WO2023077169A2 - Engineered leucine decarboxylases - Google Patents

Engineered leucine decarboxylases Download PDF

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Publication number
WO2023077169A2
WO2023077169A2 PCT/US2022/079089 US2022079089W WO2023077169A2 WO 2023077169 A2 WO2023077169 A2 WO 2023077169A2 US 2022079089 W US2022079089 W US 2022079089W WO 2023077169 A2 WO2023077169 A2 WO 2023077169A2
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Prior art keywords
amino acid
seq
engineered
leucine decarboxylase
acid sequence
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PCT/US2022/079089
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English (en)
French (fr)
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WO2023077169A3 (en
Inventor
Jonathan Benjamin ASFAHA
Subhamoy Das
Faye Loan Du
Stephan JENNE
Nikki D. KRUSE
Joyce Liu
Kerryn Mccluskie
Roasa MEHMOOD
Adam P. Silverman
Kristen Jean Vallieu
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Codexis Inc
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Codexis Inc
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Priority to JP2024526835A priority Critical patent/JP2024540358A/ja
Priority to CN202280086975.3A priority patent/CN118475692A/zh
Priority to CA3236731A priority patent/CA3236731A1/en
Priority to EP22888596.8A priority patent/EP4426827A4/en
Priority to AU2022377148A priority patent/AU2022377148A1/en
Publication of WO2023077169A2 publication Critical patent/WO2023077169A2/en
Publication of WO2023077169A3 publication Critical patent/WO2023077169A3/en
Anticipated expiration legal-status Critical
Ceased legal-status Critical Current

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    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • A61K38/16—Peptides having more than 20 amino acids; Gastrins; Somatostatins; Melanotropins; Derivatives thereof
    • A61K38/43—Enzymes; Proenzymes; Derivatives thereof
    • A61K38/51—Lyases (4)
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N9/00—Enzymes; Proenzymes; Compositions thereof; Processes for preparing, activating, inhibiting, separating or purifying enzymes
    • C12N9/88—Lyases (4.)
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P3/00—Drugs for disorders of the metabolism
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12Y—ENZYMES
    • C12Y401/00—Carbon-carbon lyases (4.1)
    • C12Y401/01—Carboxy-lyases (4.1.1)
    • C12Y401/01014—Valine decarboxylase (4.1.1.14)
    • Y—GENERAL TAGGING OF NEW TECHNOLOGICAL DEVELOPMENTS; GENERAL TAGGING OF CROSS-SECTIONAL TECHNOLOGIES SPANNING OVER SEVERAL SECTIONS OF THE IPC; TECHNICAL SUBJECTS COVERED BY FORMER USPC CROSS-REFERENCE ART COLLECTIONS [XRACs] AND DIGESTS
    • Y02—TECHNOLOGIES OR APPLICATIONS FOR MITIGATION OR ADAPTATION AGAINST CLIMATE CHANGE
    • Y02E—REDUCTION OF GREENHOUSE GAS [GHG] EMISSIONS, RELATED TO ENERGY GENERATION, TRANSMISSION OR DISTRIBUTION
    • Y02E50/00—Technologies for the production of fuel of non-fossil origin
    • Y02E50/10—Biofuels, e.g. bio-diesel

Definitions

  • the present disclosure relates to engineered leucine decarboxylase (LDC) polypeptides, compositions thereof, and polynucleotides encoding the engineered leucine decarboxylase polypeptides.
  • LDC leucine decarboxylase
  • the present disclosure also provides methods of using the engineered leucine decarboxylase polypeptides for therapeutic and industrial purposes.
  • Maple syrup urine disease also referred to as “leucineuria,” branched chain alphaleucine dehydrogenase deficiency,” and “BCKD deficiency,” is a rare inherited aminoacidopathy secondary to dysfunction in the branched chain keto acid dehydrogenase (BCKDH) complex that is involved in the catabolic pathway of leucine, isoleucine and valine (i.e., branched chain amino acids). It was first described in 1954 by Menkes et al., Pediatrics, 1954, 14:462-467, and named due to the distinctive, sweet odor of the urine of affected newborns.
  • BCKDH branched chain keto acid dehydrogenase
  • Diagnostic testing for MSUD in newborns includes blood and urine amino acid tests to determine the leucine, isoleucine, alloisoleucine, and valine concentrations in these fluids. If MSUD is identified, there will be signs of ketosis and acidosis. Upon diagnosis and during symptomatic episodes, treatment involves eating a protein-free diet and correction of the metabolic consequences associated with the elevated amino acid levels. The use of a special intravenous solution decreases the leucine level (the most toxic) and corrects energy deficits.
  • disorders of leucine metabolism and branched amino acids include isovaleric acidemia and 3-methylcrotonylglycinuria (also referred to as 3-methylcrotonyl-CoA carboxylase deficiency).
  • Current treatment for these types of disorders involves dietary restriction of leucine and other branched-chain amino acids (BCAAs) and/or restriction of protein intake.
  • BCAAs branched-chain amino acids
  • Deficient levels of enzymes involved in BCAA metabolism result in the toxic accretion of BCAAs and their related metabolites in the cerebrospinal fluid, blood, and tissues. Without treatment or constant attentive care, this leads to numerous and serious side effects (e.g., neurological dysfunction, seizures, and infant death).
  • the present invention provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase polypeptides.
  • the leucine decarboxylase polypeptides are engineered to have an improved property, including enhanced catalytic activity, reduced sensitivity to proteolysis, and/or increased tolerance to low pH environments.
  • the engineered leucine decarboxylase polypeptides are engineered to display improved storage stability.
  • the present invention also provides methods of using the engineered leucine decarboxylase polypeptides and compositions thereof for therapeutic and industrial purposes.
  • the present invention is directed to engineered leucine decarboxylase polypeptides and biologically active fragments and analogs thereof having improved properties when compared to a wild-type leucine decarboxylase enzyme or a reference leucine decarboxylase polypeptide under essentially the same conditions.
  • an engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to SEQ ID NO: 828 or 888.
  • an engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to SEQ ID NO: 828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid positions 33, 55, 64, 126, 270, or 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises substitutions on at least two amino acid positions of 33, 55, 64, 126, 270, and 357, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the substitutions at amino acid positions 33, 55, 64, 126, 270, and/or 357 are selected from 33L, 551, 64N, 126A, 270L, and 357S.
  • the engineered leucine decarboxylase comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 828, and wherein the amino acid sequence comprises at least a substitution or substitution set at amino acid position 170/270/383, 270, 41/173, 272, 5/141/272/383, 41/383, 41/141/187/272/290, 41/141/173/290, 5/272/383, 5/41/173/272/383, 41/141, 141/272, 353/384, 272/383, 41/141/173, 41/272/383, 41/141/187/200/202/272, 33/55/64/126/270/357, 33/126/353/357, 55/64/267/35/384, 33/64/357, 126/267, 64/267/353/384
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises amino acid residue 5V, 19L, 33L, 41D, 47F, 5 IE, 551, 64S/N, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L/T, 272A, 2901, 312T, 353E, 357S/C, 383S, or 384W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence comprises at least a substitution or substitution set at amino acid position 64/173/202/353/384, 41/141/272/353, 141/202/272/353/357, 173/202/357, 202/353, 5/51/173/272/353/384, 51/202/272/357, 141/173/272, 272, 41/173/384, 41/64/141/353/357/383, 141/173/202, 5/51/64/202/353, 357, 64, 5/41/141, 41/141/173/202/353, 353, 202/357, 51/141/202/272/353, 202, 51/141
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least one substitution or substitution set of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least one substitution or substitution set of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the engineered leucine decarboxylase polypeptide comprises a amino acid sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-1064.
  • the engineered leucine decarboxylase polypeptide described herein exhibits one or more improved properties as compared to the wild-type Planctomycetaceae species leucine decarboxylase or the engineered leucine decarboxylase having the sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide described herein exhibits an improved property selected from (i) increased activity on leucine, (ii) increased resistance to proteolysis, (iii) increased tolerance to low pH environments, or (iv) increased thermostability, or any combinations thereof, compared to the wild-type Planctomycetaceae species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • the present disclosure also provides recombinant polynucleotides encoding at least one engineered leucine decarboxylase polypeptide described herein.
  • the recombinant polynucleotide comprises a polynucleotide sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to the polynucleotide sequence corresponding to an odd-numbered SEQ ID NO.
  • the recombinant polynucleotide encodes a polypeptide having leucine decarboxylase activity.
  • the recombinant polynucleotide comprises the polynucleotide sequence comprising any of the odd-numbered SEQ ID NO. of SEQ ID NOS: 15-851.
  • the recombinant polynucleotide sequence is operably linked to a control sequence. In some embodiments, the recombinant polynucleotide sequence is codon-optimized.
  • the present disclosure also provides expression vectors comprising at least one recombinant polynucleotide sequence provided herein.
  • the expression vectors further comprise at least one control sequence.
  • the control sequence comprises a promoter.
  • the promoter is a heterologous promoter.
  • the present further provides host cells transformed with at least one polynucleotide sequence and/or comprising an expression vector provided herein.
  • the host cells are transformed with a polynucleotide sequence provided herein.
  • the host cells comprise an expression vector provided herein.
  • the host cell is E. coli.
  • the present disclosure also provides methods of producing an engineered leucine decarboxylase polypeptide in a host cell comprising 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.
  • the methods further comprise recovering at least one engineered leucine decarboxylase polypeptide from the culture and/or host cells. In some additional embodiments, the methods further comprise the step of purifying said at least one engineered leucine decarboxylase polypeptide.
  • the present disclosure provides a composition comprising at least one engineered leucine decarboxylase polypeptide.
  • the composition is a pharmaceutical composition comprising at least one engineered leucine decarboxylase polypeptide.
  • the pharmaceutical composition includes at least one pharmaceutically acceptable excipient and/or carrier.
  • the pharmaceutical composition comprising at least one engineered leucine decarboxylase polypeptide is suitable for oral or parenteral administration.
  • the composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion.
  • the composition is suitable for parenteral administration into a mammal, particularly a human patient.
  • the composition comprises at least one additional therapeutically effective compound.
  • the present disclosure also provides a composition comprising at least one polynucleotide encoding an engineered leucine decarboxylase polypeptide disclosed herein, wherein the polynucleotide is suitable for use in gene therapy.
  • the composition comprises an expression vector comprising at least one polynucleotide encoding an engineered polypeptide disclosed herein, wherein the expression vector is a gene therapy vector suitable for use in treatment of diseases or conditions associated with elevated levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid.
  • the composition is suitable for use in mRNA therapy.
  • the present disclosure provides methods for treating and/or preventing the symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels.
  • a method of treating and/or preventing the symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels comprises administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase to decrease levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid in the subject.
  • the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels is maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.
  • the subject in need thereof has maple syrup urine disease, isovaleric acidemia, or 3-methylcrotonyl-CoA carboxylase deficiency.
  • the engineered leucine decarboxylase is administered at a dose of 1 mg/kg to 500 mg/kg, 1 mg/kg to 400 mg/kg, or 1 mg/kg to 200 mg/kg. In some embodiments, the engineered leucine decarboxylase is administered at a dose of 1 mg/kg to less than 25 mg/kg.
  • the engineered leucine decarboxylase is administered at a dose of 5 mg/kg to less than 25 mg/kg. In some embodiments, the engineered leucine decarboxylase is administered at a dose of about 6.25 mg/kg to about 12.5 mg/kg.
  • the engineered leucine decarboxylase is administered to a subject in need thereof in an effective amount to reduce plasma leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and/or ketoisocaproic acid levels. In some embodiments, the engineered leucine decarboxylase is administered to a subject in need thereof in an effective amount to reduce plasma leucine, ketoisocaproic acid, and methionine levels. In some embodiments, the engineered leucine decarboxylase is administered to the subject for at least two or more consecutive days.
  • the engineered leucine decarboxylase is administered to the subject for at least three or more consecutive days. In some embodiments, the engineered leucine decarboxylase is administered to the subject continuously as needed. In some embodiments, the engineered leucine decarboxylase is administered immediately before, concurrently with, and/or immediately following the ingestion of a meal containing protein.
  • the subject for treatment with an engineered leucine decarboxylase has maple syrup urine disease, and wherein the symptoms of maple syrup urine disease are ameliorated.
  • the subject for treatment with an engineered leucine decarboxylase has isovaleric acidemia, and wherein the symptoms of isovaleric acidemia are ameliorated.
  • the subject for treatment with an engineered leucine decarboxylase has 3-methylcrotonyl-CoA carboxylase deficiency, and wherein the symptoms of 3-methylcrotonyl-CoA carboxylase deficiency are ameliorated.
  • the subject is able to eat a diet that is less restricted in leucine, isoleucine, and/or valine content compared to diets required by subjects who are afflicted with the disease.
  • the subject is an infant, child, young adult, or adult.
  • the subject is an infant.
  • the subject is a child.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, and/or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 2, 3, 5, 12, 14, 16, 19, 33, 34, 38, 39, 41, 47, 48, 51, 55, 63, 64, 66, 69, 76, 77, 80, 87, 89, 91, 92, 102, 106, 109, 118, 123, 126, 127, 132, 134, 135, 139, 140, 141, 156, 161, 164, 168, 170, 173, 181, 187, 189, 193, 194, 196, 198, 200,
  • amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 12, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 34/38/39/39/127/245/349/350/357, 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, 34/39/102/264/275/350
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 38, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 48/64/164/324/343/353/364, 48/64/164/324/343/364, 48/64/164/353/357/364, 48/64/357/364, 64/164/324/343/353/364, 64/164/324/343/357/364, 64/164/324/343/357/364, 64/164/318/324/357/364, 64/324/353/364, 132/255/339/379/395, 164/196/324/357/364, 164/318/324/324/364, 164/318/324/3
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 234, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 324/394/395, 324/395, 339, 340, 380, 382, 389, 389/394,
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 284, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 12/135/263/382, 12/259/263/304, 48/64/255, 64/69, 64/69/189/259/263/304,
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 484, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 3/304, 3/304, 3/304, 3/304, 3/304, 3/304, 3/304/324/339, 3/263/304/324/382, 3/304, 3/304, 3/304, 3/304
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 594, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 16/80/324, 16/91/126/168/324/366, 16/126/168/366, 16/168/259/366, 16/
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 686, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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/
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 686, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 688, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19/109/123/141/170/198/200/211/270/312, 19/109/123/141/170/198/211, 19/109/123/141/170/198/211/270/312, 19/109/123/170/211/270/312, 19/109/123/198/200/211/270/312, 19/109/170/173/211/270/312, 19/109/211/270/312, 109/170/211/270/312, or 109/211/270/312, wherein the amino acid positions
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 5/41, 5/41/228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 66, 66/118, 66/118/296, 66/118/296/300, 66/118/300, 66/296, 66/296/300, 66/300, 118, 118/296, 118/296/300, 118/300, 296, 296/300, or 300, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • 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 the reference sequence of SEQ ID NO: 828, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 170/270/383, 270, 41/173, 272, 5/141/272/383, 41/383, 41/141/187/272/290, 41/141/173/290, 5/272/383, 5/41/173/272/383, 41/141, 141/272, 353/384, 272/383, 41/141/173, 41/272/383, 41/141/187/200/202/272, 33/55/64/126/270/357, 33/126/353/357, 55/64/267
  • 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 the reference sequence of SEQ ID NO: 888, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 64/173/202/353/384, 41/141/272/353, 141/202/272/353/357, 173/202/357, 202/353, 5/51/173/272/353/384, 51/202/272/357, 141/173/272, 272, 41/173/384, 41/64/141/353/357/383, 141/173/202, 5/51/64/202/353, 357, 64, 5/41/141, 41/141/173/202/353, 353, 202/3
  • the engineered leucine decarboxylase polypeptide exhibits one or more improved properties as compared to the wild-type Planctomycetaceae species leucine decarboxylase or the engineered leucine decarboxylase having the sequence corresponding to SEQ ID NO: 12.
  • the improved property is selected from (i) increased activity on leucine, (ii) increased resistance to proteolysis, (iii) increased tolerance to low pH environments, or (iv) increased thermostability, or any combinations thereof, compared to the wild-type Planctomycetaceae species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase comprises at least one leucine decarboxylase polypeptide provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8- 2, 10-1, 11-1, 11-2, 12-1, and/or 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 2-1064.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 16- 1064.
  • FIGS. 1A and IB show results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylases in an intermediate MSUD mouse model. Treatment with engineered leucine decarboxylases following administration of whey protein meal results in suppression of plasma leucine levels.
  • FIG. 1A shows the time course of plasma leucine levels in animals treated with Vehicle, LDC polypeptide of SEQ ID NO: 484, LDC polypeptide of SEQ ID NO: 686, or LDC polypeptide of SEQ ID NO: 766. Data is represented as mean ⁇ SEM.
  • FIG. IB shows plasma leucine AUC. Data is represented as mean ⁇ SEM.
  • FIGS. 2A and 2B show results of pharmacodynamic (PD) dose-response study of LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model.
  • FIG. 2A shows the time course of plasma leucine levels in animals treated with Vehicle or three different doses of LDC of SEQ ID NO: 766. Data is represented as mean ⁇ SEM. Multiple t-test compared to vehicle: *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • FIG. 2B shows plasma leucine AUC.
  • One-way ANOVA compared to Vehicle: **, p ⁇ 0.01; **, p ⁇ 0.001; ***, p ⁇ 0.0001.
  • FIGS. 3A and 3B show results of a pharmacodynamic (PD) dose-response study comparing engineered LDC polypeptide of SEQ ID NO: 828 to engineered LDC polypeptide of SEQ ID NO: 766 in the iMSUD mouse model.
  • FIG. 3A shows plasma leucine time course for treatment with LDC polypeptide of SEQ ID NO: 766 or LDC polypeptide of SEQ ID NO: 828 following a whey protein meal. Data is represented as mean ⁇ SEM; Multiple t-tests compared to Vehicle: *, p ⁇ 0.05; **, p ⁇ 0.01; ***, pO.OOl l .
  • FIGS. 4A-4D shows pharmacodynamic (PD) dose-response study of engineered leucine decarboxylases in healthy cynomolgus monkeys.
  • FIG. 4A shows time course of plasma leucine levels following administration of an engineered LDC polypeptide of SEQ ID NO: 484 at three different doses. Data is shown as mean ⁇ SEM. Multiple t-tests compared to Vehicle: *, p ⁇ 0.05.
  • FIG. 4B shows time course of plasma leucine levels following administration of LDC polypeptide of SEQ ID NO: 686 at three different doses. Data is shown as mean ⁇ SEM. Multiple t-tests compared to Vehicle: *, p ⁇ 0.05.
  • FIG. 4C shows time course of plasma leucine levels following administration of LDC polypeptide of SEQ ID NO: 766 at three different doses. Data is shown as mean ⁇ SEM.
  • FIG. 4D show the corresponding plasma leucine AUC for the engineered leucine decarboxylases administered at different doses. Data is shown as mean ⁇ SEM.
  • FIGS. 5A-5D show results of pharmacodynamic (PD) dose-response and 3-day repeat dose studies with an engineered LDC polypeptide of SEQ ID NO: 766 in healthy cynomolgus monkeys.
  • FIG. 5A shows plasma leucine time course following treatment with three different doses of engineered LDC polypeptide of SEQ ID NO: 766. Data is shown as mean ⁇ SEM. Multiple t-tests compared to Vehicle: *, p ⁇ 0.05.
  • FIG. 5B shows corresponding plasma leucine iAUC. Data is shown as mean ⁇ SEM.
  • One- Way ANOVA vs Vehicle *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 5C show plasma ketoisocaproic acid (KIC) time course for three different doses of the engineered LDC polypeptide of SEQ ID NO: 766. Data is shown as mean ⁇ SEM. Multiple t-tests compared to Vehicle: *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 5D shows corresponding plasma KIC iAUC for the different doses of the engineered LDC polypeptide of SEQ ID NO: 766. Data is shown as mean ⁇ SEM. One-Way ANOVA vs Vehicle: **, p ⁇ 0.01.
  • FIGS. 6A and 6B show results of study examining efficacy of engineered LDC polypeptide of SEQ ID NO: 766 against leucine in a whey protein meal in healthy cynomolgus monkeys in a consecutive 3-day challenge.
  • FIG. 6A shows plasma leucine time course following treatment with engineered LDC polypeptide of SEQ ID NO: 766. Data is shown as mean ⁇ SEM; Multiple t-test *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • FIG. 6B show corresponding plasma leucine AUC. Data is shown as mean ⁇ SEM; Unpaired t-test; Vehicle v.
  • FIG. 6C shows plasma leucine time course with baseline subtracted. Data is shown as mean ⁇ SEM; Multiple t-test *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001; ****, p ⁇ 0.0001.
  • FIG. 6D shows corresponding plasma leucine iAUC (baseline subtracted). Data is shown as mean ⁇ SEM; Unpaired t-test; Vehicle v. Treatment *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIGS. 7A and 7B show results of study examining efficacy of the engineered LDC polypeptide of SEQ ID NO: 766 against methionine in a whey protein meal in healthy cynomolgus monkeys in a consecutive 3-day challenge.
  • FIG. 7A shows plasma methionine time course with baseline subtracted following treatment with engineered LDC polypeptide of SEQ ID NO: 766. Data is shown as mean ⁇ SEM; Multiple t-test *, p ⁇ 0.05; **, p ⁇ 0.01; ***, p ⁇ 0.001.
  • FIG. 7B shows corresponding plasma methionine iAUC (baseline subtracted). Data is shown as mean ⁇ SEM; Unpaired t-test; Vehicle v. Treatment *, p ⁇ 0.05; **, p ⁇ 0.01.
  • FIGS. 8A and 8B show results of a pharmacodynamic (PD) dose-response study of the effect of engineered LDC polypeptide in an intermediate MSUD mouse model.
  • FIG. 8A shows the time course of normalized plasma leucine levels in mice treated with Vehicle and LDC polypeptide of SEQ ID NO: 828 at 25, 50 and 100 mg/kg. Data is represented as mean ⁇ SEM. Multiple unpaired t-tests with Welch’s correction, compared to vehicle: *p ⁇ 0.05; **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • FIG. 8B shows plasma leucine iAUC. Data is represented as mean ⁇ SEM. One-way AN OVA with Tukey post-test compared to vehicle: **p ⁇ 0.01; ***p ⁇ 0.001.
  • FIGS. 9A and 9B show results of a pharmacodynamic (PD) study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys.
  • PD pharmacodynamic
  • FIG. 9A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and LDC polypeptide of SEQ ID NO: 828 at 6.25, 12.5 and 25 mg/kg. Data is represented as mean ⁇ SEM. Multiple unpaired t-tests with Welch’s correction, compared to vehicle: *p ⁇ 0.05;
  • FIG. 9B shows plasma leucine iAUC. Data is represented as mean ⁇ SEM. One-way ANOVA with Tukey post-test compared to Vehicle: *p ⁇ 0.05; ***p ⁇ 0.001.
  • FIGS. 10A, 10B, and 10C show results of a pharmacodynamic (PD) study of the effect of engineered LDC in healthy cynomolgus monkeys.
  • FIG. 10A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and LDC polypeptide of SEQ ID NO: 828 at 3.125, 6.25, 12.5 and 25 mg/kg. Data is represented as mean ⁇ SEM. Multiple unpaired t-tests with Welch’s correction, compared to vehicle: **p ⁇ 0.01; ***p ⁇ 0.001; ****p ⁇ 0.0001.
  • FIG. 10B shows plasma leucine iAUC. Data is represented as mean ⁇ SEM. One-way ANOVA with Tukey post-test compared to Vehicle: *p ⁇ 0.05; **p ⁇ 0.01.
  • FIG. 10C shows plasma KIC iAUC. Data is represented as mean ⁇ SEM. One-way ANOVA with Tukey posttest compared to Vehicle: *p ⁇ 0.05.
  • FIGS. 11A, 11B, and 11C show results of a pharmacodynamic (PD) 3 -day repeat dose study of the effect of engineered leucine decarboxylase (LDC) in healthy cynomolgus monkeys.
  • LDC leucine decarboxylase
  • FIG. 11A shows the time course of normalized plasma leucine levels in monkeys treated with vehicle and LDC polypeptide of SEQ ID NO: 828 at 6.25 mg/kg. Data is represented as mean ⁇ SEM. Multiple unpaired t-tests with Welch’s correction, compared to vehicle.
  • FIG. 11B shows plasma leucine iAUC. Data is represented as mean ⁇ SEM. One-way ANOVA with Tukey post-test compared to Vehicle: *p ⁇ 0.05; **, p ⁇ 0.01.
  • FIG. 11C shows plasma KIC iAUC. Data is represented as mean ⁇ SEM. One-way ANOVA with Tukey post-test compared to Vehicle: *p ⁇ 0.05.
  • the present disclosure provides engineered leucine decarboxylase (LDC) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered leucine decarboxylase polypeptides.
  • the engineered leucine decarboxylase polypeptides are engineered to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis, increased tolerance to low pH environments, and/or increased thermostability.
  • the engineered leucine decarboxylase polypeptides are evolved to provide improved storage stability.
  • the present disclosure also provides methods for the use of the engineered leucine decarboxylase polypeptides and compositions thereof for therapeutic and industrial purposes.
  • Numeric 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. It is also intended that every maximum (or minimum) numerical limitation disclosed herein includes every lower (or higher) numerical limitation, as if such lower (or higher) numerical limitations were expressly written herein.
  • the term “about” means an acceptable error 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.
  • 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.
  • ‘EC” number refers to the Enzyme Nomenclature of the Nomenclature Committee of the International Union of Biochemistry and Molecular Biology (NC-IUBMB).
  • the IUBMB biochemical classification is a numerical classification system for enzymes based on the chemical reactions they catalyze.
  • ATCC refers to the American Type Culture Collection whose biorepository collection includes genes and strains.
  • NCBI refers to National Center for Biological Information and the sequence databases provided therein.
  • LDC leucine decarboxylase polypeptide
  • PBP pyridoxal 5 ’-phosphate
  • Protein “Protein,” “polypeptide,” and “peptide” are used interchangeably herein to denote a polymer of at least two amino acids covalently linked by an amide bond, regardless of length or post- translational modification (e.g., glycosylation or phosphorylation).
  • Polynucleotide is used herein to denote a polymer comprising at least two nucleotides where the nucleotides are either deoxyribonucleotides or ribonucleotides.
  • amino acids are referred to herein by either their commonly known three-letter symbols or by the one-letter symbols recommended by IUPAC-IUB Biochemical Nomenclature Commission.
  • Nucleotides likewise, may be referred to by their commonly accepted single letter codes, as indicated
  • the abbreviations used for the genetically encoded amino acids are conventional and are as follows: alanine (Ala or A), arginine (Arg or R), asparagine (Asn or N), aspartate (Asp or D), cysteine (Cys or C), glutamate (Glu or E), glutamine (Gin or Q), glycine (Gly or G), histidine (His or H), isoleucine (He or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Vai or V).
  • a polynucleotide or a polypeptide refers to a material or a material corresponding to the natural or native form of the material that has been modified in a manner that would not otherwise exist in nature or is identical thereto but produced or derived from synthetic materials and/or by manipulation using recombinant techniques.
  • “recombinant LDC polypeptides” also referred to herein as “engineered LDC polypeptides,” “variant LDC enzymes,” and “LDC variants” are leucine decarboxylases made using recombinant techniques.
  • wild-type and “naturally-occurring” refer to the form found in nature.
  • a wild-type polypeptide or polynucleotide sequence is a sequence present in an organism that can be isolated from a source in nature and which has not been intentionally modified by human manipulation.
  • Coding sequence refers to that part of a nucleic acid (e.g., a gene) that encodes an amino acid sequence of a protein.
  • percent (%) sequence identity is used herein to refer to comparisons among polynucleotides and polypeptides, and are determined by comparing two optimally aligned sequences over a comparison window, wherein the portion of the polynucleotide or amino acid sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences.
  • the percentage may be calculated by determining the number of positions at which the identical nucleic acid base or amino acid residue occurs in both sequences to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • the percentage may be calculated by determining the number of positions at which either the identical nucleic acid base or amino acid residue occurs in both sequences or a nucleic acid base or amino acid residue is aligned with a gap to yield the number of matched positions, dividing the number of matched positions by the total number of positions in the window of comparison and multiplying the result by 100 to yield the percentage of sequence identity.
  • Optimal alignment of sequences for comparison can be conducted (e.g., by the local homology algorithm of Smith and Waterman; Smith and Waterman, Adv. Appl. Math., 1981, 2:482), by the homology alignment algorithm of Needleman and Wunsch (Needleman and Wunsch, J.
  • HSPs high scoring sequence pairs
  • Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always ⁇ 0).
  • a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative -scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the BLASTP program uses as defaults a wordlength (W) of 3, an expectation (E) of 10, and the BLOSUM62 scoring matrix (See e.g., Henikoff and Henikoff, Proc. Natl. Acad. Sci. USA, 1989, 89: 10915).
  • Exemplary determination of sequence alignment and % sequence identity can employ the BESTFIT or GAP programs in the GCG Wisconsin Software package (Accelrys, Madison WI), using default parameters provided.
  • reference sequence refers to a defined sequence used as a basis for a sequence comparison.
  • a reference sequence may be a subset of a larger sequence, for example, a segment of a full-length gene or amino acid sequence.
  • a reference sequence is at least 20 nucleotide 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 the nucleic acid or polypeptide.
  • two polynucleotides or polypeptides may each (1) comprise a sequence (i.e., a portion of the complete sequence) that is similar between the two sequences, and (2) may further comprise a sequence that is divergent between the two sequences
  • sequence comparisons between two (or more) polynucleotides or polypeptide are typically performed by comparing sequences of the two polynucleotides or polypeptides over a “comparison window” to identify and compare local regions of sequence similarity.
  • a “reference sequence” can be based on a primary amino acid sequence, where the reference sequence is a sequence that can have one or more changes in the primary sequence.
  • the phrase “reference sequence based on SEQ ID NO: 686 having a valine at the residue corresponding to “X123” refers to a reference sequence in which the corresponding residue at position X123 in SEQ ID NO: 686 (e.g., a tyrosine), has been changed to valine.
  • Comparison window refers to a conceptual segment of at least about 20 contiguous nucleotide positions or amino acids residues wherein a sequence may be compared to a reference sequence of at least 20 contiguous nucleotides or amino acids and wherein the portion of the sequence in the comparison window may comprise additions or deletions (i.e., gaps) of 20 percent or less as compared to the reference sequence (which does not comprise additions or deletions) for optimal alignment of the two sequences.
  • the comparison window can be longer than 20 contiguous residues, and includes, optionally 30, 40, 50, 100, or longer windows.
  • “Corresponding to”, “reference to,” and “relative to” when used in the context of the numbering of a given amino acid or polynucleotide sequence refer to the numbering of the residues of a specified reference sequence when the given amino acid or polynucleotide sequence is compared to the reference sequence.
  • the residue number or residue position of a given polymer is designated with respect to the reference sequence rather than by the actual numerical position of the residue within the given amino acid or polynucleotide sequence.
  • a given amino acid sequence such as that of an engineered leucine decarboxylase, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences. In these cases, although the gaps are present, the numbering of the residue in the given amino acid or polynucleotide sequence is made with respect to the reference sequence to which it has been aligned.
  • amino acid difference and “residue difference” refer to a difference in the amino acid residue at a position of an amino acid sequence relative to the amino acid residue at a corresponding position in a reference sequence.
  • the positions of amino acid differences generally are referred to herein as “Xn,” where n refers to the corresponding position in the reference sequence upon which the residue difference is based.
  • a “residue difference at position X123 as compared to SEQ ID NO:686” refers to a difference of the amino acid residue at the polypeptide position corresponding to position 123 of SEQ ID NO: 686.
  • a “residue difference at position X123 as compared to SEQ ID NO:686” refers to an amino acid substitution of any residue other than tyrosine at the position of the polypeptide corresponding to position 123 of SEQ ID NO: 686.
  • the specific amino acid residue difference at a position is indicated as “XnY” where “Xn” specified the corresponding residue and position of the reference polypeptide (as described above), and “Y” is the single letter identifier of the amino acid found in the engineered polypeptide (i.e., the different residue than in the reference polypeptide).
  • the original amino acid is not indicated (e.g., 123F).
  • the present disclosure also provides specific amino acid differences denoted by the conventional notation “AnB”, where A is the single letter identifier of the residue in the reference sequence, “n” is the number of the residue position in the reference sequence, and B is the single letter identifier of the residue substitution in the sequence of the engineered polypeptide.
  • a polypeptide of the present disclosure can include one or more amino acid residue differences relative to a reference sequence, which is indicated by a list of the specified positions where residue differences are present relative to the reference sequence.
  • 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 comprising one or more amino acid differences that include either/or both conservative and non-conservative amino acid substitutions.
  • amino acid substitution set and “substitution set” refers to a group of amino acid substitutions within an amino acid sequence.
  • substitution sets comprise 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, or more amino acid substitutions.
  • a substitution set refers to the set of amino acid substitutions that is present in any of the variant leucine decarboxylase polypeptides listed in any of the Tables in the Examples and Appendix (i.e., Tables 1-2, 2-1, 3-2, 4-1, 5- 1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and 12-1).
  • Constant amino acid substitution refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • an amino acid with an aliphatic side chain may be substituted with another aliphatic amino acid (e.g., alanine, valine, leucine, and isoleucine); an amino acid with hydroxyl side chain is substituted with another amino acid with a hydroxyl side chain (e.g., serine and threonine); an amino acid having aromatic side chains is substituted with another amino acid having an aromatic side chain (e.g., phenylalanine, tyrosine, tryptophan, and histidine); an amino acid with a basic side chain is substituted with another amino acid with a basic side chain (e.g., lysine and arginine); an amino acid with an acidic side chain is substituted with another aliphatic amino acid (e.g
  • Exemplary conservative substitutions include the 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).
  • other aliphatic residues e.g., A, L, V, I
  • Non-conservative substitution refers to substitution of an amino acid in the polypeptide with an amino acid with significantly differing side chain properties. Non-conservative substitutions may use amino acids between, rather than within, the defined groups and affect: (a) the structure of the peptide backbone in the area of the substitution (e.g., proline for glycine); (b) the charge or hydrophobicity; and/or (c) the bulk of the side chain.
  • 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.
  • ‘Deletion” refers to modification to the polypeptide by removal of one or more amino acids from the reference polypeptide.
  • Deletions can comprise removal of 1 or more amino acids, 2 or more amino acids, 5 or more amino acids, 10 or more amino acids, 15 or more amino acids, or 20 or more amino acids, up to 10% of the total number of amino acids, or up to 20% of the total number of amino acids making up the reference enzyme while retaining enzymatic activity and/or retaining the improved properties of an engineered leucine decarboxylase enzyme.
  • Deletions can be directed to the internal portions and/or terminal portions of the polypeptide.
  • the deletion can comprise a continuous segment or can be discontinuous.
  • Insertions refers to modification to the polypeptide by addition of one or more amino acids from the reference polypeptide. Insertions can be in the internal portions of the polypeptide, or to the carboxy or amino terminus. Insertions as used herein include fusion proteins as is known in the art. The insertion can be a contiguous segment of amino acids or separated by one or more of the amino acids in the naturally occurring polypeptide.
  • polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) 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 present invention) and that retains substantially all of the activity of the full-length polypeptide.
  • substantially all of the activity of the full-length polypeptide refers to at least 90% activity of the recombinant polypeptide from which it was derived.
  • isolated polypeptide refers to a polypeptide which is substantially separated from other contaminants that naturally accompany it (e.g., protein, lipids, and polynucleotides).
  • the term embraces polypeptides which have been removed or purified from their naturally-occurring environment or expression system (e.g., host cell or in vitro synthesis).
  • the recombinant leucine decarboxylase polypeptides may be present within a cell, present in the cellular medium, or prepared in various forms, such as lysates or isolated preparations.
  • the recombinant leucine decarboxylase polypeptides provided herein are isolated polypeptides.
  • substantially pure polypeptide refers to a composition in which the polypeptide species is the predominant species present (i.e., on a molar or weight basis it is more abundant than any other individual macromolecular species in the composition), and is generally a substantially purified composition when the object species comprises at least about 50 percent of the macromolecular species present by mole or % weight.
  • a substantially pure leucine decarboxylase composition will comprise about 60% or more, about 70% or more, about 80% or more, about 90% or more, about 95% or more, and about 98% or more of all macromolecular species by mole or % weight present in the composition.
  • the object species is purified to essential homogeneity (i.e., contaminant species cannot be detected in the composition by conventional detection methods) wherein the composition consists essentially of a single macromolecular species. Solvent species, small molecules ( ⁇ 500 Daltons), and elemental ion species are not considered macromolecular species.
  • the isolated recombinant leucine decarboxylase polypeptides are substantially pure polypeptide compositions.
  • “Improved enzyme property” in the context of an engineered leucine decarboxylase polypeptide refers to improvement in any enzyme property as compared to a reference leucine decarboxylase polypeptide, such as a wild-type leucine decarboxylase polypeptide (e.g., wild-type LDC having SEQ ID NO: 2) or another engineered leucine decarboxylase polypeptide.
  • a reference leucine decarboxylase polypeptide such as a wild-type leucine decarboxylase polypeptide (e.g., wild-type LDC having SEQ ID NO: 2) or another engineered leucine decarboxylase polypeptide.
  • Improved properties include but are not limited to such properties as increased protein production, increased serum stability, increased serum half-life in vivo, increased thermoactivity, 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 tolerance to proteolytic activity (i.e., reduced sensitivity to proteolysis), reduced aggregation, increased solubility, reduced immunogenicity (i.e., reduced capability of inducing and/or eliciting an immune response), and altered temperature profile.
  • properties include but are not limited to such properties as increased protein production, increased serum stability, increased serum half-life in vivo, increased thermoactivity, 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 chemose
  • ‘Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered leucine decarboxylase polypeptides, which can be represented by an increase in specific activity (e.g., product produced/time/weight protein) and/or an increase in percent conversion of the substrate to the product (e.g., percent conversion of starting amount of substrate to product in a specified time period using a specified amount of leucine decarboxylase) as compared to the reference leucine decarboxylase enzyme (e.g., wild-type leucine decarboxylase and/or another engineered leucine decarboxylase). Exemplary methods to determine enzyme activity are provided in the Examples.
  • Any property relating to enzyme activity may be affected, including the classical enzyme properties of Km, Vmax or kcat, changes of which can lead to increased enzymatic activity. Improvements in enzyme activity can be from about 1.1 fold the 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 than the naturally occurring leucine decarboxylase or another engineered leucine decarboxylase from which the leucine decarboxylase polypeptides were derived.
  • the engineered leucine decarboxylase polypeptides have a specific activity of at least 0.01 pmol/min-mg, at least 0.02/ pmol/min-mg, at least 0.03/ pmol/min-mg, at least 0.05/ pmol/min-mg, at least 1.0/ pmol/min-mg, and in some preferred embodiments greater than 2.0/ pmol/min-mg.
  • the Km is in the range of about 1 m to about 5mM; in the range of about 5pm to about 2mM; in the range of about 10pm to about 2mM; or in the range of about 10 pm to about ImM.
  • the engineered leucine decarboxylase enzyme exhibits improved enzymatic activity in the range of 1.5 to 10 fold, 1.5 to 25 fold, 1.5 to 50 fold, 1.5 to 100 fold or greater, than that of the reference leucine decarboxylase enzyme.
  • Leucine decarboxylase activity can be measured by any standard assay known in the art (e.g., by monitoring depletion of reactants or formation of products).
  • the amount of products produced or the amount of substrate consumed is measured by High-Performance Liquid Chromatography (HPLC) separation combined with UV absorbance or mass spectra detection.
  • HPLC High-Performance Liquid Chromatography
  • comparisons of enzyme activities are made using a defined preparation of enzyme, a defined assay under a set condition, and one or more defined substrates, as further described in detail herein.
  • a defined preparation of enzyme e.g., a defined assay under a set condition
  • one or more defined substrates e.g., one or more defined substrates
  • the phrase “increased storage stability” means that an engineered leucine decarboxylase polypeptide according to the invention will retain more activity compared to a reference leucine decarboxylase in a standard assay (e.g., as described in the Examples) after it has been produced in a dried form (e.g., by lyophilization or spray-drying), and stored for a period of time ranging from a few days to multiple months at a temperature above room temperature (e.g., 30 °C, 37 °C, 45 °C, 55 °C, etc.).
  • a standard assay e.g., as described in the Examples
  • a dried form e.g., by lyophilization or spray-drying
  • conversion refers to the enzymatic conversion (or biotransformation) of substrate(s) to the corresponding product(s).
  • Percent conversion refers to the percent of the substrate that is converted to the product within a period of time under specified conditions.
  • the “enzymatic activity” or “activity” of a leucine decarboxylase polypeptide can be expressed as “percent conversion” of the substrate to the product in a specific period of time.
  • Hybridization stringency relates to hybridization conditions, such as washing conditions, in the hybridization of nucleic acids. Generally, hybridization reactions are performed under conditions of lower stringency, followed by washes of varying but higher stringency.
  • hybridization refers to conditions that permit target-DNA to bind a complementary nucleic acid that has about 60% identity, preferably about 75% identity, about 85% identity to the target DNA, with greater than about 90% identity to target-polynucleotide.
  • Exemplary moderately stringent conditions are conditions equivalent to hybridization in 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in 0.2> ⁇ SSPE, 0.2% SDS, at 42 °C.
  • “High stringency hybridization” refers generally to conditions that are about 10 °C or less from the thermal melting temperature Tm as determined under the solution condition for a defined polynucleotide sequence.
  • a high stringency condition refers to conditions that permit hybridization of only those nucleic acid sequences that form stable hybrids in 0.018M NaCl at 65 °C (i.e., if a hybrid is not stable in 0.018M NaCl at 65 °C, it will not be stable under high stringency conditions, as contemplated herein).
  • High stringency conditions can be provided, for example, by hybridization in conditions equivalent to 50% formamide, 5x Denhart's solution, 5xSSPE, 0.2% SDS at 42 °C, followed by washing in O.l xSSPE, and 0.1% SDS at 65 °C.
  • Another high stringency condition is hybridizing in conditions equivalent to hybridizing in 5X SSC containing 0.1% (w:v) SDS at 65 °C and washing in O.lx SSC containing 0.1% SDS at 65 °C.
  • Other high stringency hybridization conditions, as well as moderately stringent conditions, are described in the references cited above.
  • a polynucleotide encoding a leucine decarboxylase hybridizes under high stringency conditions to an recombinant polynucleotide disclosed herein encoding an engineered decarboxylase polypeptide.
  • Codon optimized refers to changes in the codons of the polynucleotide encoding a protein to those preferentially used in a particular organism such that the encoded protein is more efficiently expressed in that organism.
  • the genetic code is degenerate, in that most amino acids are represented by several codons, called “synonyms” or “synonymous” codons, it is well known that codon usage by particular organisms is nonrandom and biased towards particular codon triplets. This codon usage bias may be higher in reference to a given gene, genes of common function or ancestral origin, highly expressed proteins versus low copy number proteins, and the aggregate protein coding regions of an organism's genome.
  • control sequence refers herein to include all components that are necessary or advantageous for the expression of a polynucleotide and/or polypeptide of the present disclosure.
  • Each control sequence may be native or foreign to the nucleic acid sequence encoding the polypeptide.
  • control sequences include, but are not limited to, leaders, polyadenylation sequences, propeptide sequences, promoter sequences, signal peptide sequences, initiation sequences, and transcription terminators.
  • the control sequences include a promoter, and transcriptional and translational stop signals.
  • the control sequences are provided with linkers for the purpose of introducing specific restriction sites facilitating ligation of the control sequences with the coding region of the nucleic acid sequence encoding a polypeptide.
  • “Operably linked” is defined herein as a configuration in which a control sequence is appropriately placed (i.e., in a functional relationship) at a position relative to a polynucleotide of interest such that the control sequence directs or regulates the expression of the polynucleotide encoding a polypeptide of interest.
  • ‘ ‘Promoter sequence” refers to a nucleic acid sequence that is recognized by a host cell for expression of a polynucleotide of interest, such as a coding sequence. The promoter sequence contains transcriptional control sequences that mediate the expression of a polynucleotide of interest.
  • the promoter may be any nucleic acid sequence which shows transcriptional activity in the host cell of choice including mutant, truncated, and hybrid promoters, and may be obtained from genes encoding extracellular or intracellular polypeptides either homologous or heterologous to the host cell.
  • Substrate in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the leucine decarboxylase polypeptide.
  • Process in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the leucine decarboxylase polypeptide on the substrate.
  • culturing refers to the growing of a population of cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).
  • suitable medium e.g., liquid, gel, or solid.
  • Recombinant polypeptides can be produced using any suitable methods known in the art. For example, there is a wide variety of different mutagenesis techniques well known to those skilled in the art. In addition, mutagenesis kits are also available from many commercial molecular biology suppliers. Methods are available to make specific substitutions at defined amino acids (site-directed), specific or random mutations in a localized region of the gene (regio-specific), or random mutagenesis over the entire gene (e.g., saturation mutagenesis).
  • the variants After the variants are produced, they can be screened for any desired property (e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability, and/or acidic pH stability, etc.).
  • desired property e.g., high or increased activity, or low or reduced activity, increased thermal activity, increased thermal stability, and/or acidic pH stability, etc.
  • suitable reaction conditions refers to those conditions in the enzymatic conversion reaction solution (e.g., ranges of enzyme loading, substrate loading, temperature, pH, buffers, co-solvents, etc.) under which a leucine decarboxylase polypeptide of the present application is capable of converting a substrate to the desired product compound.
  • exemplary “suitable reaction conditions” are provided in the present application and illustrated by the Examples.
  • “Loading”, such as in “compound loading” or “enzyme loading” refers to the concentration or amount of a component in a reaction mixture at the start of the reaction.
  • Substrate in the context of an enzymatic conversion reaction process refers to the compound or molecule acted on by the leucine decarboxylase polypeptide.
  • Process in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the leucine decarboxylase polypeptide on a substrate.
  • a "vector” is a DNA construct for introducing a DNA sequence into a cell.
  • the vector is an expression vector that is operably linked to a suitable control sequence capable of effecting the expression in a suitable host of the polypeptide encoded in the DNA sequence.
  • an "expression vector” has a promoter sequence operably linked to the DNA sequence (e.g., transgene) to drive expression in a host cell, and in some embodiments, also comprises a transcription terminator sequence.
  • the term "expression” includes any step involved in the production of the polypeptide including, but not limited to, transcription, post-transcriptional modification, translation, and post-translational modification. In some embodiments, the term also encompasses secretion of the polypeptide from a cell.
  • an amino acid or nucleotide sequence e.g., a promoter sequence, signal peptide, terminator sequence, etc.
  • a promoter sequence e.g., a promoter sequence, signal peptide, terminator sequence, etc.
  • the terms “host cell” and “host strain” refer to suitable hosts for expression vectors comprising DNA provided herein (e.g., a polynucleotide sequences encoding at least one LDC variant).
  • the host cells are prokaryotic or eukaryotic cells that have been transformed or transfected with vectors constructed using recombinant DNA techniques as known in the art.
  • analogue means a polypeptide having more than 70% sequence identity but less than 100% sequence identity (e.g., more than 75%, 78%, 80%, 83%, 85%, 88%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% sequence identity) with a reference polypeptide.
  • analogues refer to non-naturally occurring amino acid residues including, but not limited, to homoarginine, ornithine and norvaline, as well as naturally occurring amino acids.
  • analogues also include one or more D-amino acid residues and non-peptide linkages between two or more amino acid residues.
  • terapéutica refers to a compound administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.
  • composition refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of an engineered leucine decarboxylase polypeptide encompassed by the invention and an acceptable carrier.
  • genes i.e., genetic material
  • 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 specific method(s) or composition(s) useful for gene therapy.
  • 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., human).
  • mRNA messenger RNA
  • 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 specific method(s) or composition(s) useful for mRNA therapy.
  • the term “effective amount” means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount in view of the guidance in the specification.
  • isolated and purified are used to refer to a molecule (e.g., an isolated nucleic acid, polypeptide, etc.) or other component that is removed from at least one other component with which it is naturally associated.
  • purified does not require absolute purity, rather it is intended as a relative definition.
  • subject encompasses mammals such as humans, non-human primates, livestock, companion animals, and laboratory animals (e.g., rodents and lagomorphs). It is intended that the term encompass females as well as males.
  • patient means any subject that is being assessed for, treated for, or is experiencing disease.
  • infant refers to a child in the period of the first month after birth to approximately one (1) year of age.
  • newborn refers to child in the period from birth to the 28th day of life.
  • premature infant refers to an infant bom after the twentieth completed week of gestation, yet before full term, generally weighing -500 to -2499 grams at birth.
  • a “very low birth weight infant” is an infant weighing less than 1500 g at birth.
  • the term “child” refers to a person who has not attained the legal age for consent to treatment or research procedures. In some embodiments, the term refers to a person between the time of birth and adolescence.
  • the term “adult” refers to a person who has attained legal age for 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 less than 18 years of age, but who has reached sexual maturity.
  • compositions and “formulation” encompass products comprising at least one engineered leucine decarboxylase of the present invention, intended for any suitable use (e.g., pharmaceutical compositions, dietary/nutritional supplements, feed, etc.).
  • administration and “administering” a composition mean providing a composition of the present invention to a subject (e.g., to a person suffering from the effects of MSUD).
  • carrier when used in reference to a pharmaceutical composition means any of the standard pharmaceutical carrier, buffers, and excipients, such as stabilizers, preservatives, and adjuvants.
  • pharmaceutically acceptable means a material that can be administered to a subject without causing any undesirable biological effects or interacting in a deleterious manner with any of the components in which it is contained and that possesses the desired biological activity.
  • excipient refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the engineered leucine decarboxylase polypeptides of the present invention). Excipients are typically included for formulation and/or administration purposes.
  • terapéuticaally effective amount when used in reference to symptoms of disease/condition refers to the amount and/or concentration of a compound (e.g., engineered LDC polypeptides) that ameliorates, attenuates, or eliminates one or more symptom of a disease/condition or prevents or delays the onset of symptom(s) (e.g., MSUD).
  • a compound e.g., engineered LDC polypeptides
  • the term is use in reference to the amount of a composition that elicits the biological (e.g., medical) response by a tissue, system, or animal subject that is sought by the researcher, physician, veterinarian, or other clinician.
  • terapéuticaally effective amount when used in reference to a disease/condition refers to the amount and/or concentration of a composition that ameliorates, attenuates, or eliminates the disease/condition.
  • treating encompass preventative (e.g., prophylactic), as well as palliative treatment.
  • the term “at least one” is not intended to limit the invention to any particular number of items. It is intended to encompass one, two, three, four, five, six, seven, eight, nine, ten, or more items, as desired.
  • the present disclosure provides engineered leucine decarboxylases (LDC) and their use as a pharmaceutical, nutraceutical, and/or industrial applications.
  • LDC leucine decarboxylases
  • a particular leucine decarboxylase variant i.e., an engineered LDC polypeptide
  • LDC polypeptide i.e., an engineered LDC polypeptide
  • variants of another leucine decarboxylase modified in the equivalent position(s) are encompassed herein.
  • the parent leucine decarboxylase polypeptides from which the engineered leucine decarboxylase polypeptides of the invention are derived from include bacterial strains such as those in the Planctomycetaceae family bacteria. Leucine decarboxylase from different sources and applicable to the uses herein are provided in Table 1-2.
  • the engineered leucine decarboxylase polypeptides are produced by cultivating cells or microorganisms comprising at least one polynucleotide sequence encoding at least one engineered leucine decarboxylase polypeptide under conditions which are conducive for producing the engineered leucine decarboxylase polypeptide. In some embodiments, the engineered leucine decarboxylase polypeptide is subsequently recovered from the resulting culture medium and/or cells.
  • the engineered leucine decarboxylase polypeptides of the present disclosure comprise amino acid sequences having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828 or 888, wherein the amino acid sequence comprises one or more substitutions in its amino acid sequence.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to SEQ ID NO: 828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to SEQ ID NO: 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution 5V, 19L, 33L, 41D, 47F, 5 IE, 551, 64S/N, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L/T, 272A, 2901, 312T, 353E, 357S/C, 383S, or 384W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at position 33, 55, 64, 126, 270, or 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution 33L, 551, 64N, 126A, 270L, or 357S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at position 33, 55, 64, 126, 270, and 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution 33L, 551, 64N, 126A, 270L, and 357S, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 170/270/383, 270, 41/173, 272, 5/141/272/383, 41/383, 41/141/187/272/290, 41/141/173/290, 5/272/383, 5/41/173/272/383, 41/141, 141/272, 353/384, 272/383, 41/141/173, 41/272/383, 41/141/187/200/202/272, 33/55/64/126/270/357, 33/126/353/357, 55/64/267/35/384, 33/64/357, 126/267, 64/267/353/384, 33/55/64/357, 19/64/126/267, 55/267, 33/126/267/270/312/357, 19/33/55/353/357/384, 19/33/126, 126/312, 126/198/202/267/312,
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 170P/270L/383S, 270L, 41D/173I, 272A, 5V/141P/272A/383S, 41D/383S, 41D/141P/187L/272A/290I, 41D/141P/173I/290I, 5V/272A/383S, 5V/41D/173I/272A/383S, 41D/141P, 141P/272A, 353E/384W, 272A/383S, 41D/141P/173I, 41D/272A/383S, 41D/141P/187L/200S/202H/272A, 33L/55I/64N/126A/270L/357S, 33L/126A/353E/357S, 55I/64N/267L/353E/384W, 33L/64N/357S, 126A/267L, 64
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set A170P/R270L/A383S, R270L, H41D/F173I, T272A, K5V/R141P/T272A/A383S, H41D/A383S, H41D/R141P/V187L/T272A/V290I, H41D/R141P/F173I/V290I, K5V/T272A/A383S, K5V/H41D/F173I/T272A/A383S, H41D/R141P, R141P/T272A, D353E/P384W, T272A/A383S, H41D/R141P/F173I, H41D/T272A/A383S, H41D/R141P/V187L/H200S/S202H/
  • the engineered leucine decarboxylase comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises amino acid residue 5V, 19L, 33L, 41D, 47F, 5 IE, 551, 64S/N, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L/T, 272A, 2901, 312T, 353E, 357S/C, 383S, or 384W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position: 64/173/202/353/384, 41/141/272/353, 141/202/272/353/357, 173/202/357, 202/353, 5/51/173/272/353/384, 51/202/272/357, 141/173/272, 272, 41/173/384, 41/64/141/353/357/383, 141/173/202, 5/51/64/202/353, 357, 64, 5/41/141, 41/141/173/202/353, 353, 202/357, 51/141/202/272/353, 202, 51/141/173/353/384, 41/141/173/202/272/353/383/384, 64/202/357, 5/64/353/383/384, 41/272/353/383, 41/173/272/353/357, 51/141/272/353/357/383/384
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 64S/173I/202H/353E/384W, 41D/141P/272A/353E, 141P/202H/272A/353E/357C, 173I/202H/357C, 202H/353E, 5V/51E/173I/272A/353E/384W, 51E/202H/272A/357C, 141P/173I/272A, 272A, 41D/173I/384W, 41D/64S/141P/353E/357C/383S, 141P/173I/202H, 5V/51E/64S/202H/353E, 357C, 64S, 5V/41D/141P, 41D/141P/173I/202H/353E, 353E, 202H/357C, 51E/141P/202H/272A/353E, 202H, 51
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set N64S/F173I/S202H/D353E/P384W, H41D/R141P/T272A/D353E, R141P/S202H/T272A/D353E/S357C, F173I/S202H/S357C, S202H/D353E, K5V/L51E/F173I/T272A/D353E/P384W, L51E/S202H/T272A/S357C, R141P/F173I/T272A, T272A, H41D/F173I/P384W, H41D/N64S/R141P/D353E/S357C/A383S, R141P/F173I/S202H, K5V/L51E/N64S/F173I/S202H
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising a substitution or substitution set of a variant provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising a substitution in at least one amino acid position provided in Tables 12-1 and 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least one substitution provided in Tables 12-1 and 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising at least a substitution or substitution set of a variant provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising the sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • SEQ ID NOS: 854-1064 e.g., 854, 856, 858, 860, 862, 864, 868, 870, 872, 874, 876, 878, , 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOS: 854-948.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOS: 950-1604.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-1064. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-1064, wherein the amino acid sequence optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10. In some embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-948.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-948, wherein the polypeptide optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 950- 1604.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO.
  • the polypeptide optionally has 1, 2, 3, 4, 5, 6, 7, 8, 9, or up to 10 substitutions in the amino acid sequence.
  • the engineered leucine decarboxylase polypeptide has 1, 2, 3, 4, or up to 5 substitutions in the amino acid sequence.
  • the engineered leucine decarboxylase polypeptide has 1, 2, 3, or 4 substitutions in the amino acid sequence.
  • the substitutions comprise non-conservative of conservative substitutions.
  • the substitutions comprise conservative substitutions.
  • the substitutions comprise nonconservative and conservative substitutions.
  • guidance on non-conservative and conservative substitutions are provided by the variants disclosed herein.
  • the engineered leucine decarboxylase polypeptide of the present disclosure exhibits one or more improved properties as compared to the wild-type Planctomycetaceae species leucine decarboxylase or the leucine decarboxylase having the sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide exhibits an improved property selected from (i) increased activity on leucine, (ii) increased resistance to proteolysis, increased tolerance to low pH environments, and (iii) increased thermostability compared to the wildtype Planctomycetaceae species leucine decarboxylase or the leucine decarboxylase having the sequence corresponding to SEQ ID NO: 12.
  • This structure-function correlation information is provided in the form of specific amino acid residue differences relative to the reference engineered polypeptide of SEQ ID NO: 12, as well as associated experimentally determined activity data for the exemplary engineered leucine decarboxylase polypeptides, thereby providing guidance on preparation of engineered leucine decarboxylase polypeptides having the enzyme properties described herein.
  • the engineered leucine decarboxylase polypeptide is purified. In some embodiments, the engineered leucine decarboxylase polypeptide has leucine decarboxylase activity, particularly with the improved or enhanced properties described herein.
  • the present invention provides functional fragments or biologically active fragments of an engineered leucine decarboxylase polypeptide.
  • the 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 leucine decarboxylase polypeptide from which it was derived (i.e., the parent engineered LDC).
  • 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 leucine decarboxylase.
  • the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, and less than 50 amino acids.
  • functional fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered leucine decarboxylase.
  • the functional fragment is truncated by less than 5, less than 10, less than 15, less than 10, less than 25, less than 30, less than 35, less than 40, less than 45, less than 50, less than 55, less than 60, less than 65, or less than 70 amino acids.
  • the functional fragments or biologically active fragments of the engineered leucine decarboxylase polypeptide described herein include at least a mutation or mutation set in the amino acid sequence of the engineered leucine decarboxylase described herein. Accordingly, in some embodiments, the functional fragments or biologically active fragments of the engineered leucine decarboxylase displays the enhanced or improved property associated with the mutation or mutation set in the parent leucine decarboxylase.
  • the present disclosure provides engineered or recombinant polynucleotides encoding the engineered leucine decarboxylase polypeptides described herein.
  • the polynucleotides are operatively linked to one or more heterologous regulatory sequences that control gene expression to create a recombinant polynucleotide capable of expressing the polypeptide.
  • expression constructs containing at least one heterologous polynucleotide encoding the engineered leucine decarboxylase polypeptide(s) is introduced into appropriate host cells to express the corresponding leucine decarboxylase polypeptide(s).
  • the present invention provides methods and compositions for the production of each and every possible variation of leucine decarboxylase polynucleotides that could be made that encode the leucine decarboxylase polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations of polynucleotides are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Tables 12-1 and/or 12-2).
  • the codons are preferably optimized for utilization by the chosen host cell for protein production.
  • preferred codons used in bacteria are typically used for expression in bacteria. Consequently, codon optimized polynucleotides encoding the engineered leucine decarboxylase polypeptides contain preferred codons at about 40%, 50%, 60%, 70%, 80%, 90%, or greater than 90% of the codon positions in the full-length coding region.
  • the recombinant polynucleotide encodes an engineered polypeptide having leucine decarboxylase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to SEQ ID NO: 828 or 888, wherein the amino acid sequence comprises one or more substitutions relative to a reference sequence corresponding to SEQ ID NO: 828 or 888, or the amino acid sequence of any variant as disclosed in the Examples.
  • the recombinant polynucleotide encodes an engineered polypeptide having leucine decarboxylase activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%,
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%,
  • the recombinant polynucleotide encodes the engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%,
  • the recombinant polynucleotide encodes the engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the recombinant polynucleotide encodes the engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least a substitution or substitution set at amino acid position 170/270/383, 270, 41/173, 272, 5/141/272/383, 41/383, 41/141/187/272/290, 41/141/173/290, 5/272/383, 5/41/173/272/383, 41/141, 141/272, 353/384, 272/383, 41/141/173, 41/272/383, 41/141/187/200/202/272, 33/55/64/126/270/357, 33/126/353/357, 55/64/267/35/384, 33/64/357, 126/267, 64/267/353/384, 33/55/64/357, 19/64/126/267, 55/267, 33/126/267/270/312/357, 19/33/55/353/357/384, 19/33/126,
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase comprising an amino acid sequence comprising at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set at amino acid position 64/173/202/353/384, 41/141/272/353, 141/202/272/353/357, 173/202/357, 202/353, 5/51/173/272/353/384, 51/202/272/357, 141/173/272, 272, 41/173/384, 41/64/141/353/357/383, 141/173/202, 5/51/64/202/353, 357, 64, 5/41/141, 41/141/173/202/353, 353, 202/357, 51/141/202/272/353, 202, 51/141/173/353/384, 41/141/173/202/272/353/383/384, 64/202/357, 5/64/353/383/384, 41/272/353/383, 41/173/272/353
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising a substitution or substitution set of a variant provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising a substitution in at least one amino acid position provided in Tables 12-1 and 12-2.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least one substitution provided in Tables 12-1 and 12-2.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising at least a substitution or substitution set provided in Tables 12-1 and 12-2, wherein the substitution or substitution set is relative to SEQ ID NO: 828 or 888.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence comprising a sequence of an engineered leucine decarboxylase provided in Tables 12-1 and 12-2.
  • the recombinant polynucleotide encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence corresponding to an even-numbered SEQ ID NO.
  • SEQ ID NOS: 854-1064 e.g., 854, 856, 858, 860, 862, 864, 868, 870, 872, 874, 876, 878, , 880, 882, 884, 886, 888, 890, 892, 894, 896, 898, 900, 902, 904, 906, 908, 910, 912, 914, 916, 918, 920, 922, 924, 926, 928, 930, 932, 934, 936, 938, 940, 942, 944, 946, 948, 950, 952, 954, 956, 958, 960, 962, 964, 966, 968, 970, 972, 974, 976, 978, 980, 982, 984, 986, 988, 990, 992, 994, 996, 998, 1000, 1002, 1004, 1006, 1008, 1010, 1012, 1014, 1016, 1018, 1020, 1022
  • the recombinant polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93% ;
  • the recombinant polynucleotide comprises a nucleic acid sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to an odd numbered SEQ ID NO.
  • SEQ ID NOS: 853-1063 e.g., 853, 855, 857, 859, 861, 863, 867, 869, 871, 873, 875, 877, 879, 881, 883, 885, 887, 889, 891, 893, 895, 897, 899, 901, 903, 905, 907, 909, 911,
  • polynucleotide encodes a polypeptide with leucine decarboxylase activity.
  • the recombinant polynucleotide comprises a nucleic acid sequence comprising an odd numbered SEQ ID NO. of SEQ ID NOS: 853-1063 (e.g., 853, 855, 857, 859, 861,
  • the recombinant polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence encoding an engineered leucine decarboxylase polypeptide.
  • the reference sequence is selected from SEQ ID NOS: 827 or 887, or a complement thereof, or a polynucleotide sequence encoding any of the engineered leucine decarboxylase polypeptides provided herein.
  • the recombinant polynucleotide capable of hybridizing under highly stringent conditions encodes an engineered leucine decarboxylase polypeptide comprising an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 828 or 888, at residue positions selected from any positions as set forth in Tables 12-1 and 12-2.
  • the recombinant polynucleotides are capable of hybridizing under highly stringent conditions to a reference recombinant polynucleotide selected from those provided in Tables 12-1 and 12-2, or comprises a polynucleotide having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference nucleic acid sequence corresponding to SEQ ID NO: 827 or 887.
  • the recombinant polynucleotide hybridizing under highly stringent conditions comprises a sequence having at least 70%, 75%, 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to at least one polynucleotide reference sequence provided in Tables 12-1 and 12-2 (e.g., a nucleic acid sequence comprising an odd-numbered SEQ ID NO, of SEQ ID NOS: 853-1063), wherein the recombinant polynucleotide encodes a polypeptide having leucine decarboxylase activity.
  • Tables 12-1 and 12-2 e.g., a nucleic acid sequence comprising an odd-numbered SEQ ID NO, of SEQ ID NOS: 853-1063
  • an isolated polynucleotide encoding any of the engineered leucine decarboxylase polypeptides herein is manipulated in a variety of ways to facilitate expression of the leucine decarboxylase polypeptide.
  • the polynucleotides encoding the leucine decarboxylase polypeptides comprise expression vectors where one or more control sequences is present to regulate the expression of the leucine decarboxylase polynucleotides and/or polypeptides. Manipulation of the isolated polynucleotide prior to its insertion into a vector may be desirable or necessary depending on the expression vector utilized.
  • control sequences include among others, promoters, leader sequences, polyadenylation sequences, propeptide sequences, signal peptide sequences, and transcription terminators.
  • suitable promoters are selected based on the host cells selection.
  • suitable promoters for directing transcription of the nucleic acid constructs of the present disclosure include, but are not limited to promoters obtained from the E.
  • Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc. Natl Acad. Sci.
  • promoters for filamentous fungal host cells include, but are not limited to promoters obtained from the genes for Aspergillus oryzae TAKA amylase, Rhizomucor miehei aspartic proteinase, Aspergillus niger neutral alpha-amylase, Aspergillus niger acid stable alphaamylase, Aspergillus niger or Aspergillus awamori glucoamylase (glaA), Rhizomucor miehei lipase, Aspergillus oryzae alkaline protease, Aspergillus oryzae triose phosphate isomerase, Aspergillus nidulans acetamidase, and Fusarium oxysporum
  • Exemplary yeast cell promoters can be from the genes can be from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae galactokinase (GALI), Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase (ADH2/GAP), and Saccharomyces cerevisiae 3 -phosphoglycerate kinase.
  • ENO-1 Saccharomyces cerevisiae enolase
  • GALI Saccharomyces cerevisiae galactokinase
  • ADH2/GAP Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3-phosphate dehydrogenase
  • Saccharomyces cerevisiae 3 -phosphoglycerate kinase Other useful promoters for yeast host cells are known in the art (See e.g
  • control sequence is also a suitable transcription terminator sequence (i.e., a sequence recognized by a host cell to terminate transcription).
  • the terminator sequence is operably linked to the 3' terminus of the nucleic acid sequence encoding the leucine decarboxylase polypeptide.
  • Any suitable terminator which is functional in the host cell of choice finds use in the present invention.
  • the transcription terminators can be a Rho- dependent terminators that rely on a Rho transcription factor, or a Rho-independent, or intrinsic terminators, which do not require a transcription factor. Exemplary bacterial transcription terminators are described in Peters et al., J Mol Biol., 2011, 412(5):793-813.
  • 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 alphaglucosidase, 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).
  • control sequence is also a suitable leader sequence (i.e., a nontranslated region of an mRNA that is important for translation by the host cell).
  • the leader sequence is operably linked to the 5' terminus of the nucleic acid sequence encoding the leucine decarboxylase polypeptide.
  • Any suitable leader sequence that is functional in the host cell of choice find use in the present invention.
  • Exemplary leaders for filamentous fungal host cells are obtained from the genes for Aspergillus oryzae TAKA amylase, and Aspergillus nidulans triose phosphate isomerase.
  • Suitable leaders for yeast host cells are obtained from the genes for Saccharomyces cerevisiae enolase (ENO-1), Saccharomyces cerevisiae 3 -phosphoglycerate kinase, Saccharomyces cerevisiae alpha-factor, and Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase (ADH2/GAP).
  • ENO-1 Saccharomyces cerevisiae enolase
  • Saccharomyces cerevisiae 3 -phosphoglycerate kinase Saccharomyces cerevisiae alpha-factor
  • Saccharomyces cerevisiae alcohol dehydrogenase/glyceraldehyde-3 -phosphate dehydrogenase ADH2/GAP
  • control sequence is also a polyadenylation sequence (i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA).
  • a polyadenylation sequence i.e., a sequence operably linked to the 3' terminus of the nucleic acid sequence and which, when transcribed, is recognized by the host cell as a signal to add polyadenosine residues to transcribed mRNA.
  • 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 alphaglucosidase.
  • Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Bio., 1995, 15:5983-5990).
  • control sequence is also a signal peptide (i.e., a coding region that codes for an amino acid sequence linked to the amino terminus of a polypeptide and directs the encoded polypeptide into the cell's secretory pathway).
  • the 5' end of the coding sequence of the nucleic acid sequence inherently contains a signal peptide coding region naturally linked in translation reading frame with the segment of the coding region that encodes the secreted polypeptide.
  • 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 which directs the expressed polypeptide into the secretory pathway of a host cell of choice finds use for expression of the engineered polypeptide(s).
  • Effective signal peptide coding regions for bacterial host cells are the signal peptide coding regions include, but are not limited to those obtained from the genes for Bacillus NC1B 11837 maltogenic amylase, Bacillus stearothermophilus alpha- amylase, Bacillus licheniformis subtilisin, Bacillus licheniformis beta-lactamase, Bacillus stearothermophilus neutral proteases (nprT, nprS, nprM), and Bacillus subtilis prsA.
  • effective signal peptide coding regions for filamentous fungal host cells include, but are not limited to the signal peptide coding regions obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger neutral amylase, Aspergillus niger glucoamylase, Rhizomucor miehei aspartic proteinase, Humicola insolens cellulase, and Humicola lanuginosa lipase.
  • Useful signal peptides for yeast host cells include, but are not limited to those from the genes for Saccharomyces cerevisiae alpha-factor and Saccharomyces cerevisiae invertase.
  • control sequence is also a propeptide coding region that codes for an amino acid sequence positioned at the amino terminus of a polypeptide.
  • the resultant polypeptide is referred to as a “proenzyme,” “propolypeptide,” or “zymogen.”
  • a propolypeptide can be converted to a mature active polypeptide by catalytic or autocatalytic cleavage of the propeptide from the propolypeptide.
  • the propeptide coding region may be obtained from any suitable source, including, but not limited to the genes for Bacillus subtilis alkaline protease (aprE), Bacillus subtilis neutral protease (nprT), Saccharomyces cerevisiae alpha-factor, Rhizomucor miehei aspartic proteinase, and Myceliophthora thermophila lactase (See e.g., WO 95/33836). Where both signal peptide and propeptide regions are present at the amino terminus of a polypeptide, the propeptide region is positioned next to the amino terminus of a polypeptide and the signal peptide region is positioned next to the amino terminus of the propeptide region.
  • aprE Bacillus subtilis alkaline protease
  • nprT Bacillus subtilis neutral protease
  • Saccharomyces cerevisiae alpha-factor e.g., Rhizomucor miehe
  • regulatory sequences are also utilized. These sequences facilitate the regulation of the expression of the polypeptide relative to the growth of the host cell. Examples of regulatory systems are those that cause the expression of the gene to be turned on or off in response to a chemical or physical stimulus, including the presence of a regulatory compound.
  • suitable regulatory sequences include, but are not limited to the lac, tac, and trp operator systems.
  • suitable regulatory systems include, but are not limited to the ADH2 system or GALI system.
  • suitable regulatory sequences include, but are not limited to the TAKA alpha-amylase promoter, Aspergillus niger glucoamylase promoter, and Aspergillus oryzae glucoamylase promoter.
  • the present invention is directed to a recombinant expression vector comprising a polynucleotide encoding an engineered leucine decarboxylase polypeptide, and one or more expression regulating regions such as a promoter and a terminator, a replication origin, etc., depending on the type of hosts into which they are to be introduced.
  • the various nucleic acid and control sequences described herein are joined together to produce recombinant expression vectors which include one or more convenient restriction sites to allow for insertion or substitution of the nucleic acid sequence encoding the leucine decarboxylase polypeptide at such sites.
  • the nucleic acid sequence of the present invention is expressed by inserting the nucleic acid sequence or a nucleic acid construct comprising the sequence into an appropriate vector for expression.
  • the coding sequence is located in the vector so that the coding sequence is operably linked with the appropriate control sequences for expression.
  • the recombinant expression vector may be any suitable vector (e.g., a plasmid or virus), that can be conveniently subjected to recombinant DNA procedures and bring about the expression of the leucine decarboxylase polynucleotide sequence.
  • a suitable vector e.g., a plasmid or virus
  • the choice of the vector typically depends on the compatibility of the vector with the host cell into which the vector is to be introduced.
  • the vectors may be linear or closed circular plasmids.
  • the expression vector is an autonomously replicating vector (i.e., a vector that exists as an extra-chromosomal entity, the replication of which is independent of chromosomal replication, such as a plasmid, an extra-chromosomal element, a minichromosome, or an artificial chromosome).
  • the vector may contain any means for assuring self-replication.
  • the vector is one in which, when introduced into the host cell, it is integrated into the genome and replicated together with the chromosome(s) into which it has been integrated.
  • a single vector or plasmid, or two or more vectors or plasmids which together contain the total DNA to be introduced into the genome of the host cell, and/or a transposon is utilized.
  • the expression vector contains one or more selectable markers, which permit easy selection of transformed cells.
  • a “selectable marker” is a gene, the product of which provides for biocide or viral resistance, resistance to heavy metals, prototrophy to auxotrophs, and the like.
  • Examples of bacterial selectable markers include, but are not limited to the dal genes from Bacillus subtilis or Bacillus licheniformis, or markers, which confer antibiotic resistance such as ampicillin, kanamycin, chloramphenicol or tetracycline resistance.
  • Suitable markers for yeast host cells include, but are not limited to ADE2, HIS3, LEU2, LYS2, MET3, TRP1, and URA3.
  • Selectable markers for use in filamentous fungal host cells include, but are not limited to, amdS (acetamidase; e.g., from A. nidulans or A. orzyae).
  • argB ornithine carbamoyltransferases
  • bar phosphinothricin acetyltransferase; e.g., from .S'. hygroscopicus
  • hph hygromycin phosphotransferase
  • niaD nitrate reductase
  • pyrG orotidine-5 1 - phosphate decarboxylase; e.g., from A. nidulans or A. orzyae
  • sC sulfate adenyltransferase
  • trpC anthranilate synthase
  • the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered leucine decarboxylase enzyme(s) in the host cell.
  • Host cells suitable for use in expressing the polypeptides encoded by the expression vectors of the present 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 (AfhuA) and BL21).
  • the present invention provides methods of producing the engineered leucine decarboxylase polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered leucine decarboxylase polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise recovering the engineered leucine decarboxylase polypeptide from the culture and/or host cells. In some embodiments, the methods further comprise the steps of isolating and/or purifying the leucine decarboxylase polypeptides, as described herein.
  • Suitable culture media and growth conditions for host cells are well known in the art. It is contemplated that any suitable method for introducing polynucleotides for expression of the leucine decarboxylase polypeptides into cells will find use in the present invention. Suitable techniques include, but are not limited to electroporation, biolistic particle bombardment, liposome mediated transfection, calcium chloride transfection, and protoplast fusion.
  • Engineered leucine decarboxylase polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered leucine decarboxylase polypeptide to any suitable mutagenesis and/or directed evolution methods known in the art, and/or as described herein.
  • An exemplary directed evolution technique is mutagenesis and/or DNA shuffling (See e.g., Stemmer, Proc. Natl. Acad. Sci. USA, 1994, 91: 10747-10751; WO 95/22625; WO 97/0078; WO 97/35966; WO 98/27230; WO 00/42651; WO 01/75767 and U.S.
  • Mutagenesis and directed evolution methods can be readily applied to leucine decarboxylase - encoding polynucleotides to generate variant libraries that can be expressed, screened, and assayed. Any suitable mutagenesis and directed evolution methods find use in the present invention and are well known in the art (See e.g., US 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, 6,277,638, 6,287,861, 6,287,862, 6,291,242, 6,297,053, 6,303,344, 6,309,883, 6,319,713, 6,319,714, 6,323,030, 6,326,204, 6,335,160, 6,335,198, 6,344,356, 6,352,859, 6,355,484, 6,358,740,
  • the enzyme clones obtained following mutagenesis treatment are screened by subjecting the enzyme preparations to a defined temperature (or other assay conditions) and measuring the amount of enzyme activity remaining after heat treatments or other suitable assay conditions.
  • Clones containing a polynucleotide encoding a leucine decarboxylase polypeptide are then isolated from the gene, sequenced to identify the nucleotide sequence changes (if any), and used to express the enzyme in a host cell.
  • Measuring enzyme activity from the expression libraries can be performed using any suitable method known in the art (e.g., standard biochemistry techniques, such as HPUC analysis).
  • the polynucleotides encoding the enzyme can be prepared by standard solid-phase methods, according to known synthetic methods. In some embodiments, fragments of up to about 100 bases can be individually synthesized, then joined (e.g., by enzymatic or chemical litigation methods, or polymerase mediated methods) to form any desired continuous sequence.
  • polynucleotides and oligonucleotides disclosed herein can be prepared by chemical synthesis using the classical phosphoramidite method (See e.g., Beaucage et al., Tet.
  • oligonucleotides are synthesized (e.g., in an automatic DNA synthesizer, purified, annealed, ligated and cloned in appropriate vectors).
  • a method for preparing the engineered leucine decarboxylase polypeptide can comprise: (a) synthesizing a polynucleotide encoding a polypeptide comprising an amino acid sequence selected from the amino acid sequence of any variant as described herein, and (b) expressing the leucine decarboxylase polypeptide encoded by the polynucleotide.
  • 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.
  • the amino acid sequence has optionally 1-2, 1-3, 1-4, 1-5, 1-6, 1-7, 1-8, 1-9, 1-10, 1-15, 1-20, 1-21, 1-22, 1-23, 1-24, 1-25, 1-30, 1-35, 1-40, 1-45, or 1-50 amino acid residue deletions, insertions and/or substitutions.
  • the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 21, 22, 23, 24, 25, 30, 30, 35, 40, 45, or 50 amino acid residue deletions, insertions and/or substitutions.
  • the amino acid sequence has optionally 1, 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 18, 20, 21, 22, 23, 24, or 25 amino acid residue deletions, insertions and/or substitutions.
  • the substitutions are conservative or non-conservative substitutions.
  • the expressed engineered leucine decarboxylase polypeptide 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.
  • any of the engineered leucine decarboxylase polypeptides expressed in a host cell are recovered from the cells and/or the culture medium using any one or more of the well- known techniques for protein purification, including, among others, lysozyme treatment, sonication, filtration, salting-out, ultra-centrifugation, and chromatography.
  • Chromatographic techniques for isolation of the leucine decarboxylase polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography. Conditions for purifying a particular enzyme depends, in part, on factors such as net charge, hydrophobicity, hydrophilicity, molecular weight, molecular shape, etc., and will be apparent to those having skill in the art. In some embodiments, affinity techniques may be used to isolate the improved leucine decarboxylase enzymes.
  • any antibody that specifically binds a leucine decarboxylase polypeptide of interest may find use.
  • various host animals including but not limited to rabbits, mice, rats, etc., are immunized by injection with a leucine decarboxylase polypeptide, or a fragment thereof.
  • the leucine decarboxylase polypeptide or fragment is attached to a suitable carrier, such as BSA, by means of a side chain functional group or linkers attached to a side chain functional group.
  • the engineered leucine decarboxylase polypeptide is produced in a host cell by a method comprising culturing a host cell (e.g., an E. coli strain) comprising a polynucleotide sequence encoding an engineered leucine decarboxylase polypeptide as described herein under conditions conducive to the production of the engineered leucine decarboxylase polypeptide and recovering the engineered leucine decarboxylase polypeptide from the cells and/or culture medium.
  • the host cell produces more than one engineered leucine decarboxylase polypeptide.
  • the engineered leucine decarboxylase polypeptides of the disclosure are prepared as compositions for various uses. These compositions find use in many fields, including but not limited to pharmaceuticals, dietary/nutritional supplements, food, feed, and fine chemical production.
  • the present invention provides food and/or feeds comprising at least one engineered leucine decarboxylase variant and/or at least one polynucleotide sequence encoding at least one leucine decarboxylase variant.
  • the present invention provides beverages comprising at least one engineered leucine decarboxylase variant.
  • the composition further comprises an effective amount of a coenzyme, e.g., pyridoxal 5 ’-phosphate.
  • the engineered leucine decarboxylase variant in food, feed, and/or nutritional/dietary supplement is glycosylated.
  • the engineered leucine decarboxylase variants find use in any suitable edible enzyme delivery matrix.
  • the engineered leucine decarboxylase variants are present in an edible enzyme delivery matrix designed for rapid dispersal of the leucine decarboxylase variant within the digestive tract of an animal upon ingestion of the variant.
  • the engineered leucine decarboxylase polypeptides are used in the production of fine chemicals and other industrially important compounds (See e.g., US Pat. Appln. Nos. 2013/0340119, 2013/0005012, and 2005/0260724, and WO 2012/122333).
  • the present disclosure also provides engineered leucine decarboxylase polypeptides suitable for use in pharmaceutical and other compositions, such as dietary/nutritional supplements.
  • compositions comprising a therapeutically effective amount of an engineered leucine decarboxylase according to the invention are in the form of a solid, semi- solid, or liquid.
  • the compositions include other pharmaceutically acceptable components such as diluents, buffers, excipients, salts, emulsifiers, preservatives, stabilizers, fillers, and other ingredients. Details on techniques for formulation and administration are well known in the art and described in the literature.
  • the engineered leucine decarboxylase polypeptides are formulated for use in oral pharmaceutical compositions.
  • Any suitable format for use in delivering the engineered leucine decarboxylase polypeptides find use in the present invention, including but not limited to pills, tablets, gel tabs, capsules, lozenges, dragees, powders, soft gels, sol-gels, gels, emulsions, implants, patches, sprays, ointments, liniments, creams, pastes, jellies, paints, 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.
  • the engineered leucine decarboxylase polypeptides are provided in a format suitable for injection (i.e., in an injectable formulation). In some embodiments, the engineered leucine decarboxylase polypeptides are provided in biocompatible matrices such as sol- gels, including silica-based (e.g., oxysilane) sol-gels. In some embodiments, the engineered leucine decarboxylase polypeptides are encapsulated.
  • the engineered leucine decarboxylase 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 intended that the engineered leucine decarboxylase polypeptides 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.
  • the pharmaceutical composition further comprises coenzyme pyridoxal-5 -phosphate.
  • the pyridoxal-5-phosphate may be formulated with an engineered leucine decarboxylase as a stable composition or prepared shortly before administration.
  • the engineered leucine decarboxylase polypeptides are chemically modified by glycosylation, PEGylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (See e.g., Ikeda, Amino Acids, 2005, 29:283-287; US Pat. Nos. 7,531,341, 7,534,595, 7,560,263, and 7,553,653; US Pat. Appln. Publ. 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.
  • the engineered leucine decarboxylase polypeptides are provided in formulations comprising matrix-stabilized enzyme crystals.
  • the formulation comprises a cross-linked crystalline engineered leucine decarboxylase enzyme and a polymer with a reactive moiety that adheres to the enzyme crystals.
  • the present invention also provides engineered leucine decarboxylase polypeptides in polymers.
  • compositions comprising the engineered leucine decarboxylase polypeptides of the present invention include one or more commonly used carrier compounds, including but not limited to sugars (e.g., lactose, sucrose, mannitol, and/or sorbitol), starches (e.g., com, wheat, rice, potato, or other plant starch), cellulose (e.g., methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxy- methylcellulose), gums (e.g., arabic, tragacanth, guar, etc.), and/or proteins (e.g., gelatin, collagen, etc.).
  • sugars e.g., lactose, sucrose, mannitol, and/or sorbitol
  • starches e.g., com, wheat, rice, potato, or other plant starch
  • cellulose e.g., methyl cellulose, hydroxypropyl methyl cellulose, sodium carboxy- methylcellulose
  • Additional components in oral formulations may include coloring and or sweetening agents (e.g., glucose, sucrose, and mannitol) and lubricating agents (e.g., magnesium stearate), as well as enteric coatings (e.g., methacrylate polymers, hydroxyl propyl methyl cellulose phthalate, and/or any other suitable enteric coating known in the art).
  • enteric coatings e.g., methacrylate polymers, hydroxyl propyl methyl cellulose phthalate, and/or any other suitable enteric coating known in the art.
  • disintegrating or solubilizing agents are included (e.g., cross-linked polyvinyl pyrrolidone, agar, alginic acid or salts thereof, such as sodium alginate).
  • the engineered leucine decarboxylase polypeptide are combined with various additional components, including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and/or emulsifiers, particularly in liquid formulations.
  • additional components including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and/or emulsifiers, particularly in liquid formulations.
  • the engineered leucine decarboxylase polypeptide are be combined with various additional components, including but not limited to preservatives, suspending agents, thickening agents, wetting agents, alcohols, fatty acids, and/or emulsifiers, particularly in liquid formulations.
  • the engineered leucine decarboxylase polypeptides are administered to subjects in combination with other compounds used in the treatment of MSUD, as well as any other suitable compounds.
  • the present invention provides engineered leucine decarboxylase polypeptides suitable for use in decreasing, ameliorating, or eliminating the signs and/or symptoms of MSUD, as described herein.
  • the dosage of engineered leucine decarboxylase polypeptide(s) administered to a patient depends upon the genotype of the patient, the general condition of the patient, and other factors known to those in the art.
  • the compositions are intended for single or repeat administration to a patient.
  • the concentration of engineered leucine decarboxylase polypeptide(s) in the composition(s) administered to a patient is sufficient to effectively treat, ameliorate and/or prevent the symptoms of the disease.
  • the engineered leucine decarboxylase polypeptides are administered in combination with other pharmaceutical and/or dietary compositions.
  • engineered leucine decarboxylase polypeptides of the present invention will find use in industrial compositions, including such areas as food flavorings (e.g., cheese).
  • the engineered leucine decarboxylase polypeptides are formulated for use in the food and/or feed industries.
  • the engineered leucine decarboxylase polypeptides are formulated in granulated or pelleted products which are mixed with animal feed components such as additional enzymes (for example, cellulases, laccases, and amylases).
  • animal feed components such as additional enzymes (for example, cellulases, laccases, and amylases).
  • the engineered leucine decarboxylase polypeptides are used in liquid animal feed compositions (e.g., aqueous or oil-based slurries).
  • the engineered leucine decarboxylase variants of the present invention are sufficiently thermotolerant and thermostable to withstand the treatment used to produce pellets and other processed feed/foods.
  • the engineered leucine decarboxylase polypeptides find use for treating and/or preventing the symptoms of conditions associated with dysfunction in leucine, isoleucine, and/or alloisoleucine metabolism.
  • the subject for treatment to reduce levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid has an organic acidemia or inborn error of amino acid metabolism.
  • the subject for treatment to reduce levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid has elevated leucine levels caused by unknown etiology.
  • the engineered leucine decarboxylase polypeptides are used for treating and/or preventing the symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels.
  • a method of treating and/or preventing the symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels comprises administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase to decrease levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid in the subject.
  • the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels is maple syrup urine disease.
  • the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels is isovaleric acidemia.
  • the disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels is 3- methylcrotonyl-CoA carboxylase deficiency.
  • the engineered leucine decarboxylase polypeptides are used to reduce plasma leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine, and/or ketoisocaproic acid levels in a subject.
  • the levels of leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine and/or ketoisocaproic acid can be used as indicators of disease or condition, for example, associated with dysfunction in amino acid metabolism and/or as markers for effectiveness of treatment with an engineered leucine decarboxylase polypeptide.
  • the engineered leucine decarboxylase polypeptides are used to reduce plasma leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine and/or ketoisocaproic acid levels in a subject, the method comprising administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase.
  • the engineered leucine decarboxylase polypeptides are used to reduce plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels in a subject, the method comprising administering to a subject in need thereof an effective amount of an engineered leucine decarboxylase.
  • the subject for treatment to reduce levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid has maple syrup urine disease.
  • the subject for treatment to reduce levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid has isovaleric acidemia.
  • the subject for treatment to reduce levels of plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid has 3- methylcrotonyl-CoA carboxylase deficiency.
  • the engineered leucine decarboxylase polypeptide is administered at an effective dose.
  • the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg/kg to 500 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg/kg to 400 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 1 mg/kg to 200 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 2 mg/kg to about 300 mg/kg, or about 5 mg/kg to about 200 mg/kg.
  • the engineered leucine decarboxylase polypeptide is administered at a dose of about 1 mg/kg, about 5 mg/kg, about 10 mg/kg, about 20 mg/kg, about 30 mg/kg, about 40 mg/kg, about 50 mg/kg, about 60 mg/kg, about 70 mg/kg, about 80 mg/kg, about 90 mg/kg, about 100 mg/kg, about 120 mg/kg, about 140 mg/kg, about 160 mg/kg, about 180 mg/kg, about 200 mg/kg, about 250 mg/kg, about 300 mg/kg, about 350 mg/kg, or about 400 mg/kg. .
  • the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg/kg to less than 25 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of 5 mg/kg to less than 25 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg/kg to about 12.5 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 12.5 mg/kg.
  • the engineered leucine decarboxylase polypeptide is administered at a dose of 1 mg/kg to less than 25 mg/kg. In some embodiments for the treatment of maple syrup urine disease, the engineered leucine decarboxylase polypeptide is administered at a dose of 5 mg/kg to less than 25 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg/kg to about 12.5 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 6.25 mg/kg. In some embodiments, the engineered leucine decarboxylase polypeptide is administered at a dose of about 12.5 mg/kg.
  • the engineered leucine decarboxylase polypeptides are used to reduce plasma leucine, isoleucine, valine, methionine, cysteine, phenylalanine, alloisoleucine and/or ketoisocaproic acid levels in a subject.
  • the engineered leucine decarboxylase polypeptide is administered in an effective amount to reduce plasma leucine, ketoisocaproic acid, and methionine levels in a subject in need thereof.
  • the engineered leucine decarboxylase polypeptide is administered to the subject in at least two or more consecutive days.
  • the engineered leucine decarboxylase polypeptide is administered to the subject in at least three or more consecutive days. In some embodiments, the engineered leucine decarboxylase polypeptide is administered continuously, e.g., weeks, months, years, and/or as necessary to treat the subject in need thereof.
  • the engineered leucine decarboxylase polypeptide is administered at dose to reduce plasma or serum leucine levels by about 5% or more, 10% or more, 15% or more, 20% or more, 25% or more, 30% or more, 40% or more, and/or 50% or more, as compared to their baseline leucine levels in untreated subjects with the disease or disorder.
  • the engineered leucine decarboxylase polypeptide is administered immediately before, concurrently with, and/or immediately following ingestion of a meal containing protein.
  • an engineered leucine decarboxylase polypeptide may be administered in combination with coenzyme pyridoxal-5-phosphate.
  • an engineered leucine decarboxylase polypeptide can be formulated with pyridoxal-5- phosphate prior to administration.
  • the pyridoxal-5 -phosphate is administered concurrently with the administration of an engineered leucine decarboxylase polypeptide.
  • the subject treated with the engineered leucine decarboxylase polypeptide has maple syrup urine disease, and wherein the symptoms of maple syrup urine disease are ameliorated. In some embodiments, the subject treated with the engineered leucine decarboxylase polypeptide has isovaleric acidemia, and wherein the symptoms of isovaleric acidemia are ameliorated. In some embodiments, the subject treated with the engineered leucine decarboxylase polypeptide has 3- methylcrotonyl-CoA carboxylase deficiency, and wherein the symptoms of 3-methylcrotonyl-CoA carboxylase deficiency are ameliorated.
  • the subject is able to eat a diet that is less restricted in leucine, isoleucine, and/or valine content compared to diets required by subjects who are afflicted with the disease.
  • the subject is an infant, child, young adult, or adult.
  • the engineered leucine decarboxylase polypeptides useful for the therapeutic methods and uses herein include the engineered leucine decarboxylase polypeptides described above and those described in International Patent publication WO2021158686 and its corresponding Sequence Listing, which are incorporated herein by reference in their entirety. These engineered leucine decarboxylase polypeptides are also presented in the Appendix (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 11-2), and the incorporated Sequence Listing of the present disclosure.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, and/or 888.
  • the engineered leucine decarboxylase polypeptide comprises one or more substitutions relative to the reference sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.
  • the engineered leucine decarboxylase polypeptide comprises amino acid sequences having at 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.
  • 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.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 6.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 10.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 14.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a reference sequence corresponding to SEQ ID NO: 12, 38, 234, 284, 484, 594, 686, 688, 766, 828, or 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 12.
  • the amino acid sequence of the engineered leucine decarboxylase comprises at least a substitution at amino acid position 2, 3, 5, 12, 14, 16, 19, 33, 34, 38, 39, 41, 47, 48, 51, 55, 63, 64, 66, 69, 76, 77, 80, 87, 89, 91, 92, 102, 106, 109, 118, 123, 126, 127, 132, 134, 135, 139, 140, 141, 156, 161, 164, 168, 170, 173, 181, 187, 189, 193, 194, 196, 198, 200, 201, 202, 211, 223, 228,
  • the amino acid sequence of the engineered leucine decarboxylase comprises at least a substitution 2E, 3M, 5M/V, 12G, 14I/T, 16Q/V, 19I/L, 33L, 34L, 38V, 39N/S, 41D, 47F, 48L, 51Q/E, 551, 63C, 64S/A/N/E, 66S/N, 691, 76V, 77L, 80G/K, 87R, 89P, 91A/Q, 92K, 102S, 106M, 109G, 118T/D, 123F/M/V, 126A/T, 127S, 132F, 134A/S, 135V, 139G, 140V, 141P, 156A/S, 161V, 164A/C, 168K, 170A/P, 173A/I/T, 181K/R/V, 187L, 189A/D
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 12, and wherein the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 34/38/39/39/127/245/349/350/357, 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, 34/39/102/264/275/350
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 5M, 141, 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/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/357V, 34L/39
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set K5M, Hl 41, H14T/I34L/C38V/T39N/T102S/V267I/T275S/N350E/I357V, H14T/T39N/T102S/T127S/I245M/V267I/T275S/V349T/N350E, I34L/C38V/T39N/T102S/T127S/T275S/I357V, I34L/C38V/T39N/T102S/T275S/I357V, I34L/C38V/T39N/T127S/I245M/V349I7N350E/I357V, I34L/C38V/T39N/T127S/I245M/N350E/I357V, I34L/T39N/T102S/T127S/I
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 38, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 48/64/164/324/343/353/364, 48/64/164/324/343/364, 48/64/164/353/357/364, 48/64/357/364, 64/164/324/343/353/364, 64/164/324/343/357/364, 64/164/324/343/357, 64/164/353/357, 64/318/324/357/364, 64/324/353/364, 132/255/339/379/395, 164/196/324/357/364, 164/318/324/343/353/353,
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/324M/343E/353E/357C, 164A/324M/343E/353D/357C/364R
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/1164 A/R324M/H343 E/I357C/L364R, A64E/1164C/R353 D/1357 V, A64E/R318K/R324S/I357V/L364R, A64E/R324M/R353 D/1357 V
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 234, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 324/395, 339, 340, 380, 382, 389, 389/394, 389/3
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 2E, 3M, 33L, 48F/64E/255P, 48F/255P/339A, 48F/255P/379D, 64E, 64E/255P, 64S, 691, 161V, 1931, 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, 324S/394E/395K/397A, 324S/395K, 339A, 340T, 340V, 380E, 382S, 389G, 3
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set G2E, N3M, F33L, L48F/A64E/H255P, L48F/H255P/Q339A, L48F/H255P/K379D, A64E, A64E/H255P, A64S, V69I, T161V, M193I, H255P, H255P/R318K/K379D, R259L, S263T, S263V, R318K/Q339A/K379D, M324N, M324N/K394E/R395K/T397A, M324N/R395D, M324S/K389G/K394E, M324S/K389G/K394E/R395D, M324S/K389G/K394E/R395D, M324S/K389
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 284, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 12/135/263/382, 12/259/263/304, 48/64/255, 64/69, 64/69/189/259/263/304, 64/69
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 2E/64S/69I/324S/380E/382S/388A/389G, 3M/64S/69I/263T/339A/380E/388A, 3M/64S/69I/389G, 3M/64S/69I/390*, 3M/64S/379D/380E/390*, 3M/69I/263T/380E, 3M/69I/324S, 3M/69I/324S/380E/382S/389G/390*, 12G/135V/259K/263T, 12G/135V/263T/382G, 12G/259K/263T/304R, 48L/64A/255H, 64A/255H/263T, 64S/69I, 64S/69I/189A/259Q/263T/304R/339A/340T/379N, 64S/69I/189D
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/A382S/K389
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:484, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 3/304, 3/304, 3/304, 3/304, 3/304, 3/304/324, 16, 63, 77, 80, 87/270, 87/270/365, 87/328/365, 91,
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/365E, 87R/328N/365E, 91A, 91Q, 92K, 126A, 126T, 140V, 156A, 156S, 168K/270R/328N/338S, 18
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/A304R/M324S/Q339A, N3M/S263T/A304R/M324S/A382S, N3M/A304R, N3M/A304R/M324S, R16Q, R16V, A63C, E77L, A80G, A80K, H87R/
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 594, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 16Q/63C/80K/126T/168K/366M, 16Q/63C/80K/126T/181R/194C/259K/324S/328N/366M, 16Q/63C/126T/168K/270R/328N/366M, 16Q/80K/126T/324S/366M, 16Q/80K/126T/366M, 16Q/80K/168K, 16Q/80K/168K/270R/366M, 16Q/80K/168K/324S, 16Q/80K/168K/366M, 16Q/80K/324S, 16Q/91A/126T/168K/324S/366M, 16Q/126T/168K/366M, 16Q/168K/259K/366M, 16Q/168K/270R/324S/366M, 16Q/168K/324S/328N
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set R16Q/A63C/A80K/D126T/C168K/H366M, R16Q/A63C/A80K/D126T/T181R/F194C/R259K/M324S/C328N/H366M, R16Q/A63C/D126T/C168K/L270R/C328N/H366M, R16Q/A80K/D126T/M324S/H366M, R16Q/A80K/D126I7H366M, R16Q/A80K/C168K, R16Q/A80K/C168K/L270R/H366M, R16Q/A80K/C168K/M324S, R16Q/A80K/C168K/H366M, R16Q/A80K/M324S, R16Q/A80K/
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 686, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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,
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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, 12617201D/270R/324S, 126T/270R, 141P
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set S66N/R76V/T118D/R141P/E201R/R300K, S66N/R76V/A198G/H200S/D296E/A303Q, S66N/R76V/A198G/H200S/R300K, S66N/T118D/H200S/D296E/A303Q/K317Q, S66N/T118D/D296E, S66N/T118D/D296E/R300K, S66N/H200S, R76V/T118D/R141P/H200S/D296E, R76V/R141P/A198G/H200S/E201R/R300K, A80K/E201D/L270R, A80K/L270R, A80K/L270R/M324S, A89P/T118D/H200
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 686, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 191, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 1731, 173T, 187L, 21 IS, or 312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set LI 91, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A21 IS, or T312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 688, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19/109/123/141/170/198/200/211/270/312, 19/109/123/141/170/198/211, 19/109/123/141/170/198/211/270/312, 19/109/123/170/211/270/312, 19/109/123/198/200/211/270/312, 19/109/170/173/211/270/312, 19/109/211/270/312, 109/170/211/270/312, or 109/211/270/312, wherein the amino acid positions are relative to
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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, or 109G/211S/270R/312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 688.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set L 19I/L 109G/Y 123F/P 170A/A211 S/L270R/T312A, L 19I/L 109G/Y 123F/A 198G/H200S/A211 S/L270R/T312A,
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 5/41, 5/41/228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 5V/41D, 5V/41D/228D, 33L, 4 ID, 47F, 5 IE, 5 IQ, 551, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 3531, 353L, 357S, or 384W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set K5V/H41D, K5V/H41D/T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, or P384W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 66, 66/118, 66/118/296, 66/118/296/300, 66/118/300, 66/296, 66/296/300, 66/300, 118, 118/296, 118/296/300, 118/300, 296, 296/300, or 300, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 66S, 66S/118T, 66S/118T/296D, 66S/118T/296D/300R, 66S/118T/300R, 66S/296D, 66S/296D/300R, 66S/300R, 118T, 118T/296D, 118T/296D/300R, 118T/300R, 296D, 296D/300R, or 300R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set N66S, N66S/D118T, N66S/D118T/E296D, N66S/D118I7E296D/K300R, N66S/D118I7K300R, N66S/E296D, N66S/E296D/K300R, N66S/K300R, D118T, D118I7E296D, DI 18T/E296D/K300R, D118I7K300R, E296D, E296D/K300R, or K300R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least a substitution or substitution set at amino acid position 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, 38/39/102/127/349/350/357, 38/39/102/127/349/350/357, 38/39/102/127/349/
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 5M, 141, 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/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/357V, 34L/39
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set K5M, H14I, H 14T/I34L/C38 V/T39N/T 102S/V267I/T275 S/N350E/I357V, H14T/T39N/T102S/T127S/I245M/V267I/T275S/V349T/N350E,
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 64/164/318/324/357/364, 64/324/353/364, 132/255/339/379/395, 164/196/324/357/364, 164/318/324/343/353/357, 164/318/324/357/364, 164/324/343/353/364, 164/324/357/364, 164/353/357/364, 164/364, 196/318/324/353/357/364, 318/343/357, 324/364, 318/343/357, 324/364, 364, 324/343/364, 318
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/324M/343E/353E/357C, 164A/324M/343E/353D/357C/364R
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/1164A/R324M/H343E/R353D/I357V/L364K, A64E/1164 A/R324M/H343 E/I357C/L364R, A64E/1164C/R353 D/1357 V, A64E/R318K/R324S/I357V/L364R, A64E/R324M/R353 D/1357 V,
  • 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, 1164C/R353D/I357V/L364K, 1164C/R353D/I357V/L364R, 1164C/R353 W/I357C/L364R, K196D/R318K/R324M/R353N/I357C/L364K, R318K/H343E/I
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 401, or 405, wherein the amino acid positions are relative to the reference sequence corresponding to
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 2E, 3M, 33L, 48F/64E/255P, 48F/255P/339A, 48F/255P/379D, 64E, 64E/255P, 64S, 691, 161V, 1931, 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, 324S/394E/395K/397A, 324S/395K, 339A, 340T, 340V, 380E, 382S, 389G, 3
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set G2E, N3M, F33L, L48F/A64E/H255P, L48F/H255P/Q339A, L48F/H255P/K379D, A64E, A64E/H255P, A64S, V69I, T161V, M193I, H255P, H255P/R318K/K379D, R259L, S263T, S263V, R318K/Q339A/K379D, M324N, M324N/K394E/R395K/T397A, M324N/R395D, M324S/K389G/K394E, M324S/K389G/K394E/R395D, M324S/K389G/K394E/R395D, M324S/K389
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 12/135/263/382, 12/259/263/304, 48/64/255, 64/69, 64/69/189/259/263/304, 64/69/189/259/263/304/339/340/379, 64/69/223/388, 64/69/223/388/389/390, 64/69/304/379/382, 64/69/324, 64/69/324/339/380/389/390, 64/69/339, 64/69/339/382/388
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 2E/64S/69I/324S/380E/382S/388A/389G, 3M/64S/69I/263T/339A/380E/388A, 3M/64S/69I/389G, 3M/64S/69I/390*, 3M/64S/379D/380E/390*, 3M/69I/263T/380E, 3M/69I/324S, 3M/69I/324S/380E/382S/389G/390*, 12G/135V/259K/263T, 12G/135V/263T/382G, 12G/259K/263T/304R, 48L/64A/255H, 64A/255H/263T, 64S/69I, 64S/69I/189A/259Q/263T/304R/339A/340T/379N, 64S/69I/189D
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/A382S/K389
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 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
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/365E, 87R/328N/365E, 91A, 91Q, 92K, 126A, 126T, 140V, 156A, 156S, 168K/270R/328N/338S, 18
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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/A304R/M324S/Q339A, N3M/S263T/A304R/M324S/A382S, N3M/A304R, N3M/A304R/M324S, R16Q, R16V, A63C, E77L, A80G, A80K, H87R/
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, 16/80/168, 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, 16/168/366, 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
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 16Q/63C/80K/126T/168K/366M, 16Q/63C/80K/126T/181R/194C/259K/324S/328N/366M, 16Q/63C/126T/168K/270R/328N/366M, 16Q/80K/126T/324S/366M, 16Q/80K/126T/366M, 16Q/80K/168K, 16Q/80K/168K/270R/366M, 16Q/80K/168K/324S, 16Q/80K/168K/366M, 16Q/80K/324S, 16Q/91A/126T/168K/324S/366M, 16Q/126T/168K/366M, 16Q/168K/259K/366M, 16Q/168K/270R/324S/366M, 16Q/168K/324S/328N
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set R16Q/A63C/A80K/D126T/C168K/H366M, R16Q/A63C/A80K/D126T/T181R/F194C/R259K/M324S/C328N/H366M, R16Q/A63C/D126T/C168K/L270R/C328N/H366M, R16Q/A80K/D126T/M324S/H366M, R16Q/A80K/D126I7H366M, R16Q/A80K/C168K, R16Q/A80K/C168K/L270R/H366M, R16Q/A80K/C168K/M324S, R16Q/A80K/C168K/H366M, R16Q/A80K/M324S, R16Q/A80K/
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 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, or 270/324, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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, 141
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set S66N/R76V/T118D/R141P/E201R/R300K, S66N/R76V/A198G/H200S/D296E/A303Q, S66N/R76V/A198G/H200S/R300K, S66N/T118D/H200S/D296E/A303Q/K317Q, S66N/T118D/D296E, S66N/T118D/D296E/R300K, S66N/H200S, R76V/T118D/R141P/H200S/D296E, R76V/R141P/A198G/H200S/E201R/R300K, A80K/E201D/L270R, A80K/L270R, A80K/L270R/M324S, A89P/T118D/H200
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19, 109, 123, 134, 170, 173, 187, 211, or 312, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 191, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 1731, 173T, 187L, 21 IS, or 312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A21 IS, and T312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 686.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 19/109/123/141/170/198/200/211/270/312, 19/109/123/141/170/198/211, 19/109/123/141/170/198/211/270/312, 19/109/123/170/211/270/312, 19/109/123/198/200/211/270/312, 19/109/170/173/211/270/312, 19/109/211/270/312, 109/170/211/270/312, or 109/211/270/312, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 688.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 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, or 109G/211S/270R/312A, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 688.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set L 19I/L 109G/Y 123F/P 170A/A211 S/L270R/T312A,
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 5/41, 5/41/228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, or 384, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 5V/41D, 5V/41D/228D, 33L, 41D, 47F, 5 IE, 5 IQ, 551, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 3531, 353L, 357S, or 384W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set K5V/H41D, K5V/H41D/T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, or P384W, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set at amino acid position 66, 66/118, 66/118/296, 66/118/296/300, 66/118/300, 66/296, 66/296/300, 66/300, 118, 118/296, 118/296/300, 118/300, 296, 296/300, or 300, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 66S, 66S/118T, 66S/118T/296D, 66S/118T/296D/300R, 66S/118T/300R, 66S/296D, 66S/296D/300R, 66S/300R, 118T, 118T/296D, 118T/296D/300R, 118T/300R, 296D, 296D/300R, or 300R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the amino acid sequence of said engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set N66S, N66S/D118T, N66S/D118T/E296D, N66S/D118I7E296D/K300R, N66S/D118I7K300R, N66S/E296D, N66S/E296D/K300R, N66S/K300R, D118T, D118I7E296D, DI 18T/E296D/K300R, D118I7K300R, E296D, E296D/K300R, or K300R, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 766.
  • the engineered leucine decarboxylase polypeptides for the therapeutic uses and methods herein are the engineered leucine decarboxylase polypeptides disclosed above and herein.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to a reference sequence of SEQ ID NO: 828 or 888.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%,
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 80%, 81%, 82%, 83%, 84%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more sequence identity to the reference sequence corresponding to SEQ ID NO: 828, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid position 33, 55, 64, 126, 270, or 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828.
  • 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 the reference sequence of SEQ ID NO: 828, and wherein the amino acid sequence comprises at least a substitution or substitution set at amino acid position 170/270/383, 270, 41/173, 272, 5/141/272/383, 41/383, 41/141/187/272/290, 41/141/173/290, 5/272/383, 5/41/173/272/383, 41/141, 141/272, 353/384, 272/383, 41/141/173, 41/272/383, 41/141/187/200/202/272, 33/55/64/126/270/357, 33/126/353/357, 55/64/267/35/384, 33/64/357, 126/267, 64/2
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 170P/270L/383S, 270L, 41D/173I, 272A, 5V/141P/272A/383S, 41D/383S, 41D/141P/187L/272A/290I, 41D/141P/173I/290I, 5V/272A/383S, 5V/41D/173I/272A/383S, 41D/141P, 141P/272A, 353E/384W, 272A/383S, 41D/141P/173I, 41D/272A/383S, 41D/141P/187L/200S/202H/272A, 33L/55I/64N/126A/270L/357S, 33L/126A/353E/357S, 55I/64N/267L/353E/384W, 33L/64N/357S, 126A/267L, 64N/267
  • amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set A170P/R270L/A383S, R270L, H41D/F173I, T272A, K5V/R141P/T272A/A383S, H41D/A383S, H41D/R141P/V187L/T272A/V290I, H41D/R141P/F173I/V290I, K5V/T272A/A383S, K5V/H41D/F173I/T272A/A383S, H41D/R141P, R141P/T272A, D353E/P384W, T272A/A383S, H41D/R141P/F 1731, H41D/T272A/A383S, H41D/R141P/V187L/H200S/S202H/T272A, F
  • amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution at amino acid positions 33, 55, 64, 126, 270, or 357, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least two or more substitutions of amino acid positions 33, 55, 64, 126, 270, and 357, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least three or more substitutions of amino acid positions 33, 55, 64, 126, 270, and 357, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least substitutions at amino acid positions 33/55, 33/64, 33/126, 33/270, 33/357, 55/64, 55/126, 55/270, 55/357, 64/126, 64/270, 64/357, 126/270, 126/357, or 270/357, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the engineered leucine decarboxylase polypeptide comprises at least a substitution set at amino acid position 33/55/64, 33/55/126, 33/64/126, 55/64/270, 55/64/357, or 64/126/270, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least substitutions at amino acid positions 33, 55, 64, 126, 270, and 357, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 828 or 888.
  • the substitutions at amino acid positions 33, 55, 64, 126, 270, and 357 are selected from 33L, 551, 64N, 126A, 270L, and 357S. In some embodiments, the substitutions at amino acid positions 33, 55, 64, 126, 270, and 357 are selected from: F33L, V55I, S64N, D126A, R270L, and C357S.
  • the engineered leucine decarboxylase comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 888, wherein the amino acid sequence comprises one or more substitutions relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase comprises at least a substitution at amino acid position 5, 19, 33, 41, 47, 51, 55, 64, 141, 170, 173, 187, 198, 200, 202, 267, 270, 272, 290, 312, 353, 357, 383, or 384, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • the amino acid sequence of the engineered leucine decarboxylase comprises amino acid residue 5V, 19L, 33L, 41D, 47F, 5 IE, 551, 64S/N, 141P, 170P, 1731, 187L, 198G, 200S, 202H, 267L, 270L/T, 272A, 2901, 312T, 353E, 357S/C, 383S, or 384W, or combinations thereof, wherein the amino acid positions are relative to the reference sequence corresponding to SEQ ID NO: 888.
  • amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 64/173/202/353/384, 41/141/272/353, 141/202/272/353/357, 173/202/357, 202/353, 5/51/173/272/353/384, 51/202/272/357, 141/173/272, 272, 41/173/384, 41/64/141/353/357/383, 141/173/202, 5/51/64/202/353, 357, 64, 5/41/141, 41/141/173/202/353, 353, 202/357, 51/141/202/272/353, 202, 51/141/173/353/384, 41/141/173/202/272/353/383/384, 64/202/357, 5/64/353/383/384, 41/272/353/383, 41/173/272/353/357, 51/141/272/353/357/383/384, 41/353/357
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set 64S/173I/202H/353E/384W, 41D/141P/272A/353E, 141P/202H/272A/353E/357C, 173I/202H/357C, 202H/353E, 5V/51E/173I/272A/353E/384W, 51E/202H/272A/357C, 141P/173I/272A, 272A, 41D/173I/384W, 41D/64S/141P/353E/357C/383S, 141P/173I/202H, 5V/51E/64S/202H/353E, 357C, 64S, 5V/41D/141P, 41D/141P/173I/202H/353E, 353E, 202H/357C, 51E/141P/202H/272A/353E, 202H, 51E/141P
  • amino acid sequence of the engineered leucine decarboxylase polypeptide comprises at least a substitution or substitution set N64S/F173I/S202H/D353E/P384W, H41D/R141P/T272A/D353E, R141P/S202H/T272A/D353E/S357C, F173I/S202H/S357C, S202H/D353E, K5V/L51E/F173I/T272A/D353E/P384W, L51E/S202H/T272A/S357C, R141P/F173I/T272A, T272A, H41D/F173I/P384W, H41D/N64S/R141P/D353E/S357C/A383S, R141P/F173I/S202H, K5V/L51E/N64S/S202H/D
  • the amino acid sequence of the engineered leucine decarboxylase polypeptide comprises a substitution or substitution set of an engineered leucine decarboxylase provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and/or 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence of an engineered leucine decarboxylase provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, 11-2, 12-1, and/or 12-2.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to a sequence corresponding to an even-numbered SEQ ID NO. of SEQ ID NOS: 2-1064.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 2-1064.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 16-1064.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 16-852. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even- numbered SEQ ID NO. of SEQ ID NOS: 16-204. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 206-278.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 280-390. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 392- 484. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 486-636.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 638-686. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even- numbered SEQ ID NO. of SEQ ID NOS: 688-736. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 738-762.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 764-780. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 782- 822. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 824-852. In some embodiments of the therapeutic uses and methods, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence comprising an even-numbered SEQ ID NO. of SEQ ID NOS: 854-1064.
  • the leucine decarboxylase for the therapeutic uses and methods herein exhibits at least one improved property as compared to wild-type Planctomycetaceae bacteria species leucine decarboxylase.
  • the engineered leucine decarboxylase polypeptide exhibits more activity on leucine than the wild-type Planctomycetaceae species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide is more thermostable than wild-type Planctomycetaceae bacteria species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide more resistant to proteolysis than wild-type Planctomycetaceae bacteria species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide has increased tolerance to low pH environments than wild-type Planctomycetaceae bacteria species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12. In some embodiments, the engineered leucine decarboxylase polypeptide is less immunogenic than wild-type Planctomycetaceae bacteria species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • the engineered leucine decarboxylase polypeptide is more serum stable than wild-type Planctomycetaceae bacteria species leucine decarboxylase or the leucine decarboxylase having the amino acid sequence corresponding to SEQ ID NO: 12.
  • a polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used for gene therapy for treating and/or preventing the symptoms of disease or conditions associated with dysfunction in leucine, isoleucine, and/or alloisoleucine metabolism.
  • the polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used for gene therapy for treating and/or preventing the symptoms of a disease or condition associated with elevated plasma leucine, isoleucine, alloisoleucine and/or ketoisocaproic acid levels.
  • polynucleotide encoding an engineered leucine decarboxylase polypeptide can be used for gene therapy to treat a subject with maple syrup urine disease, isovaleric acidemia, or 3- methylcrotonyl-CoA carboxylase deficiency.
  • the polynucleotide encoding an engineered leucine decarboxylase polypeptide is codon-optimized for expression in a human patient.
  • the polynucleotide for use in gene therapy is DNA or RNA.
  • the polynucleotide or composition thereof for gene therapy comprises mRNA.
  • ppm parts per million
  • M molar
  • mM millimolar
  • uM and pM micromolar
  • nM nanomolar
  • mol molecular weight
  • gm and g gram
  • mg milligrams
  • ug and pg micrograms
  • L and 1 liter
  • ml and m milliliter
  • cm centimeters
  • mm millimeters
  • um and pm micrometers
  • coli strain available from the Coli Genetic Stock Center [CGSC], New Haven, CT); iMSUD (Intermediate Maple Syrup Urine Disease); HTP (high throughput); HPLC (high pressure liquid chromatography); LC (liquid chromatography); MS (mass spectroscopy); LC-MS/MS (liquid chromatography with two mass spectrometers); SPE (solid phase extraction); KIC (ketoisocaproate); IPTG (isopropyl [3-D-l -thiogalactopyranoside); PLP (pyridoxal 5’- phosphate); BSA (bovine serum albumin); BW (body weight); MSUD (maple syrup urine disease); FIOPC (fold improvements over positive control); LB (Luria broth); TB (Terrific broth); Alternative Research (Innovative Research, Novi, MI); Microfluidics (Microfluidics Corp., Newton, MA);
  • Thermotron (Thermotron, Holland, MI); Waters (Waters Corp., Milford, MA); Infers (Infers AG, Bottmingen, Switzerland); Cambridge Isotope Laboratories (Cambridge Isotope Laboratories, Inc., Tewksbury, MA); Sigma-Aldrich (Sigma-Aldrich, St.
  • EXAMPLE 1 High-Throughput (HTP) Growth of Escherichia coli (E. coli) expressing Leucine Decarboxylase (LDC) Variants and LDC Screening Conditions
  • Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose with selection. After overnight incubation at 37 °C, colonies were placed into the wells of 96-well shallow flat bottom plates (NuncTM, Thermo-Scientific) filled with 180 pl/well LB supplemented with 1% glucose and selection. The cultures were allowed to grow overnight for 18-20 hours in a shaker (200 rpm, 30 °C, and 85% relative humidity; Kuhner). Overnight growth samples (20 pL) were transferred into Costar® 96-well deep plates (Coming) filled with 380 pL of Terrific Broth supplemented with a selection compound.
  • the plates were incubated for approximately 2 hours in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner).
  • the cells were then induced with 40 pL of 10 mM IPTG and incubated overnight for 20-24 hours in a shaker (250 rpm, 30 °C, and 85% relative humidity; Kuhner).
  • the cells were pelleted (4000 rpm x 20 min), the supernatants were discarded, and the cells were frozen at -80 °C prior to lysis.
  • E. coli cell pellets were lysed with 400 pL of lysis buffer (20 mM sodium phosphate pH 7, 0.04 mM pyridoxal 5’-phosphate (PLP), 1 mg/ml lysozyme, 0.5 g/L polymyxin B sulfate (PMBS)). The mixture was agitated for 1.5 h at room temperature and pelleted (4000 rpm x 10 min) after which the clarified lysates were preincubated for 1 h at 60 °C in a Multitron plate shaker (250 or 400 rpm; Infers HT). The heat-treated lysates were pelleted (4000 rpm x 10 min), and the supernatants were used in HTP assays.
  • lysis buffer 20 mM sodium phosphate pH 7, 0.04 mM pyridoxal 5’-phosphate (PLP), 1 mg/ml lysozyme, 0.5 g/L polymyxin B sulfate (
  • LDC activity was assessed by adding diluted heat-treated clarified lysate to a reaction mix for a final concentration of 3 mM leucine in 20 mM sodium phosphate, pH 7.0 or in a mixture of simulated intestinal fluid salts.
  • reaction mix resulting in a final concentration of 3 mM of leucine, isoleucine-dlO, valine, methionine, and cysteine (5 AA mix) was used.
  • Reactions were incubated for 1 h at 37 °C at 250 rpm in a Multitron plate shaker (Infers HT) before being quenched with 3 volumes of acetonitrile with 0.1% formic acid, centrifuged for 10 min at 4 °C at 4000 rpm, and diluted 50-fold in water.
  • the resulting samples were analyzed for isopentylamine, the decarboxylation product of leucine, by RapidFire®-MS/MS (Agilent), and relative activities were determined by dividing the isopentylamine peak area of samples by the isopentylamine peak area of the backbone to compute FIOP (fold improvement over parent) values.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin to simulate the gastric environment. Specifically, heat-treated clarified lysate was preincubated 1: 1 with Mcllvaine buffer pH 2.8-3 and a final concentration of 0.8 g/L pepsin in Costar® 96-well round bottom plates (Coming). The plates were sealed and incubated for 2 h at 37 °C in a Multitron plate shaker (250 rpm; Infers HT).
  • the simulated gastric challenged lysates were preincubated 1: 1 with a final concentration of 4 g/L trypsin and 1.5 g/L chymotrypsin for 2 h at 37 °C in a Multitron plate shaker (250 rpm; Infers HT) to simulate the intestinal environment. After this incubation, the samples were centrifuged, and 40 pL of sample was added to 60 pL of reaction mix for a final concentration of 3 mM leucine in 20 mM sodium phosphate, pH 7 or in a mixture of simulated intestinal fluid salts.
  • reaction mix resulting in a final concentration of 3 mM of leucine, isoleucine-dlO, valine, methionine, and cysteine was used. Activities of LDC variants were then measured as described above in Example 1.3.
  • Library variants were generated by recombining beneficial mutations into LDC polypeptide of SEQ ID NO: 828. HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 1. Variants were screened for LDC activity in 5 AA mix dissolved in simulated intestinal fluid salt solution after a 2 h simulated gastric (50% Mcllvaine buffer pH 3, 0.8 g/L pepsin) and a 2 h intestinal challenge (200 mM sodium phosphate, pH 8, 4 g/L trypsin, 1.5 g/L chymotrypsin). LDC activity was measured as described in Example 1, and analysis of the data relative to SEQ ID NO: 828 is listed in Table 12-1.
  • SEQ ID NO: 888 was chosen as the backbone. Beneficial mutations identified from Table 12-1 were recombined into the backbone. The resulting variants were screened for unchallenged activity on leucine by incubating 20 pL of 80x diluted heat-treated clarified lysate with 80 pL of reaction mix for a final concentration of 3 mM leucine and 0.01 mM PLP in 20 mM sodium phosphate, pH 7.
  • Variants were also screened for LDC activity in 5 AA mix with 0.01 mM PLP dissolved in 20 mM sodium phosphate, pH 7 after a 2 h simulated gastric (50% Mcllvaine buffer pH 2.8, 0.8 g/L pepsin) and a 2 h intestinal challenge (200 mM sodium phosphate, pH 8, 4 g/L trypsin, 1.5 g/L chymotrypsin).
  • LDC activity was measured as described in Example 1, and analysis of the data relative to SEQ ID NO: 888 is listed in Table 12-2.
  • mice had ad libitum access to leucine-free diet and leucine-supplemented water throughout the study. Approximately 45 mg of whey (Grass Fed Whey Protein; BN Labs Lot # U0637AL; whey protein powder with 8.57 % w/w leucine), suspended in 100 pL of water, was administered to each mouse by oral gavage. Following the whey protein meal, mice received either vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or enzyme (LDC of SEQ ID NO: 484, SEQ ID NO: 686, or SEQ ID NO: 766, dosed at 200 mg/kg, diluted in vehicle) via oral gavage (100 pL/animal).
  • vehicle 20 mM sodium phosphate + 0.4 mM PLP, pH 7.2
  • enzyme enzyme
  • mice Following administration of the whey protein meal and vehicle, the mice had a significant increase in plasma leucine.
  • mice from the prior study were randomized into groups (mixed-sex, 2.5 - 4 months old, ⁇ 20 g BW) after a minimum 2-week washout. Mice had ad libitum access to leucine- free diet and leucine -supplemented water (5.75 g/L leucine) throughout the study. Approximately 45 mg of whey (Grass Fed Whey Protein; BN Labs Lot # U0637AL; whey protein powder with 8.57 % w/w leucine) suspended in 100 pL water was administered to each mouse by oral gavage.
  • whey Grass Fed Whey Protein; BN Labs Lot # U0637AL; whey protein powder with 8.57 % w/w leucine
  • mice received either vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or LDC of SEQ ID NO: 766 (50, 100, or 200 mg/kg diluted in vehicle) via oral gavage (100 pL/animal).
  • Blood was drawn through the tail vein at scheduled time points (pre-dose, and 15, 30, 60, 120, and 240 minutes post-dose).
  • Plasma was extracted from the collected blood samples and then analyzed by LC- MS to determine leucine, isoleucine, valine, phenylalanine, and methionine levels using respective standard curves (LOQ: 2 pM). Background (pre-dose) subtraction was done to correct for high baseline variance between animals.
  • Naive iMSUD mice were subjected to leucine restriction (Research Diets #A05080202i leucine- free diet supplemented with 5.75 g/L leucine; Sigma #L8912) upon weaning to enhance health and extend survival. Twenty-four iMSUD mice, at least 20 g BW, were randomized into groups (mixed-sex, 2.5 - 6 months old) for study. Leucine -supplemented water (5.75 g/L) was changed to regular water ⁇ 40- 60 minutes prior to the first blood collection and replaced following the final blood collection. Mice had ad libitum access to leucine-free diet and water throughout the study.
  • mice Approximately 45 mg of whey (Grass Fed Whey Protein; BN Labs Lot # U0637AL; whey protein powder with 8.57 % w/w leucine) suspended in 100 pL water was administered to each mouse by oral gavage. Following the whey protein meal, the mice received either vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or engineered leucine decarboxylase (SEQ ID NO: 766 or SEQ ID NO: 828 dosed at 100 mg/kg, diluted in vehicle) via oral gavage (100 pL/animal). Blood was drawn through the tail vein at scheduled time points (pre-dose, and 15, 30, 60, 120, and 240 minutes post-dose). Plasma was extracted from the collected blood samples and then analyzed by LC-MS to determine leucine, isoleucine, valine, phenylalanine, and methionine levels using respective standard curves (LOQ: 2 pM).
  • LOQ 2 pM
  • whey whey (Grass Fed Whey Protein, BN Labs Lot # U0637AL; whey protein powder containing 8.57 % w/w leucine) suspended in 20 mL water was administered via oral gavage to each monkey.
  • Cynomolgus monkeys received either vehicle (20 mM sodium phosphate + 0.4 mM PLP, pH 7.2) or an engineered leucine decarboxylase (SEQ ID NO: 484, SEQ ID NO: 686, or SEQ ID NO: 766 dosed at 25, 50, 100 mg/kg diluted in vehicle) via oral gavage as a suspension (2.5 mL/kg, 7.5 - 10 mL/animal).
  • the standard diet (Certified Primate Chow 2055C; Envigo) was offered to all animals approximately 8 hours after treatment. Blood was collected via the femoral vein twice pre-dose (- 1.5 hours and - 30 minutes) and then at 5, 15, and 30 minutes, and 1, 2, 4, 8, 12, and 24 hours post-dose. Plasma was extracted from the collected blood samples and analyzed by LC-MS for levels of leucine, isoleucine, valine, phenylalanine, methionine, and tyrosine, against respective standard curves (LOQ: 4, 8, 8.5, 6, 7, and 11 pM, respectively).
  • the standard diet (Certified Primate Diet 5048; PMI) was offered to all animals approximately 8 hours after treatment. Blood was collected via the femoral vein at pre-dose and then at 5, 15, and 30 minutes, and 1, 2, 4, 8, 12, and 24 hours post-dose. Plasma was extracted from the collected blood samples and analyzed by LC-MS for levels of leucine, ketoisocaproic acid (KIC), isoleucine, valine, methionine, and phenylalanine against respective standard curves (LOQ: 15, 10, 10, 15, 5, and 10 pM, respectively).
  • KIC ketoisocaproic acid
  • LOQ phenylalanine
  • mice were subjected to leucine restriction (Research Diets #A05080202i leucine-free diet supplemented with 5.75 g/L leucine; Sigma #L8912) upon weaning to enhance health and extend survival.
  • leucine restriction Research Diets #A05080202i leucine-free diet supplemented with 5.75 g/L leucine; Sigma #L8912
  • mice had ad libitum access to leucine-free diet and leucine-supplemented water throughout the study.
  • whey protein powder (Grass Fed Whey Protein; BN Labs Lot # X01010AL0R; with 8.613 % w/w leucine, 0% free amino acids), suspended in 100 pL of water, was administered to each mouse by oral gavage.
  • mice received either vehicle (1 mM sodium phosphate + 0.4 mM PLP, pH 7.5) or enzyme (SEQ ID NO: 828 dosed at 25, 50 and 100 mg/kg, diluted in vehicle without PLP) via oral gavage.
  • the study design followed a 2-phase crossover with a two week washout in between.
  • the standard diet (Certified Primate Diet 5048, PMI Inc.) was offered to all animals approximately 8 hours after treatment. Blood was collected via the femoral vein twice pre-dose (1 and 2 hours) and then post-dose 5, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hours post-dose. Plasma was extracted from the collected blood samples and analyzed by LC-MS/MS for levels of leucine, isoleucine, valine, phenylalanine, and methionine, against respective standard curves (LOQ: 4, 8, 8.5, 6, and 7 pM respectively).
  • LDC did not induce a statistically significant effect in iAUC in plasma levels of isoleucine, valine, methionine, or phenylalanine with the treatment (Table 18). Statistical calculations and significance were determined using GraphPad Prism 9 (GraphPad Software).
  • the study design followed a 2-phase crossover with a one week washout in between.
  • the standard diet (Certified Primate Diet 5048, PMI Inc.) was offered to all animals approximately 8 hours after treatment.
  • Blood was collected via the femoral vein twice pre-dose (1 and 2 hours) and then post-dose 5, 15, and 30 minutes, and 1, 2, 4, 8, and 24 hours post-dose.
  • Plasma was extracted from the collected blood samples and analyzed by LC-MS/MS for levels of leucine, isoleucine, valine, phenylalanine, and methionine, against respective standard curves (LOQ: 4, 8, 8.5, 6, and 7 pM respectively).
  • the standard diet (Certified Primate Diet 5048; PMI Inc.) was offered to all animals approximately 8 hours after treatment. This protocol was repeated for three consecutive days, with animals keeping the same group designation throughout the study. On each dosing day, blood was collected via the femoral vein at 1 hour pre-dose and then at 1, 2, 4, 8, 12, and 24 (day 3 only) hours post-dose. Plasma was extracted from the collected blood samples and analyzed by LC-MS/MS for levels of leucine, isoleucine, valine, methionine, and phenylalanine against respective standard curves (LOQ: 15, 10, 15, 5, and 10 DM, respectively).

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