W / I357C / L364R, K196D / R318K / R324M / R353N / I357C / L364K, R318K / H343E / I357C, R318K / H343E / I357M, R324M / H343E / I 357V / L364K, R324M / I357M / L364R, R324N / R353W / I357C / L364K, Q339A / K379D / K389G / K394E / R395D, Q339A / K389G / R395K, Q339A / A391*, Q3 and K394E / T397A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 38.

[0014] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is selected from the group consisting of 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, and at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO:234.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 ... 94E / 395D, 324S / 389G / 394E / 397A, 324S / 394E, 324S / 394E / 395K, 324S / 3 94E / 395K / 397A, 324S / 395K, 339A, 340T, 340V, 380E, 382S, 389G, 389G / 39 4E, 389G / 394E / 395D, 389G / 394E / 395D / 397A, 389G / 394E / 395K, 389G / 394 E / 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, and 405H, wherein the amino acid positions are numbered with reference to SEQ ID NO: 234. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from G2E, N3M, F33L, L48F / A64E / H255P, L48F / H255P / Q339A, L48F / H255P / K379D, A64E, A64E / H255P, A64S, V69I, T161 V, M193I, H255P, H255P / R318K / K379D, R259L, S263T, S263V, R318K / Q339A / K379D, M324 N, M324N / K394E / R395K / T397A, M324N / R395D, M324S / K389G / K394E, M324S / K389G / K394 E / R395D, M324S / K389G / K394E / T397A, M324S / K394E, M324S / K394E / R395K, M324S / K394 E / R395K / T397A, M324S / R395K, Q339A, S340T, S340V, A380E, A382S, K389G, K389G / K394 E, K389G / K394E / R395D, K389G / K394E / R395D / T397A, K389G / K394E / R395K, K389G / K394 E / 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, and T405H, wherein the amino acid positions are numbered with reference to SEQ ID NO:234.

[0015] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is selected from the group consisting of 2 / 64 / 69 / 324 / 380 / 382 / 388 / 389, 3 / 64 / 69 / 263 / 339 / 380 / 388, 3 / 64 / 69 / 389, 3 / 64 / 69 / 324 / 380 / 382 / 38 ... 9 / 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 / 25 5, 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 / 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, and 304 / 340 / 379 / 380 / 382, wherein the amino acid positions are numbered with reference to SEQ ID NO:284.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 64 A / 255H, 64A / 255H / 263T, 64S / 69I, 64S / 69I / 189A / 259Q / 263T / 304R / 33 9A / 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 / 3 24S, 64S / 69I / 324S / 339A / 380E / 389G / 390*, 64S / 69I / 339A, 64S / 69I / 3 39A / 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*, 69 I / 223M / 324S / 379D / 380E / 382S / 388A / 390*, 69I / 263T, 69I / 263T / 324S , 69I / 263T / 339A, 69I / 263T / 388A, 69I / 263T / 389G / 390*, 69I / 324S / 37 9D / 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 *, and 304R / 340T / 379D / 380E / 382G, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from 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 / T189A / R259Q / S263T / A304R / Q339A / S340T / K379N, E64S / V69I / T189D / R259K / S263T / 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 / Q 339A / A382S / Q388A / K389G, E64S / V69I / Q339A / K389G / P390*, E64S / V69I / K379D / A380E, E64S / V69I / A380E / Q388A / P390*, E64S / V 69I / 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 / A 380E / Q388A, V69I / M324S / A380E, V69I / Q339A / P390*, V69I / A382S / P390*, R259K / S263T / A 304R, R259K / S263T / A304R / Q339A / S340T / K379N, S263T / Q339A / K389G / P390*, S263T / P390, * and A304R / S340T / K379D / A380E / A382G, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284.

[0016] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is selected from the group consisting of 3 / 194 / 304, 3 / 259 / 263 / 304, 3 / 263 / 304, 3 / 319 / 304, 3 / 419 / 304, 3 / 59 / 304, 3 / 619 / 304, 3 / 719 / 304, 3 / 819 / 304, 3 / 919 / 304, 3 / 194 / 304, 3 / 259 / 263 / 304, 3 / 309 / 309, 3 / 419 / 304, 3 / 519 / 304, 3 / 619 / 304, 3 / 719 / 304, 3 / 819 / 304, 3 / 919 / 304, 3 / 194 / 304, 3 / 259 / 263 / 304, 3 / 263 / 304, 3 / 319 / 304, 3 / 263 / 304, 3 / 319 / 304, 3 / 419 / 304, 3 / 519 / 304, 3 / 194 / 304, 3 / 259 / 263 / 304, 3 / 263 / 304, 3 / 319 / 304, 3 / 419 / 304, 3 / 519 / 304, 3 / 263 / 304, 3 , 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 / 304, 263 / 30 and at least one substitution or set of substitutions at one or more amino acid positions selected from: 4 / 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, and 382, ​​wherein the amino acid positions are numbered with reference to SEQ ID NO:484. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 324S, 16Q, 16V, 63C, 77L, 80G, 80K, 87R / 270R, 87R / 270R / 3 65E, 87R / 328N / 365E, 91A, 91Q, 92K, 126A, 126T, 140V, 156A, 156S, 168K / 270R / 328N / 338S, 181K, 181R, 18 1V, 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 / 324S, 259K / 304R / 32 4S / 339A, 259K / 304R / 324S / 339A / 382S, 259K / 304R / 382S, 262D, 262G, 262H, 262I, 262S, 262T, 263T / 304R , 263T / 304R / 324S, 263T / 304R / 324S / 339A, 263T / 304R / 324S / 382S, 263T / 324S, 270R, 270R / 319A, 270R / 3 In some additional embodiments, the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 28N / 338S, 270R / 328N / 338S / 365E, 304R, 304R / 324S, 324S, 328N, 352A, 365E, 366A, 366L, 366M, 366Q, 366T, 366V, and 382S, wherein the amino acid positions are numbered with reference to SEQ ID NO: 484. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of N3M / F194L / A304R, N3M / R259K / S263T / A304R, N3M / R259K / A304R, N3M / R259K / A304R / M324S / Q339A ... 24S / A382S, N3M / R259K / A304R / A382S, N3M / S263T / A304R / M324S, N3M / S263T / A304R / M324S / Q339A, N3M / 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, G 156A, G156S, C168K / L270R / C328N / P338S, T181K, T181R, T181V, F194C, F194L, E201D, Y256W, R259K, R259K / S263T, R259K / S263T / A304R, R259K / S263T / A304R / M324S, R259K / S263T / A304R / M324S / A382S, R259K / S263T / A304R / K379 D, R259K / S263T / A304R / A382S, R259K / A304R, R259K / A304R / M324S, R259K / A304R / M324S / Q339A, R259K / A304R / M32 4S / Q339A / A382S, R259K / A304R / A382S, R262D, R262G, R262H, R262I, R262S, R262T, S263T / A304R, S263T / A304R / M 324S, S263T / A304R / M324S / Q339A, S263T / A304R / M324S / A382S, S263T / M324S, L270R, L270R / I319A, L270R / C328N / and at least one substitution or set of substitutions at one or more amino acid positions selected from P338S, L270R / C328N / P338S / Q365E, A304R, A304R / M324S, M324S, C328N, D352A, Q365E, H366A, H366L, H366M, H366Q, H366T, H366V, and A382S, wherein the amino acid positions are numbered with reference to SEQ ID NO: 484.

[0017] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide of said engineered leucine decarboxylase polypeptide The peptide sequences were 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 / 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, and 168 / 366, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 16Q / 63C / 80K / 126T / 168K / 366M, 16Q / 63C / 80K / 126T / 181R / 194C / 259K / 324S / 328N / 366M, 16Q / 63C / 126T / 168K / 270R / 328N / 366M, ...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 / 328N / 366M, 16Q / 168K / 324S / 366M, 16Q / 168K / 366M, 16Q / 259K / 263T / 328N, 16Q / 324S / 328N / 366M, 16Q / 328N / 366M, 80K / 126T / 168 and 168K / 366M, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is R16Q / A63C / A80K / D126T / C168K / H366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A80K / D126T / C168K / L270R / C328N / H366M, R16Q / A63C ... 6T / M324S / H366M, R16Q / A80K / D126T / H366M, R16Q / A80K / C168K, R16Q / A80K / C168K / L270R / H366M, R16Q / A80K / C168K / M324S, R16Q / A80K / C168K / H366M, R16Q / A80K / M324S, R16Q / E91A / D126T / C168K / M324S / H366M, R16Q / D126T / C168K / H 366M, 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, and C168K / H366M, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.

[0018] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide is selected from the group consisting of 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 / and at least one substitution or set of substitutions at one or more amino acid positions selected from 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, 126 / 270, 141 / 144 / 198 / 200 / 300, 156 / 270, 156 / 270 / 324, 201 / 270, 201 / 270 / 352, 270, and 270 / 324, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 300K, 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, and 270R / 324S, wherein the amino acid positions are numbered with reference to SEQ ID NO:686.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is S66N / R76V / T118D / R141P / E201R / R300K, S66N / R76V / A198G / H200S / D296E / A303Q, S66N / R76V / A198G / H200S / R300K, ...T118D / H200S / D296E / A303Q / K317Q, S66N / T118D / D296E, S66N / T118D / D296E / R300K, S66N / H200S, R7 6V / T118D / R141P / H200S / D296E, R76V / R141P / A198G / H200S / E201R / R300K, A80K / E201D / L270R, A80K / L270R, A80K / L270R / M324S, A89P / T118D / H200S, L106M / L270R / M324S / D352A, T118D / R14 1P / H200S, D126T, D126T / E201D / L270R / M324S, D126T / L270R, R141P / M144V / A198G / H200S / R30 0K, G156A / L270R, G156A / L270R / M324S, E201D / L270R, E201D / L270R / D352A, L270R, and L270R / M324S, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.

[0019] In some further embodiments, the engineered leucine decarboxylase polypeptide sequence has 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 the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 19, 109, 123, 134, 170, 173, 187, 211, and 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 19I, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 173I, 173T, 187L, 211S, and 312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A211S, and T312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.

[0020] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 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, 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 / 2 45 / 264 / 275 / 350 / 357, 39 / 127 / 245 / 357, 39 / 127 / 267 / 275 / 350 / 357, 39 / 127 / 267 / 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,and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO: 12. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from 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 / 275S / 357V, 34L / 38V / 39N / 102S / 245M / 34 9T / 350E / 357V, 34L / 38V / 39N / 127S / 245M / 350E / 357V, 34L / 39N / 102S / 127S / 264V / 275S / 357V, 34L / 39N / 102S / 127S / 275S / 349T / 357V, 3 4L / 39N / 102S / 264V / 275S / 350E / 357V, 34L / 39N / 275S / 349T / 350E / 357V, 38V / 39N / 102S / 127S / 264V / 267I / 350E / 357V, 38V / 39N / 102S / 1 27S / 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 / 275S, 38V / 39N / 127S / 264V / 350E / 357V, 38V / 39N / 127S / 350E / 357V, 38V / 39N / 1 27S / 357V, 38V / 39N / 245M / 275S / 357V, 38V / 39N / 264V / 267I / 275S / 350E, 38V / 39N / 264V / 275S / 357V, 38V / 39N / 275S, 38V / 39N / 275S / 350 E、39N / 102S / 127S / 264V / 275S / 357V、39N / 102S / 264V / 275S / 357V、39N / 102S / 267I / 275S / 357V、39N / 127S / 245M / 264V / 267I / 275S / 350E、39N / 127S / 245M / 264V / 275S / 350E / 357V, 39N / 127S / 245M / 357V, 39N / 127S / 267I / 2 75S / 350E / 357V, 39N / 127S / 267I / 350E / 357V, 39N / 127S / 357V, 39N / 245M / 264V / 26 7I / 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, 33 and at least one substitution or set of substitutions at one or more amino acid positions selected from 9A, 339D, 343A, 343E, 350S, 353D, 353E, 353L, 353N, 353S, 353W, 357C, 357M, 364K, 364R, 365E, 379D, 379P, 381D, 381E, 386*, 389E, 389G, 389P, 389Q, 391*, 391E, 393T, 394E, 395A, 395D, 395G, 395K, 395S, 397A, 398*, 405D, 405E, 405H, and 405L, wherein the amino acid positions are numbered with reference to SEQ ID NO:12. In some further embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / T10 2S / T275S / I357V, I34L / C38V / T39N / T127S / I245M / V349T / N350E / I357V, I34L / C38V / T39N / T127S / I245M / N350E / I357V, I34L / T39N / T102S / T127S / I264V / T275S / I357V, I34L / T39N / T102S / T127S / T275S / V349T / I357V, I34L / T39N / T102S / I264V / T275S / N350E / I357V,<h2 style=";text-align:left;direction:ltr">I34L / T39N / T275S / V349T / N350E / I357V、C38V / T39N / T102S / T127S / I264V / V267I / N350E / I357V、C38V / T39N / T102S / T127S / V267I / T275S / V349T / N350 E / I357V、C38V / T39N / T102S / T127S / V349T / N350E / I357V、C38V / T39N / T102 S / T127S / N350E、C38V / T39N / T102S / T127S / N350E / I357V、C38V / T39N / T127S / I245M / V267I / I357V、C38V / T39N / T127S / I264V / T275S、C38V / T39N / T127S / I264V / N350E / I357V、C38V / T39N / T127S / N350E / I357V、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 / T27 5S / N350E、T39N / T102S / T127S / I264V / T275S / I357V、T39N / T102S / I264V / T275S / I357V、T39N / T102S / V267I / T275S / I357V、T39N / T127S / I245M / I264V / V267I / T275S / N350E、T39N / T127S / I245M / I264V / T275S / N350E / I357V、T39N / T127S / I245M / I357V、T39N / T127S / V267I / T275S / N350E / I357V、T39N / T1 27S / V267I / N350E / I357V、T39N / T127S / I357V、T39N / I245M / I264V / V267I / T275S / I357V、T39N / I264V / V267I / T275S / N350E、T39N / T275S / N350E / I357V 、T39S、L48F、N139G、I164A、I164C、K196D、K196R、H255G、H255N、H255P、K299A、K299V、R318K、R324M、R324S、R324T、Q339A、Q339D、H343A、H343E、N350S、and at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and T405L, wherein the amino acid positions are numbered with reference to SEQ ID NO: 12.

[0021] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 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 / 389 / 394 / 395, 339 / 389 / 395, 339 / 391, 339 / 394 In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 48F / 64E / 164A / 324M / 343E / 353E / 357C / 364K, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164A / 324M / 343E / 364K, 48F / 64E / 164A / 324M / 343E / 364R, 48F / 64E / 164C / 353N / 357V ... F / 64E / 357M / 364K, 64E / 164A / 324M / 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 / 343E / 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 / 364R, 164C / 353W / 357C / 364R, 196D / 318K / 324M / 353N / 357C / 364K, 318K / 343E / 357C, 318K / 343E / 357M, 324M / 343E / 357V / 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*, and comprising at least one substitution or set of substitutions at one or more amino acid positions from 379D / 394E / 395D / 397A / 404I / 405H, 379D / 394E / 395K / 397A / 405D, 389G / 394E / 395D / 397A / 405D, and 394E / 397A, where the amino acid positions are numbered with reference to SEQ ID NO:38. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is L48F / A64E / I164A / R324M / H343E / R353E / I357C / L364K, L48F / A64E / I164A / R324M / H343E / L364R, L48F / A64E / I164C / R353N / I357V / L364R, L48F / A64E / I164A / R324M / H343E ... 4E / I357M / L364K, A64E / I164A / R324M / H343E / R353D / I357V / L364K, A64E / I164A / R324M / H343E / I357C / L 364R, 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 / R353E / I357 C, I164A / R324M / H343E / R353D / I357C / L364R, I164A / R324M / I357C / L364K, I164A / R353W / I357C / L364R, I164A / L3 64R, I164C / 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 / I357V / L364K, R324M / I357M / L364R, R324N / R353W / I357C / L364K, Q33 9A / K379D / K389G / K394E / R395D, Q339A / K389G / R395K, Q339A / A391*, Q339A / K394E / R395K / T405D, I357V / L364R, K3 and K394E / T397A / T405D, K389G / K394E / R395D / T397A / T405D, and K394E / T397A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 38.

[0022] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO:234. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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, 324S / 389G / 394E / 395D, 324S / 389G / 394E / 397A, 324S / 394E, 324S / 394E / 395K, 324S / 394E / 395K / 397A, 324S / 395K, 339A, 340T, 340V, 380E, 382S, 389G, 389G / 3 94E, 389G / 394E / 395D, 389G / 394E / 395D / 397A, 389G / 394E / 395K, 389G / 39 4E / 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, and 405H, wherein the amino acid positions are numbered with reference to SEQ ID NO: 234. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from G2E, N3M, F33L, L48F / A64E / H255P, L48F / H255P / Q339A, L48F / H255P / K379D, A64E, A64E / H255P, A64S, V69I, T161 V, M193I, H255P, H255P / R318K / K379D, R259L, S263T, S263V, R318K / Q339A / K379D, M324 N, M324N / K394E / R395K / T397A, M324N / R395D, M324S / K389G / K394E, M324S / K389G / K394 E / R395D, M324S / K389G / K394E / T397A, M324S / K394E, M324S / K394E / R395K, M324S / K394 E / R395K / T397A, M324S / R395K, Q339A, S340T, S340V, A380E, A382S, K389G, K389G / K394 E, K389G / K394E / R395D, K389G / K394E / R395D / T397A, K389G / K394E / R395K, K389G / K394 E / 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, and T405H, wherein the amino acid positions are numbered with reference to SEQ ID NO:234.

[0023] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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, 12 / 135 / 26 3 / 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 / 263 / 324, 69 / 263 / 339, 69 / 263 / 388, 69 / 263 / 3 and at least one substitution or set of substitutions at one or more amino acid positions selected from 89 / 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, and 304 / 340 / 379 / 380 / 382, wherein the amino acid positions are numbered with reference to SEQ ID NO:284.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 135 V / 259K / 263T, 12G / 135V / 263T / 382G, 12G / 259K / 263T / 304R, 48L / 64A / 2 55H, 64A / 255H / 263T, 64S / 69I, 64S / 69I / 189A / 259Q / 263T / 304R / 339A / 3 40T / 379N, 64S / 69I / 189D / 259K / 263T / 304R, 64S / 69I / 223M / 388A, 64S / 6 9I / 223M / 388A / 389G / 390*, 64S / 69I / 304R / 379E / 382G, 64S / 69I / 324S, 6 4S / 69I / 324S / 339A / 380E / 389G / 390*, 64S / 69I / 339A, 64S / 69I / 339A / 3 82S / 388A / 389G, 64S / 69I / 339A / 389G / 390*, 64S / 69I / 379D / 380E, 64S / 6 9I / 380E / 388A / 390*, 64S / 69I / 389G, 64S / 69I / 390*, 64S / 263T, 64S / 324 S / 339A / 389G / 390*, 69I / 223M / 263T / 324S / 382S / 388A / 390*, 69I / 223M / 324S / 379D / 380E / 382S / 388A / 390*, 69I / 263T, 69I / 263T / 324S, 69I / 263 T / 339A, 69I / 263T / 388A, 69I / 263T / 389G / 390*, 69I / 324S / 379D / 380E / 3 88A, 69I / 324S / 380E, 69I / 339A / 390*, 69I / 382S / 390*, 259K / 263T / 304R , 259K / 263T / 304R / 339A / 340T / 379N, 263T / 339A / 389G / 390*, 263T / 390. *, and 304R / 340T / 379D / 380E / 382G, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from 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 / T189A / R259Q / S263T / A304R / Q339A / S340T / K379N, E64S / V69I / T189D / R259K / S263T / 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 / Q 339A / A382S / Q388A / K389G, E64S / V69I / Q339A / K389G / P390*, E64S / V69I / K379D / A380E, E64S / V69I / A380E / Q388A / P390*, E64S / V 69I / 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 / A 380E / Q388A, V69I / M324S / A380E, V69I / Q339A / P390*, V69I / A382S / P390*, R259K / S263T / A 304R, R259K / S263T / A304R / Q339A / S340T / K379N, S263T / Q339A / K389G / P390*, S263T / P390, * and A304R / S340T / K379D / A380E / A382G, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284.

[0024] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 324, 25 9 / 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, 263 / 304 / 3 and at least one substitution or set of substitutions at one or more amino acid positions selected from 24 / 382, 263 / 324, 270, 270 / 319, 270 / 328 / 338, 270 / 328 / 338 / 365, 304, 304 / 324, 324, 328, 352, 365, 366, and 382, ​​wherein the amino acid positions are numbered with reference to SEQ ID NO:484. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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, 1 6V, 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, 256W, 259K, 259K / 263T, 259K / 263T / 304R, 259K / 263T / 304R / 324S, 259K / 263T / 304R / 324S / 382S, 2 59K / 263T / 304R / 379D, 259K / 263T / 304R / 382S, 259K / 304R, 259K / 304R / 324S, 259K / 30 4R / 324S / 339A, 259K / 304R / 324S / 339A / 382S, 259K / 304R / 382S, 262D, 262G, 262H, 262 and 382S, wherein the amino acid positions are numbered with reference to SEQ ID NO:484. In some further embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / L 270R, 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, R259K / S263T / A304R, R259K / S263T / A304R / M324S, R259K / S263T / A304R / M324S / A382S, R259K / S263T / A304R / K379D, R 259K / S263T / A304R / A382S, R259K / A304R, R259K / A304R / M324S, R259K / A304R / M324S / Q339A, R 259K / A304R / M324S / Q339A / A382S, R259K / A304R / A382S, R262D, R262G, R262H, R262I, R262S, R2 62T, S263T / A304R, S263T / A304R / M324S, S263T / A304R / M324S / Q339A, S263T / A304R / M324S / A3 82S, S263T / M324S, L270R, L270R / I319A, L270R / C328N / P338S, L270R / C328N / P338S / Q365E, A30 and at least one substitution or set of substitutions at one or more amino acid positions selected from: 4R, A304R / M324S, M324S, C328N, D352A, Q365E, H366A, H366L, H366M, H366Q, H366T, H366V, and A382S, wherein the amino acid positions are numbered with reference to SEQ ID NO: 484.

[0025] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / and 168 / 366, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 16Q / 63C / 80K / 126T / 168K / 366M, 16Q / 63C / 80K / 126T / 181R / 194C / 259K / 324S / 328N / 366M, 16Q / 63C / 126T / 168K / 270R / 328N / 366M, ...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 / 328N / 366M, 16Q / 168K / 324S / 366M, 16Q / 168K / 366M, 16Q / 259K / 263T / 328N, 16Q / 324S / 328N / 366M, 16Q / 328N / 366M, 80K / 126T / 168 and 168K / 366M, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is R16Q / A63C / A80K / D126T / C168K / H366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A63C / A80K / D126T / T181R / F194C / R259K / M324S / C328N / H366M, R16Q / A63C / D126T / C168K / L270R / C328N / H366M, R16Q / A80K / D126T / C168K / L270R / C328N / H366M, R16Q / A63C ... 6T / M324S / H366M, R16Q / A80K / D126T / H366M, R16Q / A80K / C168K, R16Q / A80K / C168K / L270R / H366M, R16Q / A80K / C168K / M324S, R16Q / A80K / C168K / H366M, R16Q / A80K / M324S, R16Q / E91A / D126T / C168K / M324S / H366M, R16Q / D126T / C168K / H 366M, 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, and C168K / H366M, wherein the amino acid positions are numbered with reference to SEQ ID NO:594.

[0026] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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, 126 / 270, 141 / 144 / 198 / 200 / 300, 156 / 270, 156 / 270 / 324, 201 / 270, 201 / 270 / 352, 270, and 270 / 324, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 300K, 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, and 270R / 324S, wherein the amino acid positions are numbered with reference to SEQ ID NO:686.In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is S66N / R76V / T118D / R141P / E201R / R300K, S66N / R76V / A198G / H200S / D296E / A303Q, S66N / R76V / A198G / H200S / R300K, ...T118D / H200S / D296E / A303Q / K317Q, S66N / T118D / D296E, S66N / T118D / D296E / R300K, S66N / H200S, R7 6V / T118D / R141P / H200S / D296E, R76V / R141P / A198G / H200S / E201R / R300K, A80K / E201D / L270R, A80K / L270R, A80K / L270R / M324S, A89P / T118D / H200S, L106M / L270R / M324S / D352A, T118D / R14 1P / H200S, D126T, D126T / E201D / L270R / M324S, D126T / L270R, R141P / M144V / A198G / H200S / R30 0K, G156A / L270R, G156A / L270R / M324S, E201D / L270R, E201D / L270R / D352A, L270R, and L270R / M324S, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.

[0027] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 19, 109, 123, 134, 170, 173, 187, 211, and 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 191, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 1731, 173T, 187L, 211S, and 312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A211S, and T312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.

[0028] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 141 / 170 / 198 / 211 / 270 / 312, 19 / 109 / 123 / 170 / 211 / 270 / 312, and at least one substitution or set of substitutions at one or more amino acid positions selected from 9 / 123 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 170 / 173 / 211 / 270 / 312, 19 / 109 / 211 / 270 / 312, 109 / 170 / 211 / 270 / 312, and 109 / 211 / 270 / 312, wherein the amino acid positions are numbered with reference to SEQ ID NO:688. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 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, and 109G / 211S / 270R / 312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 688.In some further embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / A211S ... and L109G / A211S / L270R / T312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 688.

[0029] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 5 / 41, 5 / 41 / 228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, and 384, wherein the amino acid positions are numbered with reference to SEQ ID NO:766. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 5V / 41D, 5V / 41D / 228D, 33L, 41D, 47F, 51E, 51Q, 55I, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 353I, 353L, 357S, and 384W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from K5V / H41D, K5V / H41D / T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, and P384W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.

[0030] In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 300, wherein the amino acid positions are numbered with reference to SEQ ID NO:766. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 300R, wherein the amino acid positions are numbered with reference to SEQ ID NO:766. In some additional embodiments, the polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and K300R, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.

[0031] In some additional 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, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, or a functional fragment thereof. In some additional embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, or a functional fragment thereof. In some additional embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, or a functional fragment thereof. In some embodiments, the engineered leucine decarboxylase polypeptide is a variant 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, and / or 11-2. In some further embodiments, the engineered leucine decarboxylase polypeptide is a variant enzyme of a Planctomycetaceae bacterial species. In some embodiments, the leucine decarboxylase exhibits at least one improved property compared to a wild-type leucine decarboxylase of a Planctomycetaceae bacterial species. In some embodiments, the engineered leucine decarboxylase polypeptide exhibits greater activity on leucine than the wild-type leucine decarboxylase of the Planctomycetaceae species. In yet some additional embodiments, the engineered leucine decarboxylase polypeptide is more thermostable than the leucine decarboxylase of the wild-type Planctomycetaceae bacterial species.In still some further embodiments, the engineered leucine decarboxylase polypeptide is more resistant to proteolysis than the leucine decarboxylase of a wild-type Planctomycetaceae bacterial species. In some additional embodiments, the engineered leucine decarboxylase polypeptide is less immunogenic than the leucine decarboxylase of a wild-type Planctomycetaceae bacterial species. In yet some additional embodiments, the engineered leucine decarboxylase polypeptide is more serum stable than the leucine decarboxylase of a wild-type Planctomycetaceae bacterial species. In some embodiments, the engineered leucine decarboxylase polypeptide comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the even-numbered sequences of SEQ ID NOs: 16-852. In some embodiments, the engineered leucine decarboxylase polypeptide comprises a sequence at least 90% identical to any of the even-numbered sequences of SEQ ID NOs: 16-852. In some further embodiments, the engineered leucine decarboxylase polypeptide comprises any of the even-numbered sequences of SEQ ID NOs: 16-852. In some additional embodiments, the engineered leucine decarboxylase polypeptide is purified. The present invention also provides compositions comprising at least one engineered leucine decarboxylase polypeptide provided herein. The present invention also provides compositions comprising one engineered leucine decarboxylase polypeptide provided herein.

[0032] The present invention also provides engineered polynucleotide sequences encoding at least one engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the engineered polynucleotide sequence encodes an engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the engineered polynucleotide sequence comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the odd-numbered sequences of SEQ ID NOs: 15-851. In some additional embodiments, the engineered polynucleotide sequence comprises a sequence at least 90% or more identical to any of the odd-numbered sequences of SEQ ID NOs: 15-851. In some further embodiments, the engineered polynucleotide sequence comprises any of the odd-numbered sequences of SEQ ID NOs: 15-851. In some additional embodiments, the engineered polynucleotide sequence is operably linked to a regulatory sequence. In some embodiments, the engineered polynucleotide sequence is codon-optimized.

[0033] The present invention also provides an expression vector comprising at least one engineered polynucleotide sequence provided herein. In some embodiments, the expression vector further comprises at least one regulatory sequence. In some embodiments, the regulatory sequence comprises a promoter. In some further embodiments, the promoter is a heterologous promoter.

[0034] The present invention also provides host cells transformed with at least one polynucleotide sequence provided herein and / or host cells comprising an expression vector provided herein. In some embodiments, the host cell is transformed with at least one polynucleotide sequence provided herein. In some embodiments, the host cell is transformed with a polynucleotide sequence provided herein. In some additional embodiments, the host cell comprises at least one expression vector provided herein. In some further embodiments, the host cell comprises an expression vector provided herein. In some embodiments, the host cell is E. coli.

[0035] The present invention 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 polynucleotide encoding at least one engineered leucine decarboxylase polypeptide provided herein, and / or at least one polynucleotide sequence provided herein, and / or at least one expression vector provided herein, under suitable culture conditions such that at least one engineered leucine decarboxylase polypeptide is produced. In some embodiments, the method for producing an engineered leucine decarboxylase polypeptide in a host cell comprises culturing a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide provided herein, under suitable culture conditions such that at least one engineered leucine decarboxylase polypeptide is produced. In some additional embodiments, the method for producing an engineered leucine decarboxylase polypeptide in a host cell comprises culturing a host cell comprising at least one polynucleotide sequence provided herein, under suitable culture conditions such that at least one engineered leucine decarboxylase polypeptide is produced. In some embodiments, a method for producing an engineered leucine decarboxylase polypeptide in a host cell comprises culturing a host cell comprising at least one expression vector provided herein under suitable culture conditions such that at least one engineered leucine decarboxylase polypeptide is produced. 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.

[0036] The present invention also provides compositions comprising at least one engineered polynucleotide provided herein. In some embodiments, the composition comprises at least one engineered leucine decarboxylase polynucleotide provided herein. In some embodiments, the composition comprises at least one engineered polynucleotide encoding at least one engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the composition is a pharmaceutical composition. In some additional embodiments, the composition further comprises at least one pharmaceutically acceptable excipient and / or carrier. In some embodiments, the composition is suitable for treating maple syrup urine disease. In some additional embodiments, the composition is suitable for use in gene therapy. In yet some further embodiments, the composition is suitable for use in gene therapy to treat maple syrup urine disease and / or elevated blood levels of isoleucine, leucine, alloisoleucine, and / or valine. In some additional embodiments, the composition is suitable for use in mRNA therapy. In yet some additional embodiments, the composition is suitable for oral administration to humans. In some embodiments, the composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. In some further embodiments, the pill, tablet, capsule, or gelcap further comprises an enteric coating. In yet some additional embodiments, the composition is suitable for parenteral injection into animals. In yet some additional embodiments, the composition is suitable for parenteral injection into humans. In some embodiments, the injection is administered on a daily, weekly, or monthly basis. In some additional embodiments, the composition is co-administered with at least one additional therapeutically effective compound. In some embodiments, the composition comprises at least one additional therapeutically effective compound.

[0037] The present invention also provides methods for treating and / or preventing symptoms of maple syrup urine disease in a subject, the method comprising providing a subject with maple syrup urine disease and providing the subject with a composition provided herein. In some embodiments, the symptoms of maple syrup urine disease are ameliorated. In some additional embodiments, the subject is able to eat a diet that is less restricted in isoleucine, leucine, and / or valine content than a diet required by a subject not provided with at least one composition comprising at least one engineered leucine decarboxylase polypeptide and / or polynucleotide provided herein. In some embodiments, the subject is able to eat a diet that is less restricted in isoleucine, leucine, and / or valine content than a diet required by a subject not provided with at least one composition comprising at least one engineered leucine decarboxylase polypeptide provided herein.

[0038] 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.

[0039] In some embodiments, the subject is a young adult. In some embodiments, the subject is an adult. The present invention also provides uses of the compositions provided herein. In some embodiments, the compositions comprise at least one engineered leucine decarboxylase polypeptide and / or polynucleotide provided herein. In some embodiments, the compositions comprise at least one engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise an engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise at least one engineered leucine decarboxylase polynucleotide provided herein. In some embodiments, the compositions comprise an engineered leucine decarboxylase polynucleotide provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise at least two polynucleotides encoding at least two engineered leucine decarboxylase polypeptides provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least one engineered leucine decarboxylase provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least two engineered leucine decarboxylase polypeptides provided herein. In some embodiments, the compositions comprise at least one polynucleotide encoding at least one engineered leucine decarboxylase provided herein. In some embodiments, a composition comprises a polynucleotide encoding an engineered leucine decarboxylase polypeptide provided herein. DETAILED DESCRIPTION OF THE INVENTION

[0040] Description of the Invention The present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase (LDC) polypeptides. In some embodiments, the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, reduced susceptibility to proteolysis, and / or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability. The present invention also provides methods for the use of compositions comprising engineered LDC polypeptides for therapeutic and industrial purposes. The present invention provides engineered LDC polypeptides, mutants, biologically active fragments, and analogs thereof, as well as pharmaceutical and industrial compositions comprising the same. Abbreviations and definitions:

[0041] Unless otherwise defined, all technical and scientific terms used herein generally have the same meaning as commonly understood by those skilled in the art to which this invention belongs. Generally, the nomenclature used herein and the laboratory procedures for 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 are described in numerous texts and references well known to those skilled in the art. Standard techniques, or variations thereof, are used for chemical synthesis and chemical analysis. All patents, patent applications, papers, and publications mentioned herein, both above and below, are hereby expressly incorporated by reference.

[0042] Although any suitable method and material similar or equivalent to those described herein can be used in the practice of the present invention, some methods and materials are described herein.It should be understood that the present invention is not limited to the specific methodology, protocols, and reagents described, as they may vary depending on the context in which they are used by those skilled in the art.Therefore, the terms defined immediately below are more fully described by referring to this application as a whole.All patents, patent applications, papers, and publications mentioned herein, both above and below, are hereby expressly incorporated by reference into this specification.

[0043] Also, as used herein, the singular forms "a," "an," and "the" include plural referents unless the context clearly dictates otherwise.

[0044] Numerical ranges are inclusive of the numbers defining the range. Thus, every numerical range disclosed herein is intended to encompass every narrower numerical range that falls within such broader numerical range, as if such narrower numerical ranges were all expressly written herein. Every maximum (or minimum) numerical limitation disclosed herein is also intended to include every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were all expressly written herein.

[0045] The term "about" refers to a tolerance for a particular value. In some instances, "about" means within 0.05%, 0.5%, 1.0%, or 2.0% of a given value range. In some instances, "about" means within 1, 2, 3, or 4 standard deviations of a given value.

[0046] Furthermore, the headings provided herein are not limitations of the various aspects or embodiments of the invention, which can be done by reference to the application as a whole. Accordingly, the terms defined immediately below are more fully defined by reference to the application as a whole. Nevertheless, to facilitate understanding of the invention, certain terms are defined below.

[0047] 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.

[0048] 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).

[0049] The "EC" number refers to the Nomenclature Commission of the International Union of Biochemistry and Molecular Biology (NC-IUBMB) enzyme nomenclature. The IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reaction they catalyze.

[0050] "ATCC" refers to the American Type Culture Collection, whose biorepository collection includes genes and strains.

[0051] "NCBI" refers to the National Center for Biotechnology Information and the sequence databases provided therein.

[0052] As used herein, the term "leucine decarboxylase (LDL) polypeptide" refers to a member of the class of valine decarboxylase enzymes (EC 4.1.1.14). These enzymes use a pyridoxial 5'-phosphate (PLP) cofactor to decarboxylate amino acids such as valine and leucine to give 2-methylpropanamine and isopentylamine, respectively, while releasing carbon dioxide.

[0053] "Protein," "polypeptide," and "peptide" are used interchangeably herein to refer to a polymer of at least two amino acids covalently linked by amide bonds, regardless of length or post-translational modification (e.g., glycosylation or phosphorylation).

[0054] "Polynucleotide" is used herein to refer to a polymer comprising at least two nucleotides, where the nucleotides are either deoxyribonucleotides or ribonucleotides.

[0055] "Amino acids" are referred to herein by either their commonly known three letter symbols or by the one-letter symbols recommended by the IUPAC-IUB Biochemical Nomenclature Commission. Similarly, nucleotides may be referred to by their commonly accepted single-letter codes, as indicated. Abbreviations used for 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), 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).

[0056] The terms "engineered," "recombinant," "non-naturally occurring," and "variant" when used with respect to a cell, polynucleotide, or polypeptide refer to material that has been modified in a way that would not otherwise occur in nature, or that is identical to these, but is produced or derived from synthetic material and / or by manipulation using recombinant techniques, or material that corresponds to a natural or native form of the material.

[0057] 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 that can be isolated from a natural source and is present in an organism that has not been intentionally modified by human manipulation.

[0058] "Coding sequence" refers to a portion of a nucleic acid (eg, a gene) that codes for the amino acid sequence of a protein.

[0059] The term "percent sequence identity (%)" is used herein to refer to a comparison between polynucleotides and polypeptides and is determined by comparing two optimally aligned sequences over a comparison window, where the portion of the polynucleotide or polypeptide sequence in the comparison window may contain additions or deletions (i.e., gaps) compared to the reference sequence for optimal alignment of the two sequences. The percentage may be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences to obtain 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 obtain the percentage of sequence identity. Alternatively, the percentage may be calculated by determining the number of positions where the same nucleic acid base or amino acid residue occurs in both sequences, or where the nucleic acid base or amino acid residue is aligned with a gap, to obtain 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 obtain the percentage of 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 performed, for example, by the local homology algorithm of Smith and Waterman (Smith and Waterman, Adv. Appl. Math., 2:482

[1981] ), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J. Mol. Biol., 48:443

[1970] ), by the search for similarity method of Pearson and Lipman (Pearson and Lipman, Proc. Natl. Acad. Sci. USA 85:2444

[1988] ), by computer 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 percent sequence similarity include, but are not limited to, the BLAST and BLAST 2.0 algorithms (e.g., Altschul et al., J. Mol. Biol., 215: 403-410

[1990] ); and and Altschul et al., Nucleic Acids Res., 3389-3402

[1977] . Software for performing BLAST analysis is publicly available through the website of the National Center for Biotechnology Information. This algorithm involves initially identifying high-scoring sequence pairs (HSPs) by identifying short words of length "W" in the query sequence that, when aligned with words of the same length in a database sequence, match or meet some positive threshold score "T." T is referred to as the neighborhood word score threshold (Altschul et al., supra). These initial neighborhood word hits are then compared to the initial neighbor word hits. These word hits serve 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, cumulative scores are 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. Extension of word hits in each direction is stopped when the cumulative alignment score falls by an amount "X" from its maximum achieved value; when the cumulative score falls to zero or below due to the accumulation of alignments of one or more negatively scoring residues; or when the end of either sequence is reached. The BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment. The BLASTN program (for nucleotide sequences) uses as defaults a word length (W) of 11, an expectation (E) of 10, M=5, N=-4, and a comparison of both strands. For amino acid sequences, the BLASTP program uses as defaults a word length (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (see, e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA 89:10915

[1989] ). Exemplary sequence alignments and determination of percent sequence identity can utilize the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI) using the default parameters provided.

[0060] A "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, such as a segment of a full-length gene or polypeptide sequence. Generally, a reference sequence is at least 20 nucleotides or amino acid residues in length, at least 25 residues in length, at least 50 residues in length, at least 100 residues in length, or the full length of a nucleic acid or polypeptide. Because two polynucleotides or polypeptides may each contain (1) similar sequences (i.e., a portion of the complete sequence) between the two sequences and (2) additional sequences that differ between the two sequences, sequence comparison between two (or more) polynucleotides or polypeptides is typically 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" can be based on a primary amino acid sequence, where the reference sequence 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 residue corresponding to position X123 in SEQ ID NO: 686 (e.g., a tyrosine) has been changed to a valine.

[0061] A "comparison window" refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acid residues within which a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids, and the portion of the sequence within the comparison window may contain 20 percent or less additions or deletions (i.e., gaps) compared to the reference sequence (without additions or deletions) for optimal alignment of the two sequences. The comparison window may be longer than 20 contiguous residues, optionally including windows of 30, 40, 50, 100, or longer.

[0062] "Corresponding to," "with reference to," and "compared to," when 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, residue numbers or residue positions in a given polymer are specified with respect to the reference sequence, rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence. For example, a given amino acid sequence, such as the amino acid sequence of an engineered LDC, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although gaps exist, 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.

[0063] "Amino acid difference" and "residue difference" refer to the difference in the amino acid residue at a position in a polypeptide sequence compared to the amino acid residue at the corresponding position in a reference sequence. The position of the amino acid difference is generally referred to herein as "Xn" (where n refers to the corresponding position in the reference sequence on which the residue difference is based). For example, "a residue difference at position X123 compared to SEQ ID NO:686" refers to the difference in the amino acid residue at the polypeptide position corresponding to position 123 of SEQ ID NO:686. Thus, if the 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 the amino acid substitution of any residue other than tyrosine at the polypeptide position corresponding to position 123 of SEQ ID NO:686. In most examples herein, specific amino acid residue differences at a position are designated as "XnY" (where "Xn" identifies the corresponding residue and position in the reference polypeptide (as described above), and "Y" is a single-letter identifier of the amino acid found in the engineered polypeptide (i.e., the residue that differs in the reference polypeptide). In some examples, the original amino acid is not designated (e.g., 109G). In some examples (e.g., in Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and / or 11-2), the present disclosure also provides specific amino acid differences that are designated by 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 sequence of the engineered polypeptide). In some examples, the polypeptides of the present disclosure can include one or more amino acid residue differences compared to a reference sequence, as indicated by a list of identified positions at which the residue difference occurs compared to the reference sequence. 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.

[0064] The terms "amino acid substitution set" and "substitution set" refer to a group of amino acid substitutions within a polypeptide 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 the set of amino acid substitutions present in any of the variant LDC polypeptides listed in any of the tables in the Examples (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, and / or 11-2).

[0065] " Conservative amino acid substitution " refers to the substitution of a residue with a different residue that has a similar side chain, and therefore typically includes the substitution of an amino acid in a polypeptide with an amino acid from the same or similar class of amino acids.For example, and not limited to, an amino acid with an aliphatic side chain can be substituted with another aliphatic amino acid (such as alanine, valine, leucine, and isoleucine); an amino acid with a hydroxyl side chain can be substituted with another amino acid with a hydroxyl side chain (such as serine and threonine); an amino acid with an aromatic side chain can be substituted with another amino acid with an aromatic side chain (such as phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain can be substituted with another amino acid with a basic side chain (such as lysine and arginine); an amino acid with an acidic side chain can be substituted with another amino acid with an acidic side chain (such as aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid can be substituted with another hydrophobic or hydrophilic amino acid, respectively. Exemplary conservative substitutions include substitution of 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); substitution of G or M with other non-polar residues (e.g., A, L, V, I, G, M); substitution of D or E with other acidic residues (e.g., D, E); substitution of K or R with other basic residues (e.g., K, R); substitution of N, Q, S, or T with other polar residues (e.g., N, Q, S, T); substitution of H, Y, W, or F with other aromatic residues (e.g., H, Y, W, F); or substitution of C or P with other non-polar residues (e.g., C, P).

[0066] "Non-conservative substitution" refers to the replacement of an amino acid in a polypeptide with an amino acid having significantly different side chain properties. Non-conservative substitutions may use amino acids between groups rather than within a defined group, and may affect (a) the structure of the peptide backbone in the area of ​​the substitution (e.g., proline for glycine); (b) charge or hydrophobicity; and / or (c) the bulkiness of the side chain. By way of example and not limitation, exemplary non-conservative substitutions include an acidic amino acid substituted with a basic or aliphatic amino acid; an aromatic amino acid substituted with a small amino acid; and a hydrophilic amino acid substituted with a hydrophobic amino acid.

[0067] "Deletion" refers to a modification to a polypeptide by removing one or more amino acids from a reference polypeptide. Deletions can include removing 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 constituting the reference enzyme, or up to 20% of the total number of amino acids, while retaining enzymatic activity and / or the improved properties of the engineered leucine decarboxylase enzyme. Deletions can be in internal and / or terminal portions of the polypeptide. In various embodiments, deletions can include continuous segments or can be discontinuous.

[0068] "Insertion" refers to a modification to a polypeptide by the addition of one or more amino acids from a reference polypeptide. The insertion can be in the internal portion of the polypeptide or at the carboxy or amino terminus. As used herein, an insertion includes fusion proteins known in the art. The insertion can be a continuous segment of amino acids in a naturally occurring polypeptide, or can be separated by one or more amino acids.

[0069] 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 where the remaining amino acid sequence is identical to the corresponding positions in the sequence to which it is being compared (e.g., a full-length engineered LDC of the invention), and that retains substantially all of the activity of the full-length polypeptide.

[0070] 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 polypeptides that have been removed or purified from their naturally occurring environment or expression system (e.g., a host cell or in vitro synthesis). Recombinant LDC polypeptides may be present intracellularly, in cell culture medium, or prepared in various forms, such as a lysate or isolated preparation. Thus, in some embodiments, the recombinant LDC polypeptides provided herein are isolated polypeptides.

[0071] A "substantially pure polypeptide" refers to a composition in which the polypeptide species is the predominant species present (i.e., it is more abundant than any other individual macromolecular species in the composition, on a molar or weight basis); generally, a composition is substantially purified when the desired species comprises at least about 50 percent of the macromolecular species present, on a molar or weight percent basis. Generally, a substantially pure LDC composition comprises about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species present in the composition, on a molar or weight percent basis. In some embodiments, the desired species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods), where the composition consists essentially of a single macromolecular species. Solvent species, small molecules (<500 Daltons), and elemental ion species are not considered macromolecular species. In some embodiments, an isolated recombinant LDC polypeptide is a substantially pure polypeptide composition.

[0072] "Improved enzymatic properties" refers to engineered LDC polypeptides that exhibit any improved enzymatic properties compared to a reference LDC polypeptide, such as a wild-type LDC polypeptide (e.g., a wild-type LDC having SEQ ID NO: 2) or another engineered LDC polypeptide. Improved properties include, but are not limited to, 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 resistance to substrate and / or end-product inhibition, increased chemical stability, improved chemoselectivity, improved solvent stability, increased tolerance to acidic pH, increased resistance to proteolytic activity (i.e., reduced susceptibility to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity (i.e., reduced ability to induce and / or elicit an immune response), and altered temperature profile.

[0073] "Increased enzymatic activity" and "enhanced enzymatic activity" refer to improved properties of an engineered LDC polypeptide, which can be expressed as an increase in specific activity (e.g., product produced / time / weight of protein) and / or an increase in the percent conversion of substrate to product (e.g., percent conversion of starting amount of substrate to product in a specific time using a specific amount of LDC) compared to a reference LDC enzyme (e.g., wild-type LDC and / or another engineered LDC). Exemplary methods for determining enzymatic activity are provided in the Examples. Any property related to enzymatic activity can be affected, including classical enzymatic properties such as Km, Vmax, or kcat, changes of which can result in increased enzymatic activity. Improved enzymatic activity can range from about 1.1-fold enzymatic activity of the corresponding wild-type enzyme to as much as 2-fold, 5-fold, 10-fold, 20-fold, 25-fold, 50-fold, 75-fold, 100-fold, 150-fold, 200-fold, or more enzymatic activity greater than the naturally occurring LDC from which the LDC polypeptide was derived or another engineered LDC.

[0074] In some embodiments, the engineered LDC 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, or 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; about 5 μm to about 2 mM; about 10 μm to about 2 mM; or about 10 μm to about 1 mM. In some specific embodiments, the engineered LDC enzyme exhibits an improvement in enzymatic activity of 1.5-10-fold, 1.5-25-fold, 1.5-50-fold, 1.5-100-fold, or greater than the enzymatic activity of a reference LDC enzyme. LDC activity can be measured by any standard assay known in the art (e.g., by monitoring reactant depletion or product formation). 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 absorbance or mass spectrometry detection. In some embodiments, the comparison of enzyme activity is performed using a defined preparation of enzyme, a defined assay under set conditions, and one or more defined substrates, as further described in detail herein. Generally, when lysates are compared, the same expression system and the same host cells are used, as well as the number of cells and amount of protein assayed, to minimize variations in the amount of enzyme produced by the host cells and present in the lysates.

[0075] The phrase "increased storage stability" means that an engineered LDC polypeptide according to the invention retains greater activity in standard assays (e.g., as described in the Examples) compared to a reference LDC after being produced in dry form (e.g., by lyophilization or spray drying) and stored above room temperature (e.g., 30°C, 37°C, 45°C, 55°C, etc.) for periods ranging from several days to several months.

[0076] "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 under specified conditions within a given period of time. Thus, the "enzyme activity" or "activity" of an LDC polypeptide can be expressed as the "percent conversion" of substrate to product in a specified period of time.

[0077] "Hybridization stringency" refers to hybridization conditions, such as washing conditions, in nucleic acid hybridization. Generally, hybridization reactions are performed under lower stringency conditions, followed by washing under different but higher stringency conditions. The term "moderately stringent hybridization" refers to conditions that allow target DNA to bind to complementary nucleic acids that have about 60% identity with target DNA, preferably about 75% identity, about 85% identity, and have more than about 90% identity with target polynucleotide. Exemplary moderately stringent conditions are equivalent to hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, at 42°C, followed by washing in 0.2x SSPE, 0.2% SDS, at 42°C. "High stringency hybridization" generally refers to conditions that are about 10°C or less from the thermal melting temperature (Tm) determined under solution conditions for a defined polynucleotide sequence. In some embodiments, high stringency conditions refer to conditions that allow hybridization of only nucleic acid sequences that form stable hybrids at 0.018M NaCl and 65°C (i.e., if a hybrid is not stable at 0.018M NaCl and 65°C, it is not stable under high stringency conditions as contemplated herein). High stringency conditions can be provided, for example, by hybridization in 50% formamide, 5x Denhardt's solution, 5x SSPE, 0.2% SDS, conditions equivalent to 42°C, followed by washing in 0.1x SSPE and 0.1% SDS at 65°C. Another high stringency condition is hybridization under conditions equivalent to hybridization in 5×SSC containing 0.1% (w:v) SDS at 65° C., and washing in 0.1×SSC containing 0.1% SDS at 65° C. Other high stringency hybridization conditions, and moderately stringent conditions, are described in the references cited above.

[0078] "Codon-optimized" refers to changing the codons in a polynucleotide encoding a protein to those preferentially used in that organism so that the encoded protein is more efficiently expressed in that organism. While the genetic code is degenerate in that most amino acids are represented by several codons, called "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 codon usage bias may be higher for a given gene, for common functional or ancestral genes, for highly expressed proteins versus low copy number proteins, and for aggregated protein-coding regions of an organism's genome. In some embodiments, a polynucleotide encoding an LDC enzyme is codon-optimized for optimal production from the host organism selected for expression. "Control sequences," as used herein, refer to the inclusion of all components necessary or advantageous for expression of the polynucleotides and / or polypeptides of the present disclosure. Each control sequence may be native to or exogenous to the nucleic acid sequence encoding the polypeptide. Such control sequences include, but are not limited to, a leader, polyadenylation sequence, propeptide sequence, promoter sequence, signal peptide sequence, initiation sequence, and transcription terminator. At a minimum, control sequences include a promoter, and transcriptional and translational stop signals. In some embodiments, control sequences are provided with linkers for the purpose of introducing specific restriction sites to facilitate ligation of the control sequences with the coding region of a nucleic acid sequence encoding a polypeptide.

[0079] "Operably linked" is defined herein as a configuration in which a control sequence is suitably positioned relative to a polynucleotide of interest (i.e., in a functional relationship) so that the control sequence directs or regulates the expression of the polynucleotide encoding a polypeptide of interest.

[0080] A "promoter sequence" refers to a nucleic acid sequence, e.g., a coding sequence, that is recognized by a host cell for expression of a polynucleotide of interest. The promoter sequence contains transcriptional control sequences that mediate expression of the polynucleotide of interest. The promoter may be any nucleic acid sequence that exhibits transcriptional activity in the host cell of choice, including mutant promoters, truncated promoters, and hybrid promoters, and may be derived from a gene encoding an extracellular or intracellular polypeptide, either homologous or heterologous to the host cell.

[0081] A "substrate," in the context of an enzymatic conversion reaction process, refers to a compound or molecule that is acted upon by an LDC polypeptide. A "product," in the context of an enzymatic conversion process, refers to a compound or molecule that results from the action of an LDC polypeptide on a substrate.

[0082] As used herein, the term "culturing" refers to the growth of a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).

[0083] Recombinant polypeptides (e.g., LDC enzyme variants) can be produced using any suitable method known in the art. For example, there are a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are available from many commercial molecular biology suppliers. Methods are available for specific substitutions at defined 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). Numerous suitable methods are known to those skilled in the art for generating enzyme variants, including, but 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 any other suitable method known in the art. Non-limiting examples of methods used for 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 properties (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermostability, and / or acidic pH stability, etc.). In some embodiments, "recombinant LDC polypeptides" (also referred to herein as "engineered LDC polypeptides," "variant LDC enzymes," and "LDC variants") are used.

[0084] As used herein, a "vector" is a DNA construct for introducing a DNA sequence into a cell. In some embodiments, the vector is an expression vector that is operably linked to a suitable control sequence that can cause the expression of the polypeptide encoded by the DNA sequence in a suitable host. In some embodiments, an "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 contains a transcription terminator sequence.

[0085] As used herein, the term "expression" includes any step involved in producing a polypeptide, including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from the cell.

[0086] As used herein, an amino acid sequence or a nucleotide sequence (e.g., a promoter sequence, signal peptide, terminator sequence, etc.) is "heterologous" to another sequence to which it is operably linked if the two sequences are not associated in nature.

[0087] As used herein, the terms "host cell" and "host strain" refer to a suitable host for an expression vector containing DNA (e.g., a polynucleotide sequence encoding at least one LDC variant) provided herein. In some embodiments, a host cell is a prokaryotic or eukaryotic cell that has been transformed or transfected with a vector constructed using recombinant DNA techniques known in the art.

[0088] 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) to a reference polypeptide. In some embodiments, analog refers to non-naturally occurring amino acid residues, including, but not limited to, homoarginine, ornithine, and norvaline, as well as naturally occurring amino acids. In some embodiments, analog also includes one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.

[0089] The term "therapeutic agent" refers to a compound administered to a subject exhibiting signs or symptoms of a disease state that has a beneficial or desired medical effect.

[0090] The term "pharmaceutical composition" refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., a human) comprising a pharmaceutically effective amount of an engineered LDC polypeptide encompassed by the present invention and an acceptable carrier.

[0091] The term "gene therapy" is used in reference 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 introduced directly into at least some cells of the mammalian subject. It is not intended that the present invention be limited to any particular methods or compositions useful for gene therapy.

[0092] 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, 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 any particular method or composition useful for mRNA therapy.

[0093] The term "effective amount" means an amount sufficient to bring about a desired result. One of ordinary skill in the art can determine what an effective amount is by using routine experimentation.

[0094] 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 removed from at least one other component with which it is naturally associated. The term "purified" does not require absolute purity, but rather is intended as a relative definition.

[0095] The term "subject" includes mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). The term is intended to include females and males.

[0096] As used herein, the term "patient" means any subject being evaluated, treated, or experiencing a disease.

[0097] The term "infant" refers to a child between one month and approximately one year of age. As used herein, the term "newborn" refers to a child between birth and 28 days after birth. The term "premature infant" generally refers to an infant born after 20 full weeks of gestation but before full term, 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.

[0098] As used herein, the term "child" refers to a person who has not reached the legal age to consent to a treatment or research procedure. In some embodiments, the term refers to a person between birth and adolescence.

[0099] As used herein, the term "adult" refers to a person who has reached the legal age of consent in the relevant jurisdiction (e.g., 18 years of age in the United States). In some embodiments, the term refers to any fully grown, mature organism. In some embodiments, the term "young adult" refers to a person who is under the age of 18 but has reached sexual maturity.

[0100] As used herein, "composition" and "formulation" encompass products comprising at least one engineered LDC of the present invention intended for any suitable use (e.g., pharmaceutical composition, dietary / nutritional supplement, feed, etc.).

[0101] The terms "administration" of a composition and "administering" a composition mean providing a composition of the present invention to a subject (e.g., a person suffering from the effects of MSUD).

[0102] The term "carrier" when used in reference to pharmaceutical compositions means any of the standard pharmaceutical carriers, buffers and excipients, such as stabilizers, preservatives and adjuvants.

[0103] The term "pharmaceutically acceptable" means a material that may be administered to a subject without causing any undesired biological effects or interacting in a deleterious manner with any of the components it contains, and that possesses the desired biological activity.

[0104] As used herein, the term "excipient" refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient other than the active pharmaceutical ingredient (API; e.g., an engineered LDC polypeptide of the invention). Excipients are typically included for formulation and / or administration purposes.

[0105] The term "therapeutically effective amount," when 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 ameliorates, attenuates, 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 as desired by a researcher, physician, veterinarian, or other clinician.

[0106] The term "therapeutically effective amount," when used in reference to a disease / condition, refers to an amount and / or concentration of a composition that ameliorates, attenuates, or eliminates the disease / condition.

[0107] The terms "treating," "treat," and "treatment" are intended to encompass preventative (e.g., prophylactic) and palliative treatment.

[0108] As used herein, the term "at least one" is not intended to limit the invention to any particular number of items. It is intended to encompass 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, or more items, as desired. Engineered LDC polypeptides:

[0109] Parent LDC polypeptides from which the engineered LDC polypeptides of the present invention are derived include bacterial strains such as those in the bacteria family Planctomycetaceae.

[0110] Furthermore, when a particular LDC variant (i.e., an engineered LDC polypeptide) is referred to by reference to a modification of a particular amino acid residue in the sequence of a wild-type or reference LDC, it should be understood that a variant of another LDC modified at the equivalent position (as determined from an amino acid sequence alignment, if necessary, between the respective amino acid sequences) is encompassed herein.

[0111] In some embodiments, engineered LDC polypeptides are produced by culturing a microorganism comprising at least one polynucleotide sequence encoding at least one engineered LDC polypeptide under conditions conducive to the production of the engineered LDC polypeptide, which in some embodiments is then recovered from the resulting culture medium and / or cells.

[0112] The present invention provides exemplary engineered LDC polypeptides having LDC activity. The Examples provide tables (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, and / or 11-2) that display sequence-structure information correlating specific amino acid sequence features with the functional activity of the engineered LDC polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences compared to the reference engineered polypeptide of SEQ ID NO:2 and associated experimentally determined activity data for the exemplary engineered LDC polypeptides.

[0113] In some embodiments, engineered LDC polypeptides of the invention with LDC activity have: a) an amino acid sequence having at least 85% sequence identity to the reference sequence SEQ ID NO:2; b) contain an amino acid residue difference compared to SEQ ID NO:2 at one or more amino acid positions; and c) exhibit improved properties compared to the reference sequence selected from: i) enhanced catalytic activity; ii) reduced proteolytic susceptibility; iii) reduced aggregation; iv) increased stability to elevated temperatures as a lyophilized preparation; v) reduced immunogenicity; or any combination of i), ii), iii), iv), or v).

[0114] In some embodiments, the present invention provides functional fragments of engineered LDC polypeptides. In some embodiments, functional fragments comprise 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 LDC polypeptide from which it is derived (i.e., the parent engineered LDC). In some embodiments, functional fragments comprise 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 the engineered LDC. In some embodiments, functional fragments are truncated by fewer than 5, fewer than 10, fewer than 15, fewer than 10, fewer than 25, fewer than 30, fewer than 35, fewer than 40, fewer than 45, and fewer than 50 amino acids.

[0115] In some embodiments, the present invention provides functional fragments of engineered LDC polypeptides. In some embodiments, functional fragments comprise at least about 95%, 96%, 97%, 98%, or 99% of the activity of the engineered LDC polypeptide from which they are derived (i.e., the parent engineered LDC). In some embodiments, functional fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered LDC. In some embodiments, functional fragments are truncated by fewer than 5, 10, 15, 10, 25, 30, 35, 40, 45, 50, 55, 60, 65, or 70 amino acids.

[0116] In some embodiments, the engineered LDC polypeptide comprises an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, or the amino acid sequence of any variant (e.g., those provided in the Examples). In some embodiments, the reference sequence is selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766.

[0117] In some embodiments, an engineered LDC 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 SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, as well as one or more residue differences compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766. Polynucleotides encoding engineered polypeptides, expression vectors and host cells:

[0118] The present invention provides polynucleotides encoding the engineered LDC polypeptides described herein. In some embodiments, the polynucleotides are operably linked to one or more heterologous regulatory sequences that control gene expression to produce recombinant polynucleotides capable of expressing the polypeptides. In some embodiments, an expression construct containing at least one heterologous polynucleotide encoding an engineered LDC polypeptide is introduced into a suitable host cell to express the corresponding LDC polypeptide.

[0119] As will be apparent to those skilled in the art, the availability of a protein sequence and knowledge of the codons corresponding to various amino acids provides a description of all polynucleotides capable of encoding a subject polypeptide. 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 enormous number of nucleic acids, all of which encode engineered LDC polypeptides. Thus, the present invention provides methods and compositions for generating any and all possible variations of LDC polynucleotides that can be made that encode the LDC polypeptides described herein by selecting combinations based on potential codon choices; all such variations should be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and / or 11-2).

[0120] In some embodiments, codons are preferably optimized for use by the host cell selected for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria. As a result, a codon-optimized polynucleotide encoding an engineered LDC polypeptide contains preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or more than 90% of the codon positions in the full-length coding region.

[0121] In some embodiments, an LDC polynucleotide encodes an engineered polypeptide having LDC activity with the properties disclosed herein, wherein the polypeptide has at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166, 167, 168, 169, 170, 171, 172, 173, 174, 175, 176, 177, 178, 179, 180, 181, 182, 183 An amino acid sequence having 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identity, and containing one or more residue differences compared to the amino acid sequence (e.g., 1, 2, 3, 4, 5, 6, 7, 8, 9, 10 or more amino acid residue positions) of a reference polynucleotide of SEQ ID NO: 1, 3, 5, 7, 9, 11, 13, 37, 233, 283, 483, 593, 685, 687, and / or 765, or any variant disclosed in the Examples. In some embodiments, the reference sequence is selected from SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766.

[0122] In some embodiments, the LDC polynucleotide encodes an engineered polypeptide having LDC activity with the properties disclosed herein, wherein the 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 the reference sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, and one or more residue differences compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766.

[0123] In some embodiments, a polynucleotide encoding an engineered LDC polypeptide comprises a polynucleotide sequence selected from SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 37, 233, 283, 483, 593, 685, 687, and / or 765. In some embodiments, a polynucleotide encoding an engineered LDC polypeptide has at least 80%, 85%, 90%, 93%, 95%, 96%, 97%, 98%, 99% nucleotide residue identity to SEQ ID NOs: 1, 3, 5, 7, 9, 11, 13, 37, 233, 283, 483, 593, 685, 687, and / or 765.

[0124] In some embodiments, a polynucleotide is capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, or a complement thereof, or to a polynucleotide sequence encoding any of the variant LDC polypeptides provided herein. In some embodiments, a polynucleotide capable of hybridizing under highly stringent conditions encodes an LDC polypeptide comprising an amino acid sequence having one or more residue differences compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766.

[0125] In some embodiments, an isolated polynucleotide encoding any of the engineered LDC polypeptides herein is engineered in various ways to facilitate expression of the LDC polypeptide. In some embodiments, the polynucleotide encoding the LDC polypeptide comprises an expression vector in which one or more regulatory sequences are present to regulate expression of the LDC polynucleotide and / or polypeptide. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary, depending on the expression vector utilized. Techniques for modifying polynucleotides and nucleic acid sequences using recombinant DNA methods are well known in the art. In some embodiments, regulatory sequences include, among others, 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 host cell. For bacterial host cells, suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to, promoters from the E. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase genes (e.g., Villa-Kamaroff et al., Proc. Natl. Acad. Sci. USA 75:3727-3731).

[1978] ), as well as promoters derived from the tac promoter (see, e.g., DeBoer et al., Proc. Natl. Acad. Sci. USA 80: 21-25

[1983] ).Exemplary promoters for filamentous fungal host cells include, but are not limited to, Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus. Examples of suitable promoters include those obtained from the genes for Aspergillus niger acid-stable alpha-amylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triosephosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum trypsin-like protease (see, e.g., WO 96 / 00787), as well as the NA2-tpi promoter (a hybrid of the promoters from the genes for Aspergillus niger neutral alpha-amylase and Aspergillus oryzae triosephosphate isomerase), and mutant, truncated, and hybrid promoters thereof. Exemplary yeast cell promoters can be derived from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GAL1), Saccharomyces cerevisiae alcohol dehydrogenase / glyceraldehyde-3-phosphate dehydrogenase (ADH2 / GAP), and Saccharomyces cerevisiae 3-phosphoglycerate kinase. Other useful promoters for yeast host cells are known in the art (see, for example, Romanos et al., Yeast 8:423-488

[1992] ).

[0126] 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 LDC polypeptide. Any suitable terminator that is functional in the host cell of choice finds use in the present invention. Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin-like protease. Exemplary 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).

[0127] In some embodiments, the control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA 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 LDC polypeptide. Any suitable leader sequence that is functional in the host cell of choice 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 triosephosphate 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).

[0128] 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 the addition of polyadenosine residues to the transcribed mRNA). Any suitable polyadenylation sequence that is functional in the host cell of choice 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 niger 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., 15:5983-5990

[1995] ).

[0129] In some embodiments, the control sequence is also a signal peptide (i.e., a coding region encoding an amino acid sequence linked to the amino terminus of a polypeptide 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 inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes a secreted polypeptide. Alternatively, in some embodiments, the 5' end of the coding sequence contains a signal peptide coding region that is foreign to the coding sequence. Any suitable signal peptide coding region that directs the expressed polypeptide into the secretory pathway of a host cell of choice is used for expression of the engineered polypeptide. Useful signal peptide coding regions for bacterial host cells include, but are not limited to, those obtained from the genes encoding Bacillus NC1B 11837 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 (e.g., Simonen and Palva, Microbiol. Rev., 57:109-137

[1993] ). (See, for example, the Japanese Patent Application Publication No. 2004-200901104.) In some embodiments, useful signal peptide coding regions for filamentous fungal host cells include, but are not limited to, those obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase. Useful signal peptides for yeast host cells include, but are not limited to, those derived from the genes for Saccharomyces cerevisiae alpha factor and Saccharomyces cerevisiae invertase.

[0130] In some embodiments, the control sequence is also a propeptide coding region that encodes an amino acid sequence positioned 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. Propeptide coding regions include, but are not limited to, Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces The signal peptide may be obtained from any suitable source, including the genes for S. cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (see, e.g., WO 95 / 33836). When both the signal peptide and propeptide regions are present at the amino terminus of the 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.

[0131] In some embodiments, regulatory sequences are also utilized. These sequences facilitate regulation of polypeptide expression relative to host cell growth. Examples of regulatory systems are those that cause gene expression to be turned on or off in response to chemical or physical stimuli, including the presence of a regulatory compound. In prokaryotic host cells, suitable regulatory sequences include, but are not limited to, the lac, tac, and trp operator systems. In yeast host cells, suitable regulatory systems include, but are not limited to, the ADH2 system or the GAL1 system. In filamentous fungi, suitable regulatory sequences include, but are not limited to, the TAKA alpha-amylase promoter, the Aspergillus niger glucoamylase promoter, and the Aspergillus oryzae glucoamylase promoter.

[0132] In another aspect, the present invention is directed to recombinant expression vectors comprising a polynucleotide encoding an engineered LDC polypeptide and one or more expression control regions, such as a promoter and terminator, an origin of replication, and the like, depending on the type of host into which they will be introduced. In some embodiments, the various nucleic acids and control sequences described herein are joined to generate a recombinant expression vector, which contains one or more convenient restriction sites that allow for insertion or substitution of a nucleic acid sequence encoding an LDC polypeptide at such site. Alternatively, in some embodiments, the nucleic acid sequences of the present invention are expressed by inserting a nucleic acid sequence or nucleic acid construct comprising the sequence into an appropriate vector for expression. In some embodiments involving the creation of an expression vector, the coding sequence is positioned in the vector such that it is operably linked to appropriate control sequences for expression.

[0133] The recombinant expression vector may be any suitable vector (e.g., a plasmid or virus) that can be conveniently subjected to recombinant DNA procedures to bring about expression of the LDC polynucleotide sequence. The choice of vector will typically depend on the compatibility of the vector with the host cell into which the vector will be introduced. The vector may be a linear or closed circular plasmid.

[0134] In some embodiments, the expression vector is a self-replicating vector (i.e., a vector that exists as an extrachromosomal entity whose replication is independent of chromosomal replication, e.g., a plasmid, extrachromosomal element, minichromosome, or artificial chromosome). The vector may contain any means for ensuring self-replication. In some alternative embodiments, the vector, upon introduction into a host cell, is integrated into the genome and replicated together with the chromosome into which it has been integrated. Furthermore, some embodiments utilize a single vector or plasmid, or two or more vectors or plasmids, and / or transposons, which together contain the total DNA to be introduced into the genome of the host cell.

[0135] In some embodiments, the expression vector contains one or more selectable markers that allow for easy selection of transformed cells. A "selectable marker" is a gene whose product provides biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, etc. Examples of bacterial selectable markers include, but are not limited to, the dal genes from 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. nidulans or A. orzyae), 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. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), and equivalents thereof. In another aspect, the invention provides a host cell comprising at least one polynucleotide encoding at least one engineered LDC polypeptide of the invention, wherein the polynucleotide is operably linked to one or more control sequences for expression of the engineered LDC enzyme 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 and Spodoptera Sf9 cells; animal cells such as CHO, COS, BHK, 293, and Bowes melanoma cells; and plant cells. Exemplary host cells also include various Escherichia coli strains (e.g., W3110(ΔfhuA) and BL21).

[0136] Thus, in another aspect, the invention provides methods for producing an engineered LDC polypeptide, comprising culturing a host cell capable of expressing a polynucleotide encoding the engineered LDC polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the method further comprises isolating and / or purifying the LDC polypeptide as described herein.

[0137] Suitable culture medium and growth conditions for host cells are well known in the art.Any suitable method for introducing polynucleotide into cells for expressing LDC polypeptide is contemplated to be used in the present invention.Suitable techniques include, but are not limited to, electroporation, particle bombardment, liposome-mediated transfection, calcium chloride transfection, and protoplast fusion.

[0138] The engineered LDC polypeptides having the properties disclosed herein can be obtained by subjecting naturally occurring LDC polypeptides or polynucleotides encoding engineered LDC polypeptides 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, for example, Stemmer, Proc. Natl. Acad. Sci. USA 91:10747-10751

[1994] ; 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, staggered extension process (StEP), in vitro recombination (see, e.g., Zhao et al., Nat. Biotechnol., 16:258-261

[1998] ), mutagenic PCR (see, e.g., Caldwell et al., PCR Methods Appl., 3:S136-S140

[1994] ). (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ), and cassette mutagenesis (see, e.g., Black et al., Proc. Natl. Acad. Sci. USA 93:3525-3529

[1996] ).

[0139] Mutagenesis and directed evolution methods can be readily applied to LDC-encoding polynucleotides 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 is well known in the art (e.g., U.S. Patent 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 ... No. 6,277,638, No. 6,287,861, 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. 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, No. 742, No. 6,365,377, No. 6,365,408, No. 6,368,861, No. 6,372,497, No. 6,337,186, No. 6,376,246, No. 6,379,964, No. 6,387,702, No. 6, 391,552, 6,391,640, 6,395,547, 6,406,855, 6,406,910, 6,413,745, 6,413,774, 6,420,175, 6,423,542, No. 6,426,224, No. 6,436,675, No. 6,444,468, No. 6,455,253, No. 6,479,652, 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. 6,519,065, No. 6,521,453, No. 6,528,311, No. 6,537,No. 746, No. 6,573,098, No. 6,576,467, No. 6,579,678, No. 6,586,182, No. 6,602,986, No. 6,605,430, 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. 7,148,054, 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, 7,873,499, 7,904,249, 7,957,912, 7,981,614, 8,014,961, 8,029,988, 8,048,674, 8,058, 001, 8,076,138, 8,108,150, 8,170,806, 8,224,580, 8,377,681, 8,383,346, 8,457,903, 8,504,498, 8,589,085, 8,762,066, 8,768,871, 9,593,326, 9,665,694, 9,684,771, and all related U.S. and non-U.S. counterparts; Ling et al., Anal. Biochem., 254(2):157-78

[1997] ;Dale et al., Meth. Mol. Biol., 57:369-74

[1996] ;Smith, Ann. Rev. Genet., 19:423-462

[1985] ;Botstein et al., Science, 229:1193-1201

[1985] ;Carter, Biochem. Biol., 3:284-290

[1999] ;Christians et al., Nat. Biotechnol., 17:259-264

[1999] ;Crameri et al., Nature, 391:288-291

[1998] ;Crameri, et al., Nat. Biotechnol., 15:436-438

[1997] ;Zhang et al., Proc. Nat. Acad. Sci. USA, 94:4504-4509

[1997] ; Crameri et al., Nat. Biotechnol., 14:315-319

[1996] ; Stemmer, Nature, 370:389-391

[1994] ; Stemmer, Proc. Nat. Acad. Sci. USA, 91:10747-10751

[1994] ; International Publication See 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).

[0140] In some embodiments, enzyme clones obtained after 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 appropriate assay conditions. Clones containing polynucleotides encoding LDC polypeptides are then isolated from the gene, sequenced to identify nucleotide sequence changes, if any, and used to express the enzyme in host cells. Measurement of enzyme activity from expression libraries can be performed using any suitable method known in the art (e.g., standard biochemical techniques such as HPLC analysis).

[0141] For engineered polypeptides of known sequence, polynucleotides encoding the enzymes can be prepared by standard solid-phase synthesis methods according to known synthesis methods. In some embodiments, fragments of up to about 100 bases can be synthesized individually and then joined to form any desired contiguous sequence (e.g., by enzymatic or chemical ligation, or polymerase-mediated methods). For example, the polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method, as typically performed in automated synthesis methods (see, e.g., Beaucage et al., Tet. Lett., 22:1859-69

[1981] ; and Matthes et al., EMBO J., 3:801-05

[1984] ). Following the phosphoramidite method, oligonucleotides are synthesized (e.g., purified, annealed, ligated, and cloned into an appropriate vector in an automated DNA synthesizer).

[0142] Thus, in some embodiments, a method for preparing an engineered LDC polypeptide can include (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from any variant amino acid sequence described herein, and (b) expressing the LDC polypeptide encoded by the polynucleotide. In some embodiments of the method, the amino acid sequence encoded by the polynucleotide can optionally have one or several (e.g., up to 3, 4, 5, or up to 10) amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has 1 to 2, 1 to 3, 1 to 4, 1 to 5, 1 to 6, 1 to 7, 1 to 8, 1 to 9, 1 to 10, 1 to 15, 1 to 20, 1 to 21, 1 to 22, 1 to 23, 1 to 24, 1 to 25, 1 to 30, 1 to 35, 1 to 40, 1 to 45, or 1 to 50 amino acid residue deletions, insertions, and / or substitutions. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the amino acid sequence optionally has deletions, insertions, and / or substitutions of 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 residues. In some embodiments, the substitutions are conservative or non-conservative.

[0143] The expressed engineered LDC 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.

[0144] In some embodiments, any of the engineered LDC polypeptides expressed in host cells are recovered from the cells and / or culture medium using any one or more of well-known techniques for protein purification, including lysozyme treatment, sonication, filtration, salting out, ultracentrifugation, and chromatography, among others.

[0145] Chromatographic methods for isolating LDC 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. The 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 those skilled in the art. In some embodiments, affinity techniques may be used to isolate improved LDC enzymes. Any antibody that specifically binds to the LDC polypeptide of interest may be used for affinity chromatography purification. For antibody production, various host animals, including but not limited to rabbits, mice, rats, and the like, are immunized by injection with an LDC polypeptide or a fragment thereof. In some embodiments, the LDC polypeptide or fragment is conjugated to a suitable carrier, such as BSA, using a side chain functional group or a linker attached to a side chain functional group.

[0146] In some embodiments, engineered LDC polypeptides are produced in host cells by a method comprising culturing host cells (e.g., E. coli strains) containing polynucleotide sequences encoding the engineered LDC polypeptides described herein under conditions conducive to the production of the engineered LDC polypeptides, and recovering the engineered LDC polypeptides from the cells and / or culture medium. In some embodiments, the host cells produce two or more engineered LDC polypeptides.

[0147] In some embodiments, the invention provides methods for producing an engineered LDC polypeptide, the method comprising culturing a recombinant bacterial cell comprising a polynucleotide sequence encoding an engineered LDC polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to reference sequences SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, as provided herein, and one or more amino acid residue differences compared to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766, under suitable culture conditions that allow production of the engineered LDC polypeptide, and optionally recovering the engineered LDC polypeptide from the culture and / or the cultured bacterial cells. In some embodiments, the host cell produces two or more engineered LDC polypeptides.

[0148] In some embodiments, once the engineered LDC polypeptides are recovered from the recombinant host cells and / or culture medium, they are further purified by any suitable method known in the art. In some additional embodiments, the purified LDC polypeptides are combined with other components and compounds to provide compositions and formulations comprising the engineered LDC polypeptides, as needed, for different applications and uses (e.g., pharmaceutical compositions). Composition:

[0149] The present invention provides engineered LDC polypeptides suitable for use in numerous compositions. These compositions are used in many fields, including, but not limited to, pharmaceuticals, dietary / nutritional supplements, foods, feeds, and fine chemical production. For example, in some embodiments, the present invention provides foods and / or feeds comprising at least one engineered LDC variant and / or at least one polynucleotide sequence encoding at least one LDC variant. In some embodiments, the present invention provides beverages comprising at least one engineered LDC variant.

[0150] In some embodiments, the engineered LDC variant in the food, feed, and / or nutritional / nutraceutical product is glycosylated. Furthermore, the engineered LDC variant is used in any suitable edible enzyme delivery matrix. In some embodiments, the engineered LDC variant is present in an edible enzyme delivery matrix designed for rapid dispersion of the LDC variant in the animal's digestive tract upon ingestion of the variant.

[0151] The present invention also provides engineered LDC polypeptides suitable for use in the production of fine chemicals and other industrially important compounds (see, e.g., U.S. Patent Application Publication Nos. 2013 / 0340119, 2013 / 0005012, and 2005 / 0260724, and WO 2012 / 122333). Pharmaceutical and other compositions:

[0152] The present invention provides engineered LDC polypeptides suitable for use in pharmaceutical and other compositions, such as dietary / nutritional supplements.

[0153] Depending on the mode of administration, these compositions containing a therapeutically effective amount of an engineered LDC according to the present invention are in solid, semi-solid, or liquid form. In some embodiments, the compositions contain other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details regarding techniques for formulation and administration are well known in the art and described in the literature.

[0154] In some embodiments, the engineered LDC polypeptide is formulated for use in an oral pharmaceutical composition. Any suitable format for use in delivering the engineered LDC polypeptide may be used in the present invention, including, but not limited to, pills, tablets, gel tabs, capsules, lozenges, dragees, powders, softgels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, liniments, aerosols, chewing gums, demulcents, sticks, suspensions (including, but not limited to, oil-based suspensions, oil-in-water emulsions, etc.), slurries, syrups, controlled-release formulations, suppositories, etc. In some embodiments, the engineered LDC polypeptide is provided in a format suitable for injection (i.e., an injectable formulation). In some embodiments, the engineered LDC 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 LDC polypeptide is encapsulated. In some alternative embodiments, the engineered LDC polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules or microcapsules, microspheres, liposomes, etc.). Indeed, it is not intended that the present invention be limited to any particular delivery formulation and / or means of delivery. It is contemplated that the engineered LDC polypeptides will be administered by any suitable means known in the art, including, but not limited to, parenteral, oral, topical, transdermal, intranasal, intraocular, intrathecal, via implants, etc.

[0155] In some embodiments, the engineered LDC 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 29:283-287).

[2005] (See, e.g., 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.

[0156] In some additional embodiments, the engineered LDC polypeptide is provided in a formulation comprising a matrix-stabilized enzyme crystal. In some embodiments, the formulation comprises a crosslinked crystalline engineered LDC enzyme and a polymer having a reactive moiety that adheres to the enzyme crystal. The present invention also provides an engineered LDC polypeptide in a polymer.

[0157] In some embodiments, compositions comprising the engineered LDC polypeptides of the present invention comprise 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., acacia, tragacanth, 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 coating known in the art). In some embodiments, a disintegrant or solubilizer is included (e.g., cross-linked polyvinylpyrrolidone, agar, alginic acid or a salt thereof, e.g., sodium alginate). In some embodiments, the engineered LDC polypeptides are combined with a variety of additional components, including but not limited to preservatives, suspending agents, thickening agents, humectants, alcohols, fatty acids, and / or emulsifiers, particularly in liquid formulations.

[0158] In some embodiments, the engineered LDC polypeptides are combined with various additional components, 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 LDC polypeptides are administered to a subject in combination with other compounds used in the treatment of MSUD, and any other suitable compounds.

[0159] In some embodiments, the present invention provides engineered LDC polypeptides suitable for use in reducing, ameliorating, or eliminating the signs and / or symptoms of MSUD. The dosage of the engineered LDC 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, it is contemplated that the concentration of the engineered LDC polypeptide in the composition administered to the patient is sufficient to effectively treat, ameliorate, and / or prevent symptoms of the disease. In some embodiments, the engineered LDC polypeptide is administered in combination with other pharmaceutical and / or dietary compositions. Industrial composition:

[0160] The engineered LDC polypeptides of the present invention are contemplated for use in industrial compositions, including areas such as food flavorings (eg, cheese).

[0161] In some embodiments, the engineered LDC polypeptides are formulated for use in the food and / or feed industry. In some embodiments, the engineered LDC polypeptides are formulated into granulated or pelleted products that are mixed with animal feed components, such as additional enzymes (e.g., cellulases, laccases, and amylases). In some alternative embodiments, the engineered LDC polypeptides are used in liquid animal feed compositions (e.g., aqueous or oil-based slurries). Thus, in some embodiments, the engineered LDC variants of the present invention are sufficiently heat-resistant and heat-stable to withstand the processes used to produce pellets and other processed feeds / foods.

[0162] The foregoing and other aspects of the present invention may be better understood with reference to 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. [Example]

[0163] experiment The following examples, including experiments and results achieved, are provided for illustrative purposes only and should not be construed as limiting the invention.

[0164] In the experimental disclosure below, the following abbreviations apply: ppm (parts per million); M (molar); mM (millimolecular), uM and μM (micromolar); nM (nanomolar); mol (mole); gm and g (grams); mg (milligrams); ug and μg (micrograms); L and l (liters); ml and mL (milliliters); cm (centimeters); mm (millimeters); um 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); E. coli W3110 (Col. Genetic Stock Center [CGSC], New Commonly used laboratory E. coli strains available from I. coli Haven, CT); 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 with Mass Spectrometry in Two Streams); SPE (Solid Phase Extraction); KIC (Ketoisocaproate); 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); Informs (Informs AG, Bottmingen, Switzerland);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 (Thermo Fisher Scientific, part of Waltham, MA); Gibco (Thermo Fisher Scientific, a Gibco division of Waltham, MA); Khuner (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). . Example 1 Synthesis and assay of amino acid decarboxylase enzymes with leucine decarboxylase activity.

[0165] This example describes the methods used in the synthesis and assay of amino acid decarboxylase enzymes for leucine decarboxylase (LDC) activity. Obtaining amino acid decarboxylase genes and constructing expression vectors

[0166] Streptomyces sp. GP55 (SEQ ID NO: 2; Acc. No. WP_101384472.1), Saccharothrix sp. ST-888 (SEQ ID NO: 4; Acc. No. WP_052681825.1), and Kitasatospora sp. MBT63 (SEQ ID NO: 6; Acc. No. WP_051812394.1), Kitasatospora sp. MMS61-BH015 (SEQ ID NO: 8; Acc. No. WP_104818078.1), Streptomyces sp. NRRL F-6131 (SEQ ID NO: 10; Acc. No. WP_051769113.1), Planctomycetaceae bacteria (SEQ ID NO: 12; Acc. No. RPI63066.1), and Larkinella Polynucleotide sequences encoding amino acid decarboxylases from Saccharomyces arboricola (SEQ ID NO: 14; Acc No. A0A327WPB0) were synthesized as genes set forth in SEQ ID NOs: 1, 3, 5, 7, 9, 11, and 13, respectively. These synthetic genes were cloned into the pCK110900 vector system (see, e.g., U.S. Pat. No. 7,629,157 and U.S. Patent Application Publication No. 2016 / 0244787, both of which are hereby incorporated by reference), and then expressed in E. coli strains derived from W3110. In some embodiments, expression vectors lacking antimicrobial resistance markers are used. Shake flask powder (SFP) generation

[0167] E. coli cultures transformed with the amino acid decarboxylase-containing plasmids were plated onto Luria Broth-agar plates with 1% glucose and, in some cases, 30 μg / mL chloramphenicol and grown overnight at 37°C. A single colony from each culture was transferred to 5 mL of Luria Broth (LB) with 1% glucose and 30 μg / mL chloramphenicol, where appropriate. Cultures were grown at 30°C, 250 rpm for 18 hours and subcultured approximately 1:50 into 250 mL of Terrific Broth (TB) with 30 μg / mL chloramphenicol. Cultures were cultured at an OD of 0.6-0.8. 600The culture was grown at 30°C and 250 rpm for approximately 3-4 hours and induced with 1 mM IPTG. The culture was grown at 30°C and 250 rpm for 20 hours. The cells were harvested by centrifugation (7000 rpm x 10 min, 4°C) and the supernatant was discarded. The pellet was resuspended in 30 mL of 50 mM sodium phosphate, pH 7.0, with 1 mM PLP and lysed using a single pass through a Microfluidics at 110 psi. The lysate was pelleted (10,000 x rpm, 30 min, 4°C), and the resulting supernatant was frozen and lyophilized to produce a powder containing the expressed enzyme. SFP characterization assay for decarboxylase activity towards leucine at different pH

[0168] The shake flask powder was reconstituted to prepare a 40 g / L powder and serially diluted from 2.5 to 40 g / L. 25 μL of these stocks were then added to 75 μL of reaction mix for a final concentration of 10 mM leucine and 0.1 mM PLP in 50 mM sodium phosphate, pH 7.0, or McIlvaine buffer, pH 4.6. The reactions were incubated at 37°C and 250 rpm in a THERMOTRON® titer plate shaker for 3-4 hours and then quenched with 3 volumes of acetonitrile with 0.1% formic acid. The resulting samples were centrifuged at 4000 rpm at 4°C for 10 minutes, and the supernatant was analyzed by LC-MS / MS for isopentylamine, a decarboxylation product of leucine. An example of the LC-MS / MS instrument and parameters is shown in Table 1-1, and results for SEQ ID NOs: 2-14 are shown in Table 1-2. [Table 1-1] SFP characterization assay for pH tolerance

[0169] To evaluate the enzyme's relative tolerance to acidic pH, SFP was reconstituted to prepare a 20 g / L powder and diluted two-fold into McIlvaine buffer, pH 2.8-8.0. The mixture was incubated for 1.5 hours at 37°C and 250 rpm in a THERMOTRON® titer plate shaker. After incubation, 20 μL of enzyme solution was added to 90 μL of reaction mix for a final concentration of 5 mM leucine in 50 mM sodium phosphate, pH 7.0. The reaction was incubated for 2 hours at 37°C and 250 rpm in a THERMOTRON® titer plate shaker, then diluted two-fold in water and quenched with 3 volumes of acetonitrile with 0.1% formic acid. The resulting samples were processed as described above, and the supernatants were analyzed for isopentylamine by LC-MS / MS. The results obtained for SEQ ID NOs: 2-14 are shown in Tables 1-2. SFP characterization assay for thermal stability

[0170] Improved thermal stability is a valuable trait useful in the manufacture and storage of enzyme therapeutics and often occurs as a by-product of other stabilization efforts. To assess the relative stability of variants generated during the development of this invention, the enzyme's thermal stability was evaluated as follows: 100 μL of 10 g / L amino acid decarboxylase SFP was incubated in a thermocycler at 30-70°C for 1.5 hours. After incubation, the sample was briefly centrifuged, and 10 μL of the heat-treated SFP was added to 90 μL of reaction mix for a final concentration of 5 mM leucine in 50 mM sodium phosphate, pH 7.0. The reaction was incubated in a THERMOTRON® titer plate at 37°C and 250 rpm for 2 hours, processed, and analyzed by LC-MS / MS as described above. The results for SEQ ID NOs: 2-14 are shown in Tables 1-2. SFP characterization assay for resistance to proteases

[0171] To assess the relative stability of the enzyme against representative gut proteases, a mix of porcine trypsin (Sigma-Aldrich) and bovine chymotrypsin (Sigma-Aldrich) was dissolved in 50 mM sodium phosphate, pH 7.0, to a concentration of 3 g / L each and serially diluted two-fold. Then, 10 g / L of amino acid decarboxylase SFP was incubated with 0 to 1.5 g / L trypsin / chymotrypsin in a THERMOTRON® titer plate shaker at 37°C and 250 rpm for 1 hour. After incubation, 10 μL of the protease-treated SFP was added to 90 μL of reaction mix for a final concentration of 3 mM leucine in 50 mM sodium phosphate, pH 7.0. The reaction was incubated in a THERMOTRON® titer plate at 37°C and 250 rpm for 1 hour, then diluted five-fold in water and quenched with 3 volumes of acetonitrile with 0.1% formic acid. The resulting samples were processed as described above, and the supernatants were analyzed by LC-MS / MS as described above. The results for SEQ ID NOs: 2 and 12 are shown in Table 1-2. [Table 1-2] Example 2 LDC variant of SEQ ID NO: 12

[0172] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 12 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of LDC encoded by SEQ ID NO: 11 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. High-throughput (HTP) growth of cultures expressing LDC enzymes

[0173] Transformed E. coli cells were selected by plating on LB agar plates containing 1% glucose. After overnight incubation at 37°C, colonies were picked in NUNC™ (Thermo-Scientific) 96-well shallow flat-bottom plates filled with 180 μL / well of LB medium supplemented with 1% glucose. Cultures were grown overnight for 18-20 hours in a Kühner shaker (200 rpm, 30°C, and 85% relative humidity). Samples (20 μL) of the overnight growth were transferred to COSTAR® 96-well deep plates (Corning) filled with 380 μL of TB. Cultures were incubated in a Kuhner shaker (250 rpm, 30°C, and 85% relative humidity) for 2-3 hours, then induced with 40 μL of 10 mM IPTG in sterile water and incubated overnight for 20-24 hours in a Kuhner shaker (250 rpm, 30°C, and 85% relative humidity). Cells were pelleted (4000 rpm x 10 min), the supernatant discarded, and the cells frozen at -80°C prior to analysis. Lysis of HTP cell pellets

[0174] E. coli cell pellets were lysed with 400 μL of lysis buffer (1 mg / ml lysozyme + 0.5 g / L PMBS in 50 mM sodium phosphate pH 7). The mixture was agitated for 1.5 hours at room temperature and pelleted (4000 rpm × 10 minutes), after which the clarified lysate was preincubated at 60°C for 1 hour in a THERMOTRON® titer plate shaker (400 rpm). The heat-treated lysate was pelleted (4000 rpm × 10 minutes) and the supernatant was used in the HTP assay. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0175] Heat-treated lysates containing LDC variants were challenged with an acidic buffer solution simulating the gastric environment. First, the heat-treated clarified lysates were preincubated 1:1 with McIlvaine buffer, pH 3.6, in a COSTAR® 96-well round-bottom plate (Corning). The plate was sealed and incubated at 37°C for 1 hour in a THERMOTRON® titer plate shaker (250 rpm). The resulting acidic buffer-treated lysates were then preincubated with a final concentration of 0.02 g / L trypsin and chymotrypsin (1:1) at 37°C with shaking for 1 hour. After incubation, the resulting samples were centrifuged, and 50 μL of the sample was added to a 50 μL reaction mix for a final concentration of 3 mM leucine and 10 μM PLP in 50 mM sodium phosphate, pH 7.0. In some experiments, isoleucine-d was used instead of isoleucine in 50 mM sodium phosphate, pH 7.0. 10 0.6-3 mM of all 20 amino acids (Cambridge A reaction mix resulting in a final concentration of 10 μM PLP was used (Isotope Laboratories). Reactions were incubated in a THERMOTRON® titer plate at 37° C. and 250 rpm for 1 hour, then quenched with 3 volumes of acetonitrile with 0.1% formic acid, centrifuged at 4000 rpm for 10 minutes at 4° C., and diluted 50-fold in water. The resulting samples were analyzed by LC-MS / MS as described in Example 1. The results of these assays are shown in Table 2-1. [Table 2-1A] [Table 2-1B] [Table 2-1C] Example 3 LDC variant of SEQ ID NO: 38

[0176] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 38 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of LDC encoded by SEQ ID NO: 38 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0177] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2. Heat-treated lysates containing LDC variants were challenged with acidic buffer as described in Example 2. The acidic buffer-treated lysates were then preincubated with a final concentration of 0.1 g / L trypsin and chymotrypsin (1:1) at 37°C for 1 hour with shaking. After incubation, the samples were centrifuged, and 50 μL of the sample was added to 50 μL of reaction mix for a final concentration of 3 mM leucine and 10 μM PLP in 50 mM sodium phosphate, pH 7.0. In some experiments, isoleucine-d was used instead of isoleucine in 50 mM sodium phosphate, pH 7.0. 10 A reaction mix resulting in a final concentration of 0.6-3 mM total 20 amino acids (Cambridge Isotope Laboratories) with 10 μM PLP was used. Reactions were incubated in a THERMOTRON® titer plate at 37°C and 250 rpm for 1 hour, then quenched with 3 volumes of acetonitrile with 0.1% formic acid, centrifuged at 4000 rpm for 10 minutes at 4°C, and diluted 50-fold in water. The resulting samples were analyzed by LC-MS / MS as described in Example 1 or by RAPIDFIRE® mass spectrometry (Agilent) using the parameters shown in Table 3-1. The results of these assays are shown in Table 3-2. [Table 3-1] [Table 3-2A] [Table 3-2B] Example 4 LDC variant of SEQ ID NO: 234

[0178] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 234 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 234 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0179] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2. Heat-treated lysates containing LDC variants were challenged with acidic buffer as described in Example 2 under the following conditions: heat-treated clarified lysates were preincubated 1:1 with McIlvaine buffer, pH 3.3. The resulting acidic buffer-treated lysates were then preincubated 1:1 with a final concentration of 0.5 g / L trypsin and chymotrypsin (1:1) at 37°C with shaking for 1 hour. After incubation, the samples were centrifuged, and 40 μL of the sample was added to a 60 μL reaction mix for a final concentration of 3 mM leucine and 10 μM PLP in 50 mM sodium phosphate, pH 7.0. In some experiments, isoleucine-d was used instead of isoleucine in 50 mM sodium phosphate, pH 7.0. 10 0.6-3 mM of all 20 amino acids (Cambridge Isotope Laboratories) and a reaction mix resulting in a final concentration of 10 μM PLP was used. Reactions were incubated in a THERMOTRON® titer plate at 37° C. and 250 rpm for 1 hour, then quenched with 3 volumes of acetonitrile with 0.1% formic acid, centrifuged at 4000 rpm for 10 minutes at 4° C., and diluted 10- or 500-fold in water. The resulting samples were analyzed by LC-MS / MS as described in Example 1 or by RAPIDFIRE®-MS as described in Example 3. [Table 4-1A] [Table 4-1B] Example 5 LDC variant of SEQ ID NO: 284

[0180] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 284 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 284 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer, pepsin, and protease

[0181] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2. Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin to simulate the gastric environment. First, the heat-treated clarified lysates were preincubated 1:1 with McIlvaine's buffer, pH 3.2, containing 0.05 g / L pepsin from porcine gastric mucosa (Sigma) in a COSTAR® 96-well round-bottom plate (Corning). The plate was sealed and incubated at 37°C for 1 hour in a THERMOTRON® titer plate shaker (250 rpm). The samples were then briefly centrifuged, and the resulting supernatants were preincubated with a final concentration of 0.5 g / L trypsin and chymotrypsin (1:1) in a 1:1 ratio at 37°C for 1 hour with shaking. After incubation, 40 μL of the sample was added to 60 μL of reaction mix for a final concentration of 3 mM leucine and 10 μM PLP in 50 mM sodium phosphate, pH 7.0. The reaction was incubated in a THERMOTRON® titer plate at 37° C. and 250 rpm for 1 hour, then quenched with 3 volumes of acetonitrile with 0.1% formic acid, centrifuged at 4000 rpm for 10 minutes at 4° C., and diluted 500-fold in water. The resulting sample was analyzed by RAPIDFIRE®-MS as described in Example 3. [Table 5-1A] [Table 5-1B] Example 6 LDC variant of SEQ ID NO: 484

[0182] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 484 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 484 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0183] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, except that the lysis buffer contained 40 μM PLP. Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin and then challenged with a protease as described in Example 5. Specifically, the heat-treated clarified lysates were preincubated 1:1 with a final concentration of 0.375 g / L pepsin in McIlvaine's buffer, pH 3, at 37°C for 1 hour, and the resulting supernatant was preincubated 1:1 with a final concentration of 0.75 g / L trypsin and chymotrypsin (1:1) dissolved in 200 mM sodium phosphate, pH 8, at 37°C for 1 hour. After incubation, 40 μL of the sample was added to a 60 μL reaction mix for a final concentration of 3 mM leucine in 50 mM sodium phosphate, pH 7.0. Reactions were incubated, quenched, and analyzed as described in Example 5, except that samples were diluted 50-fold in water before analysis by RAPIDFIRE®-MS. The results of these assays are shown in Table 6-1. [Table 6-1A] [Table 6-1B] [Table 6-1C] Example 7 LDC variant of SEQ ID NO: 594

[0184] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 594 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 594 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0185] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, except that the lysis buffer contained 40 μM PLP. Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin and then challenged with a protease as described in Example 5. Specifically, the heat-treated clarified lysates were preincubated 1:1 with a final concentration of 0.2 g / L pepsin in McIlvaine's buffer, pH 2.8, at 37°C for 1 hour, and the resulting supernatant was preincubated 1:1 with a final concentration of 1.5 g / L trypsin and chymotrypsin (1:1) dissolved in 400 mM sodium phosphate, pH 8, at 37°C for 1 hour. After incubation, 40 μL of the sample was added to a 60 μL reaction mix for a final concentration of 2.5 mM leucine in 50 mM sodium phosphate, pH 7.0. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. The results of these assays are shown in Table 7-1. [Table 7-1] Example 8 LDC variant of SEQ ID NO: 686

[0186] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 686 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 686 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0187] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, except that the lysis buffer contained 40 μM PLP. The heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin and then challenged with a protease as described in Example 5. Specifically, the heat-treated clarified lysates were preincubated 1:1 with a final concentration of 0.4 g / L pepsin in McIlvaine's buffer, pH 2.8, at 37°C for 1-1.5 hours, and the resulting supernatant was preincubated 1:1 with a final concentration of 2-4 g / L trypsin and 1.5 g / L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, at 37°C for 1-2 hours. After incubation, 40 μL of sample was added to 60 μL of reaction mix for a final concentration of 2.5 mM leucine in 50 mM sodium phosphate, pH 7.0. In some experiments, 2.5 mM leucine, isoleucine-d in 50 mM sodium phosphate, pH 7.0. 10 A reaction mix resulting in final concentrations of 1000kJ / mL ribonucleotides (RIB), valine, asparagine, methionine, and cysteine ​​was used. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. The results of these assays are provided in Tables 8-1 and 8-2. [Table 8-1] [Table 8-2] Example 9 Amino acid activity analysis of LDC variants

[0188] This example describes experiments to evaluate the activity of SEQ ID NOs: 12 and 594 against all 20 amino acids. Shake flask powder (SFP) generation

[0189] Shake-flask powders of LDC variants were prepared as described in Example 1, with the following modifications: cell pellets were resuspended in 30 mL of 50 mM sodium phosphate, pH 7.0, with 200 μM PLP. After lysis using a single pass through a Microfluidics at 110 psi, the lysate was pelleted (10,000× rpm, 30 min, 4°C), and the resulting supernatant was heat-treated in a water bath at 60°C for 1 h. The sample was then centrifuged (10,000× rpm, 1 h, 4°C), after which the resulting supernatant was frozen and lyophilized to produce a powder containing the expressed enzyme. SFP Characterization Assay for Decarboxylase Activity Toward Different Amino Acids

[0190] The shake flask powder was reconstituted in phosphate buffered saline, pH 7.4 to prepare a stock solution of 10 g / L powder. 20 μL of these SFP solutions were then added to 50 mM sodium phosphate buffer, pH 7.0, with d-isoleucine instead of isoleucine. 10The 20 amino acids were added to 180 μL of a reaction mix containing 2 mM of all 20 amino acids (Cambridge Isotope Laboratories) with 1% ATP. The reaction was incubated for 1.5 to 2 hours at 37°C and 400 rpm in a THERMOTRON® titer plate shaker and then quenched with 2 volumes of acetonitrile with 0.1% formic acid. The resulting sample was centrifuged at 4000 rpm for 10 minutes at 4°C, and the supernatant was diluted up to six-fold in acetonitrile with 0.1% formic acid. The sample was analyzed for all 20 amino acids by LC-MS / MS; examples of the LC-MS / MS instrument and parameters are shown in Tables 9-1 and 9-2. The conversion (i.e., depletion) of each amino acid was calculated relative to the negative control, and the results for SEQ ID NOs: 12 and 594 are shown in Table 9-2. [Table 9-1] [Table 9-2] [Table 9-3] Example 10 LDC variant of SEQ ID NO: 688

[0191] This example describes experiments for the evolution and screening of LDC variants derived from SEQ ID NO: 688 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 688 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0192] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, except that the lysis buffer contained 20 mM sodium phosphate and 40 μM PLP. Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin and then challenged with a protease as described in Example 5. Specifically, the heat-treated clarified lysates were preincubated 1:1 with a final concentration of 0.4 g / L pepsin in McIlvaine's buffer, pH 3, at 37°C for 1.5 hours, and the resulting supernatant was preincubated 1:1 with a final concentration of 4 g / L trypsin and 1.5 g / L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, at 37°C for 2 hours. After incubation, 40 μL of the sample was added to 60 μL of reaction mix and incubated with 2.5 mM leucine, isoleucine-d in simulated intestinal fluid. 10 Final concentrations of valine, asparagine, methionine, and cysteine ​​were obtained. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. The results of these assays are provided in Table 10-1. [Table 10-1] Example 11 LDC variant of SEQ ID NO: 766

[0193] This example describes experiments to evolve and screen LDC variants derived from SEQ ID NO: 766 for improved leucine activity, low pH tolerance, and protease resistance. Directed evolution of the LDC encoded by SEQ ID NO: 766 was performed by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP activity assay of clarified lysates pretreated with acidic buffer and protease

[0194] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 10. Heat-treated lysates containing LDC variants were challenged with an acidic buffer containing pepsin, followed by a protease challenge, as described in Example 5. Specifically, the heat-treated clarified lysates were preincubated 1:1 with a final concentration of 0.8 g / L pepsin in McIlvaine's buffer, pH 2.6-2.8, at 37°C for 2 hours. The resulting supernatant was preincubated 1:1 with a final concentration of 4 g / L trypsin and 1.5 g / L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, at 37°C for 2 hours. After incubation, 40 μL of the sample was added to 60 μL of reaction mix and incubated with 2-3 mM leucine, isoleucine-d in simulated intestinal fluid. 10 Final concentrations of valine, asparagine, methionine, and cysteine ​​were obtained. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. The results of these assays are provided in Tables 11-1 and 11-2. HTP activity assay of clarified lysates for leucine

[0195] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 10. The heat-treated lysates were diluted 20-fold in water, and 20 μL of the sample was added to 80 μL of reaction mix to give a final concentration of 3 mM leucine in 50 mM sodium phosphate, pH 7. The reactions were incubated, quenched, diluted, and analyzed as described in Example 6. Activity toward leucine was normalized by the concentration of leucine decarboxylase in the heat-treated, clarified lysates, as determined by SDS-PAGE and size-exclusion chromatography. The results of these assays are provided in Table 11-2. [Table 11-1] [Table 11-2] Example 12 In vivo characterization of LDC variants in healthy cynomolgus monkeys

[0196] An in vivo pharmacodynamic study was conducted in healthy cynomolgus monkeys (n=12, male, BW 3-4 kg) to characterize three LDC variants (SEQ ID NO:484; SEQ ID NO:686; and SEQ ID NO:766). Each variant was dosed at either 25, 50, or 100 mg / kg. The study consisted of four dosing days, each separated by a one-week washout period, over a four-week period. Dosing groups were rotated so that animals did not receive the same dose twice. One day prior to dosing, animals were placed in individual cages and fasted overnight. On each dosing day, animals were given 10 g of whey protein powder (BN Labs Grass Fed Whey Protein; 9.14% leucine) formulated with water to a final volume of 20 mL per animal by oral gavage. Immediately following the whey protein suspension, animals received either vehicle (20 mM sodium phosphate, 0.4 mM pyridoxal phosphate, pH 7.2) or the appropriate dose of LDC at 2.5 mL / kg, followed by a 4 mL water rinse to ensure efficient delivery of all materials using the same gavage tube. On the dosing day, animals were offered their regular diet for 8 hours immediately following blood collection. Blood samples were obtained 90 and 30 minutes before feeding, and 5, 15, 30, 1, 2, 4, 8, 12, and 24 hours after feeding (the 24-hour time point was collected the following day, before feeding). Samples were transferred to tubes containing the anticoagulant K2EDTA, placed on wet ice, and centrifuged at 2500 rpm at approximately 4°C for 10 minutes while awaiting processing. The resulting plasma was collected and stored frozen (≤-60°C) until analysis. Plasma leucine, phenylalanine, tyrosine and methionine were quantified using LC-MS / MS to evaluate efficacy.A significant increase in plasma leucine was observed after the meal challenge alone compared to the baseline before intake.All LDC variants at all doses resulted in significant suppression of plasma leucine in response to the whey protein meal challenge.SEQ ID NO:484 and SEQ ID NO:686 demonstrated efficacy in a dose-response manner.All doses of SEQ ID NO:766 showed similar efficacy, which was superior to both SEQ ID NO:484 and SEQ ID NO:686.LDC had no effect on plasma phenylalanine, tyrosine, or methionine. Table 12-1 summarizes the percent reduction in the area under the curve (iAUC) of plasma leucine by treatment compared to vehicle. iAUC was reported by first subtracting the pre-feeding leucine value (fasting background) from each post-feeding time point for each animal, then performing the AUC calculation. Statistical calculations and significance were performed using GraphPad Prism 7 (GraphPad). The determination was made using the software. [Table 12-1] Example 13 In vivo characterization of LDC variants in mouse models of disease

[0197] In vivo pharmacodynamic studies were performed in a mouse model of intermediate maple syrup urine disease (iMSUD; (Dbt tm1GehThree LDC variants (SEQ ID NO: 484; SEQ ID NO: 686; and SEQ ID NO: 766) were characterized in a 100-mL iMSUD mouse (Tg(Cebpb-tTA)5Bjd Tg(tetO-DBT)A1Geh / J; JAX stock no. 6999). Each variant was dosed at 200 mg / kg. Prior to the start of dosing and weaning, iMSUD mice were maintained on leucine-free mouse chow with supplemental leucine (5.75 g leucine / L) in their drinking water to support growth and prolong survival. Animals at least 2 months old and weighing 20 g were used for the experiment. Animals were fasted overnight (approximately 15 hours) before dosing. On each dosing day, animals were given 45 mg of whey protein powder (BN Labs Grass Fed Whey Protein; 9.14% leucine) formulated with water to a total volume of 100 mL per animal by oral gavage. Immediately following the whey protein suspension, mice received either vehicle (20 mM sodium phosphate, 0.4 mM pyridoxal phosphate, pH 7.2) or the appropriate LDC variant at 5 mL / kg. Blood samples were obtained 15 minutes before feeding and 15, 30, 60, 120, and 240 minutes after feeding. Samples were transferred to tubes containing the anticoagulant K2EDTA, placed on wet ice awaiting processing, and centrifuged at 2500 rpm at approximately 4°C for 10 minutes. The resulting plasma was collected and stored frozen (≤-60°C) until analysis. Plasma leucine was quantified using LC-MS / MS to assess efficacy. A significant increase in plasma leucine was observed compared to the pre-feeding baseline. All LDC variants resulted in a significant suppression of plasma leucine in response to the whey protein meal challenge. SEQ ID NO: 484 and SEQ ID NO: 686 demonstrated similar efficacy, while SEQ ID NO: 766 produced superior efficacy. Table 13-1 summarizes the percent reduction of iAUC plasma leucine by treatment compared to vehicle. iAUC was reported by first subtracting the pre-feeding leucine value (fasting background) from each post-feeding time point for each animal, and then performing AUC calculation. Statistical calculations and significance were determined using GraphPad Prism 7 (GraphPad Software). [Table 13-1]

[0198] Although the invention has been described with reference to specific embodiments, various modifications may be made to adapt a particular situation, material, composition of matter, process, process step or steps, and equivalents may be substituted, thereby achieving the benefits of the invention without departing from the scope of the appended claims.

[0199] For all purposes in the United States, any and all publications and patent documents cited in this disclosure are incorporated herein by reference as if each such publication or document was specifically and individually indicated to be incorporated herein by reference. Citation of publications and patent documents is not intended as an indication that any such document is pertinent prior art or as constituting an admission as to the contents or date thereof. The present invention provides, for example, the following items. (Item 1) 1. An engineered leucine decarboxylase polypeptide comprising 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, and / or 766, wherein the amino acid positions of the amino acid sequence are numbered with reference to the amino acid sequence of SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766. (Item 2) 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence has 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. (Item 3) 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence has 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. (Item 4) 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence has 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. (Item 5) the polypeptide sequence is at least 85%, 86%, 87%, or 2. The engineered leucine decarboxylase polypeptide of claim 1, having 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the polypeptide. (Item 6) 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence has 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. (Item 7) 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence has 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. (Item 8) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 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 / 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 / 3 50 / 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 / 35 7, 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, 3 9 / 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, 339,2. The engineered leucine decarboxylase polypeptide of item 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from 343, 350, 353, 357, 364, 365, 379, 381, 386, 389, 391, 393, 394, 395, 397, 398, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO: 12. (Item 9) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 48 / 64 / 164 / 324 / 343 / 353 / 357 / 364, 48 / 64 / 164 / 324 / 343 ... 8 / 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 / 3 95, 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, 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from positions 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, and 394 / 397, wherein the amino acid positions are numbered with reference to SEQ ID NO: 38. (Item 10) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of: 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, 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO:234. (Item 11) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 2 / 64 / 69 / 324 / 380 / 382 / 38 8 / 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, 12 / 135 / 263 / 382, 12 / 259 / 263 / 304, 48 / 64 / 2 55, 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 / 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from 382 / 388 / 390, 69 / 263, 69 / 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, and 304 / 340 / 379 / 380 / 382, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284. (Item 12) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 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 / 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 / 304, 263 / 304 / 324, 263 / 304 / 324 / 339, 263 / 304 / 324 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from: 263 / 324, 270, 270 / 319, 270 / 328 / 338, 270 / 328 / 338 / 365, 304, 304 / 324, 324, 328, 352, 365, 366, and 382, ​​wherein the amino acid positions are numbered with reference to SEQ ID NO:484. (Item 13) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 16 / 63 / 80 / 126 / 168 / 366, 1 6 / 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 / 324 / 328 / 366, 16 / 328 / 366, 80 / 126 / 168 / 270 / 366, 80 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from positions 126 / 168 / 366, 80 / 126 / 181 / 270 / 324 / 366, 80 / 168 / 270 / 366, and 168 / 366, wherein the amino acid positions are numbered with reference to SEQ ID NO: 594. (Item 14) The polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, 100%, 101%, 102%, 103%, 104%, 105%, 106%, 107%, 108%, 109%, 1109%, 1110%, 112%, 113%, 114%, 115%, 116%, 117%, 118%, 119%, 120%, 121%, 122%, 123%, 124%, 125%, 126%, 127%, 128%, 129%, 130%, 131%, 132%, 133%, 134%, 135%, 136%, 137%, 138%, 139%, 140%, 141%, 142%, 143%, 144%, 145%, 146%, 147%, 148%, 149%, 150%, 151%, 152%, 153%, 154%, 155%, 156%, 157%, 158%, 159%, 160%, 161%, 162%, 163%, 164%, 165%, 166%, 167%, 168%, 169%, 170%, 171%, 172%, 173%, 174%, 175%, 176%, 177%, 178%, 179%, 180%, 181%, 182%, 183%, 184%, 185%, 186, 187%, 18 %, 98%, 99% or higher sequence identity, and the polypeptide sequence of the engineered leucine decarboxylase polypeptide is 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 / 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from: 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, and 270 / 324, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. (Item 15) 2. The engineered leucine decarboxylase polypeptide of item 1, wherein the polypeptide sequence has 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 polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 19, 109, 123, 134, 170, 173, 187, 211, and 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. (Item 16) the polypeptide sequence has 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 the polypeptide sequence of the engineered leucine decarboxylase polypeptide is selected from the group consisting of 19 / 109 / 123 / 141 / 170 / 198 / 200 / 211 / 270 / 312, 19 / 109 / 123 / 141 / 170 / 198 / 21 ... 2. The engineered leucine decarboxylase polypeptide of claim 1, comprising at least one substitution or set of substitutions at one or more amino acid positions selected from positions 9 / 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, and 109 / 211 / 270 / 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 688. (Item 17) 2. The engineered leucine decarboxylase polypeptide of item 1, wherein the polypeptide sequence has 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 polypeptide sequence of the engineered leucine decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 5 / 41, 5 / 41 / 228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, and 384, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766. (Item 18) the polypeptide sequence has 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 the engineered leucine 2. The engineered leucine decarboxylase polypeptide of claim 1, wherein the polypeptide sequence of the decarboxylase polypeptide comprises at least one substitution or set of substitutions at one or more amino acid positions selected from 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, and 300, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766. (Item 19) An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NOs: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766. (Item 20) 20. An engineered leucine decarboxylase polypeptide, comprising an engineered polypeptide provided in any of items 1 to 19. (Item 21) 21. The engineered leucine decarboxylase polypeptide of claim 20, comprising an amino acid sequence having at least about 95% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and / or 766. (Item 22) 19. The engineered leucine decarboxylase polypeptide of any of items 1 to 18, wherein the variant leucine decarboxylase polypeptide is a variant 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, and / or 11-2. (Item 23) 23. The engineered leucine decarboxylase polypeptide according to any one of items 1 to 22, which is a variant enzyme of Planctomycetaceae. (Item 24) 24. The engineered leucine decarboxylase polypeptide according to any of items 1 to 23, which exhibits higher activity on leucine than wild-type leucine decarboxylase of Planctomycetaceae species. (Item 25) 25. The engineered leucine decarboxylase polypeptide of any of items 1 to 24, which is more resistant to proteolysis than wild-type leucine decarboxylase of Planctomycetaceae species. (Item 26) 26. The engineered leucine decarboxylase polypeptide of any of items 1 to 25, which is more thermostable than the wild-type leucine decarboxylase of a Planctomycetaceae species. (Item 27) 27. The engineered leucine decarboxylase polypeptide of any of items 1 to 26, comprising a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or higher identical to any of the even-numbered sequences of SEQ ID NOs: 16 to 852. (Item 28) 28. The engineered leucine decarboxylase polypeptide of item 27, comprising a sequence at least 90% identical to any of the even-numbered sequences of SEQ ID NOs: 16 to 852. (Item 29) 29. The engineered leucine decarboxylase polypeptide according to Item 28, comprising any of the even-numbered sequences of SEQ ID NOs: 16 to 852. (Item 30) 30. The engineered leucine decarboxylase polypeptide of any of items 1 to 29, which is purified. (Item 31) 31. A composition comprising at least one engineered leucine decarboxylase polypeptide as provided in any of items 1 to 30. (Item 32) 32. An engineered polynucleotide sequence encoding at least one engineered leucine decarboxylase polypeptide according to any of items 1 to 31. (Item 33) 33. The engineered polynucleotide sequence of Item 32, comprising a sequence that is at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identical to any of the odd-numbered sequences of SEQ ID NOs: 15-851. (Item 34) 33. The engineered polynucleotide sequence of item 32, comprising a sequence at least 90% or higher identical to any of the odd-numbered sequences of SEQ ID NOs: 15 to 851. (Item 35) 33. The engineered polynucleotide sequence according to Item 32, comprising any of the odd-numbered sequences of SEQ ID NOs: 15 to 851. (Item 36) 36. The engineered polynucleotide sequence according to any one of items 32 to 35, operably linked to a regulatory sequence. (Item 37) 37. The engineered polynucleotide sequence according to any of items 32 to 36, wherein the polynucleotide is codon-optimized. (Item 38) 38. An expression vector comprising at least one engineered polynucleotide sequence according to any one of items 32 to 37 and at least one regulatory sequence. (Item 39) 39. The expression vector of item 38, wherein the control sequence comprises a promoter. (Item 40) 40. The expression vector of item 39, wherein the promoter is a heterologous promoter. (Item 41) A host cell transformed with at least one polynucleotide sequence according to any one of items 32 to 37 and / or comprising an expression vector according to any one of items 38 to 40. (Item 42) 42. The host cell according to item 41, which is E. coli. (Item 43) 41. A method for producing an engineered leucine decarboxylase polypeptide in a host cell, the method comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide according to any of items 1 to 30, and / or at least one polynucleotide sequence according to any of items 32 to 37, and / or at least one expression vector according to any of items 38 to 40, under suitable culture conditions such that the at least one engineered leucine decarboxylase polypeptide is produced. (Item 44) A host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide according to any one of items 1 to 30 is cultured at a low temperature. 44. The method of claim 43, comprising culturing under suitable culture conditions such that at least one engineered leucine decarboxylase polypeptide is produced. (Item 45) 45. The method of paragraph 43 and / or 44, further comprising recovering the at least one engineered leucine decarboxylase polypeptide from the culture and / or host cell. (Item 46) 46. ​​The method of any of items 43 to 45, further comprising purifying the at least one engineered leucine decarboxylase polypeptide. (Item 47) 38. A composition comprising at least one engineered polynucleotide according to any of items 32 to 37. (Item 48) 48. The composition according to item 47, which is a pharmaceutical composition. (Item 49) 49. The composition according to item 48, further comprising at least one pharmaceutically acceptable excipient and / or carrier. (Item 50) 50. The composition according to any of items 31, 48 and 49, which is suitable for the treatment of maple syrup urine disease. (Item 51) 51. The composition according to any of items 31 and 47 to 50, which is suitable for oral administration to humans. (Item 52) 52. The composition according to any of items 31 and 47 to 51, which is in the form of a pill, tablet, capsule, gelcap, liquid or emulsion. (Item 53) 53. The composition of claim 52, wherein the pill, tablet, capsule, or gelcap further comprises an enteric coating. (Item 54) 53. The composition according to any of items 31 and 47 to 52, which is suitable for parenteral injection into humans. (Item 55) 55. The composition according to any of items 31 and 47-54, co-administered with at least one additional therapeutically effective compound. (Item 56) 56. The composition according to item 55, comprising at least one additional therapeutically effective compound. (Item 57) 57. A method for treating and / or preventing symptoms of maple syrup urine disease in a subject, the method comprising the steps of providing a subject with maple syrup urine disease, and providing the subject with the composition of any of items 31 and 47-56. (Item 58) 58. The method of claim 57, wherein the symptoms of maple syrup urine disease are ameliorated. (Item 59) 59. The method of claim 57, wherein the subject is able to consume a diet that is less restricted in isoleucine, leucine, and / or valine than a diet required by a subject not provided with at least one composition comprising at least one engineered leucine decarboxylase polypeptide described in items 1-30. (Item 60) 60. The method of any of items 57-59, wherein the subject is able to consume a diet that is less restricted in isoleucine, leucine, and / or valine content than a diet required by a subject that has not been provided with at least one composition comprising at least one engineered leucine decarboxylase polypeptide produced using the method of items 43-46. (Item 61) 61. The method of any of items 57 to 60, wherein the subject is an infant, a child, a young adult or an adult. (Item 62) Use of the composition provided in any of items 31 and / or 47 to 56.

Claims

[Claim 1] The invention described in the specification.

Images