WO2021158686A1 - Engineered leucine decarboxylases - Google Patents

Engineered leucine decarboxylases Download PDF

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Publication number
WO2021158686A1
WO2021158686A1 PCT/US2021/016450 US2021016450W WO2021158686A1 WO 2021158686 A1 WO2021158686 A1 WO 2021158686A1 US 2021016450 W US2021016450 W US 2021016450W WO 2021158686 A1 WO2021158686 A1 WO 2021158686A1
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Prior art keywords
polypeptide
engineered
sequence
seq
amino acid
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English (en)
French (fr)
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WO2021158686A8 (en
Inventor
Joyce Liu
Leann Quertinmont TEADT
Nikki DELLAS
Stephan JENNE
Faye Loan DU
Kristen Jean VALLIEU
Kerryn Mccluskie
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Codexis Inc
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Codexis Inc
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Priority to PH1/2022/551776A priority Critical patent/PH12022551776A1/en
Priority to AU2021216389A priority patent/AU2021216389A1/en
Priority to CA3168922A priority patent/CA3168922A1/en
Priority to EP21751078.3A priority patent/EP4103711A4/en
Priority to JP2022547083A priority patent/JP2023512683A/ja
Priority to CN202180026927.0A priority patent/CN116113691A/zh
Priority to IL295169A priority patent/IL295169A/en
Priority to KR1020227030236A priority patent/KR20220137057A/ko
Application filed by Codexis Inc filed Critical Codexis Inc
Priority to BR112022015139A priority patent/BR112022015139A2/pt
Priority to MX2022009531A priority patent/MX2022009531A/es
Publication of WO2021158686A1 publication Critical patent/WO2021158686A1/en
Publication of WO2021158686A8 publication Critical patent/WO2021158686A8/en
Anticipated expiration legal-status Critical
Priority to JP2025114444A priority patent/JP2025143444A/ja
Ceased legal-status Critical Current

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    • 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
    • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
    • A61K38/00—Medicinal preparations containing peptides
    • 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
    • C12N15/00—Mutation or genetic engineering; DNA or RNA concerning genetic engineering, vectors, e.g. plasmids, or their isolation, preparation or purification; Use of hosts therefor
    • C12N15/09—Recombinant DNA-technology
    • C12N15/63—Introduction of foreign genetic material using vectors; Vectors; Use of hosts therefor; Regulation of expression
    • C12N15/70—Vectors or expression systems specially adapted for E. coli
    • 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 engineered LDC polypeptides are optimized to provide improved storage stability.
  • the present invention also provides methods for the use of the compositions comprising the engineered LDC polypeptides for therapeutic and industrial purposes.
  • REFERENCE TO A "SEQUENCE LISTING,” A TABLE, OR A COMPUTER PROGRAM LISTING APPENDIX SUBMITTED AS AN ASCII TEXT FILE [0003] The Sequence Listing written in file CX7-198WO2_ST25.TXT, created on February 3, 2021, with a size of 2.06 megabytes, machine format IBM-PC, MS-Windows operating system, is hereby incorporated by reference.
  • Maple syrup urine disease also referred to as “leucineuria,” branched chain alpha- leucine 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. (Menkes et al., Pediatrics 14:462-467 [1954]) and named due to the distinctive, sweet odor of the urine of affected newborns.
  • BCKDH keto acid dehydrogenase
  • BCAAs branched-chain amino acids
  • BCKDH complex enzymes results in 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).
  • BCAA turnover via renal clearance results in the typical sweet, maple syrup smell of affected patients’ urine
  • the engineered LDC polypeptides are optimized to provide improved storage stability.
  • the present invention also provides methods for the use of the compositions comprising the engineered LDC polypeptides for therapeutic and industrial purposes.
  • the present invention is directed to engineered LDC polypeptides and biologically active fragments and analogs thereof having improved properties when compared to a wild-type LDC enzyme or a reference LDC polypeptide under essentially the same conditions.
  • the invention is further directed to methods of using the engineered LDC polypeptides and biologically active fragments and analogs thereof in therapeutic and/or industrial compositions and methods of using such compositions for therapeutic and/or industrial purposes.
  • the present invention provides engineered leucine decarboxylase polypeptides comprising amino acid sequences 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, and/or 766, wherein the amino acid positions of said amino acid sequences are numbered with reference to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • the present invention also provides engineered leucine decarboxylase polypeptides wherein said polypeptide sequences have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2.
  • the present invention further provides engineered leucine decarboxylase polypeptides wherein said polypeptide sequences have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4.
  • the present invention further provides engineered leucine decarboxylase polypeptides wherein said polypeptide sequences have at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 10.
  • the present invention further provides engineered leucine decarboxylase polypeptides wherein said polypeptide sequences have 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 polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 5M, 14I, 14T/34L/38V/39N/102S/267I/275S/350E/357V, 14T/39N/102S/127S/245M/267I/275S/349T/350E, 34L/38V/39N/102S/127S/275S/357V, 34L/38V/39N/102S/275S/357V, 34L/38V/39N/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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more positions selected from K5M, H14I, H14T/I34L/C38V/T39N/T102S/V267I/T275S/N350E/I357V, H14T/T39N/T102S/T127S/I245M/V267I/T275S/V349T/N350E, I34L/C38V/T39N/T102S/T127S/T275S/I357V, I34L/C38V/T39N/T102S/T275S/I357V, I34L/C38V/T39N/T127S/I245M/V349T/N350E/I357V, I34L/C38V/T39N/T127S/I245M/V349T/N350E/I357V, I34L/C38V/T39
  • the engineered leucine decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:38, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 64/164/318/324/357/364, 64/324/353/364, 132/255/339/379/395, 164/196/324/357/364, 164/318/324/34343
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 48F/64E/164A/324M/343E/353E/357C/364K, 48F/64E/164A/324M/343E/364R, 48F/64E/164C/353N/357V/364R, 48F/64E/357M/364K, 64E/164A/324M/343E/353D/357V/364K, 64E/164A/324M/343E/353D/357V, 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/343
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from L48F/A64E/I164A/R324M/H343E/R353E/I357C/L364K, L48F/A64E/I164A/R324M/H343E/L364R, L48F/A64E/I164C/R353N/I357V/L364R, L48F/A64E/I357M/L364K, A64E/I164A/R324M/H343E/R353D/I357V/L364K, A64E/I164A/R324M/H343E/I357C/L364R, A64E/I164C/R353D/I357V, A64E/R318K/R324S/I357V/L364R,
  • the engineered leucine decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 234, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 3
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 2E, 3M, 33L, 48F/64E/255P, 48F/255P/339A, 48F/255P/379D, 64E, 64E/255P, 64S, 69I, 161V, 193I, 255P, 255P/318K/379D, 259L, 263T, 263V, 318K/339A/379D, 324N, 324N/394E/395K/397A, 324N/395D, 324S/389G/394E, 324S/389G/394E/395D, 324S/389G/394E/397A, 324S/394E, 324S/394E/395K, 324S/394E/395K, 324S/394E/395K/397A, 324S/395K, 339A, 340T, 340V, 380
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the engineered leucine decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 284, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from G2E/E64S/V69I/M324S/A380E/A382S/Q388A/K389G, N3M/E64S/V69I/S263T/Q339A/A380E/Q388A, N3M/E64S/V69I/K389G, N3M/E64S/V69I/P390*, N3M/E64S/K379D/A380E/P390*, N3M/V69I/S263T/A380E, N3M/V69I/M324S, N3M/V69I/M324S/A380E/A382S/K389G/P390*, S12G/L135V/R259K/S263T, S12G/L135V
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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/270
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the engineered leucine decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 594, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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/90A/126
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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/D126T/H366M, R16Q/A80K/C168K, R16Q/A80K/C168K/L270R/H366M, R16Q/A80K/C168K/M324S, R16Q/A80K/C168K/H366M, R16Q/A80K,
  • the engineered leucine decarboxylase polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO:686, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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 polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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,
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, A
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 19I, 109G, 123F, 123M, 123V, 134A, 134S, 170A, 173A, 173I, 173T, 187L, 211S, and 312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from L19I, L109G, Y123F, Y123M, Y123V, N134A, N134S, P170A, F173A, F173I, F173T, V187L, A211S, and T312A, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 38/39/102/127/267/275/349/350/357, 38/39/102/127/349/350/357, 38/39/102/127/349/350, 38/
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 5M, 14I, 14T/34L/38V/39N/102S/267I/275S/350E/357V, 14T/39N/102S/127S/245M/267I/275S/349T/350E, 34L/38V/39N/102S/127S/275S/357V, 34L/38V/39N/102S/275S/357V, 34L/38V/39N/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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 48/64/164/324/343/353/364, 48/64/164/324/343/364, 48/64/164/353/357/364, 48/64/357/364, 64/164/324/343/353/357/364, 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, 164/324/343/353/364, 164/324/357/364, 164/353/357/364, 164/364, 196/318/324/353/357/364, 318/357/364, 318/357/364, 318/343/357, 3
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions from L48F/A64E/I164A/R324M/H343E/R353E/I357C/L364K, L48F/A64E/I164A/R324M/H343E/L364R, L48F/A64E/I164C/R353N/I357V/L364R, L48F/A64E/I357M/L364K, A64E/I164A/R324M/H343E/R353D/I357V/L364K, A64E/I164A/R324M/H343E/I357C/L364R, A64E/I164C/R353D/I357V, A64E/R318K/R324S/I357V/L364R, A
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 64/69/339, 64
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from G2E/E64S/V69I/M324S/A380E/A382S/Q388A/K389G, N3M/E64S/V69I/S263T/Q339A/A380E/Q388A, N3M/E64S/V69I/K389G, N3M/E64S/V69I/P390*, N3M/E64S/K379D/A380E/P390*, N3M/V69I/S263T/A380E, N3M/V69I/M324S, N3M/V69I/M324S/A380E/A382S/K389G/P390*, S12G/L135V/R259K/S263T, S12G/L135V
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 259/
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, 16/80/324, 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, 80/126/168/270/366, 80/126/168/366, 80/126/181/270/3
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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/90A/126
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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/D126T/H366M, R16Q/A80K/C168K, R16Q/A80K/C168K/L270R/H366M, R16Q/A80K/C168K/M324S, R16Q/A80K/C168K/H366M, R16Q/A80K,
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 66/76/118/141/201/300, 66/76/198/200/296/303, 66/76/198/200/300, 66/118/200/296/303/317, 66/118/296, 66/118/296/300, 66/200, 76/118/141/200/296, 76/141/198/200/201/300, 80/201/270, 80/270, 80/270/324, 89/118/200, 106/270/324/352, 118/141/200, 126, 126/201/270/324, 126/270, 141/144/198/200/300, 156/270, 156/270/324, 201/270, 201/270/352, 270, and 270/324, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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,
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 19, 109, 123, 134, 170, 173, 187, 211, and 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 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, and 109/211/270/312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 688.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from L19I/L109G/Y123F/P170A/A211S/L270R/T312A, L19I/L109G/Y123F/A198G/H200S/A211S/L270R/T312A, L19I/L109G/Y123V/R141P/P170A/A198G/H200S/A211S/L270R/T312A, L19I/L109G/Y123V/R141P/P170A/A198G/A211S, L19I/L109G/Y123V/R141P/P170A/A198G/A211S, L19I/L109G/Y123V/R141P/P170A/A198G/A211S/L270R/T312A, L19I/L109G/P170A/F173
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 5/41, 5/41/228, 33, 41, 47, 51, 55, 64, 126, 265, 267, 270, 331, 353, 357, and 384, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 5V/41D, 5V/41D/228D, 33L, 41D, 47F, 51E, 51Q, 55I, 64N, 126A, 126T, 265P, 267L, 270A, 270T, 331V, 353E, 353I, 353L, 357S, and 384W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from K5V/H41D, K5V/H41D/T228D, F33L, H41D, L47F, L51E, L51Q, V55I, S64N, D126A, D126T, E265P, I267L, R270A, R270T, T331V, D353E, D353I, D353L, C357S, and P384W, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from 66S, 66S/118T, 66S/118T/296D, 66S/118T/296D/300R, 66S/118T/300R, 66S/296D, 66S/296D/300R, 66S/300R, 118T, 118T/296D, 118T/296D/300R, 118T/300R, 296D, 296D/300R, and 300R, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.
  • the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from N66S, N66S/D118T, N66S/D118T/E296D, N66S/D118T/E296D/K300R, N66S/D118T/K300R, N66S/E296D, N66S/E296D/K300R, N66S/K300R, D118T, D118T/E296D, D118T/E296D/K300R, D118T/K300R, E296D, E296D/K300R, and K300R, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, or a functional fragment thereof.
  • the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, or a functional fragment thereof. In some additional embodiments, the engineered leucine decarboxylase polypeptide comprises an amino acid sequence having at least 95% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, or a functional fragment thereof.
  • the engineered leucine decarboxylase polypeptide is a variant leucine decarboxylase polypeptide provided in any of Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and/or 11-2.
  • the engineered leucine decarboxylase polypeptide is a Planctomycetaceae bacteria species variant enzyme.
  • the leucine decarboxylase 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. In yet some additional embodiments, the engineered leucine decarboxylase polypeptide is more thermostable than wild-type Planctomycetaceae bacteria species leucine decarboxylase. In yet some further embodiments, the engineered leucine decarboxylase polypeptide more resistant to proteolysis than wild-type Planctomycetaceae bacteria species leucine decarboxylase.
  • the engineered leucine decarboxylase polypeptide comprises a sequence at least 90%, identical to any of the even-numbered sequences of SEQ ID NOS: 16-852. In some further embodiments, the engineered leucine decarboxylase polypeptide comprises any of the even-numbered sequences of SEQ ID NOS: 16-852. In some additional embodiments, the engineered leucine decarboxylase polypeptide is purified.
  • the present invention also provides compositions comprising at least one engineered leucine decarboxylase polypeptide provided herein.
  • the present invention also provides compositions comprising an engineered leucine decarboxylase polypeptide provided herein.
  • the engineered polynucleotide sequence comprises a sequence at least 90% or more identical to any of the odd- numbered sequences of SEQ ID NOS: 15-851. In some further embodiments, the engineered polynucleotide sequence comprises any of the odd-numbered sequences of SEQ ID NOS: 15-851. In some additional embodiments, the engineered polynucleotide sequence is operably linked to a control sequence. In some embodiments, the engineered polynucleotide sequence is codon-optimized. [0032]
  • the present invention also provides expression vectors comprising at least one engineered polynucleotide sequence provided herein. In some embodiments, the expression vectors further comprise at least one control sequence.
  • control sequence comprises a promoter. In some further embodiments, the promoter is a heterologous promoter.
  • the present invention also provides host cells transformed with at least one polynucleotide sequence and/or comprising an expression vector provided herein. In some embodiments, the host cells are transformed with at least one polynucleotide sequence provided herein. In some embodiments, the host cells are transformed with a polynucleotide sequence provided herein. In some additional embodiments, the host cell comprise at least one expression vector provided herein. In some further embodiments, the host cells comprise an expression vector provided herein. In some embodiments, the host cell is E. coli.
  • the methods of producing an engineered leucine decarboxylase polypeptide in a host cell comprise culturing a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide provided herein, under suitable culture conditions, such that at least one engineered leucine decarboxylase polypeptide is produced.
  • the methods of producing an engineered leucine decarboxylase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide sequence provided herein, under suitable culture conditions, such that at least one engineered leucine decarboxylase polypeptide is produced.
  • the composition is suitable for use in gene therapy to treat maple syrup urine disease, and/or elevated blood levels of isoleucine, leucine, alloisoleucine and/or valine. In some additional embodiments, the composition is suitable for use in mRNA therapy. In yet some additional embodiments, the composition is suitable for oral administration to a human. In some embodiments, the composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. In some further embodiments, the pill, tablet, capsule, or gelcap further comprises an enteric coating. In yet some additional embodiments, the composition is suitable for parenteral injection into an animal. In yet some additional embodiments, the composition is suitable for parenteral injection into a human.
  • the injections are administered on a daily, weekly, or monthly basis.
  • composition is coadministered with at least one additional therapeutically effective compound.
  • the composition comprises at least one additional therapeutically effective compound.
  • the present invention also provides methods for treating and/or preventing the symptoms of maple syrup urine disease in a subject, comprising providing a subject having maple syrup urine disease, and providing the composition provided herein to said subject. In some embodiments, the symptoms of maple syrup urine disease are ameliorated.
  • the subject is able to eat a diet that is less restricted in its in isoleucine, leucine, and/or valine content than diets required by subjects who have not been provided at least one composition comprising at least one engineered leucine decarboxylase polypeptide and/or polynucleotide provided herein. In some embodiments, the subject is able to eat a diet that is less restricted in its in isoleucine, leucine, and/or valine content than diets required by subjects who have not been provided at least one composition comprising at least one engineered leucine decarboxylase polypeptide provided herein. [0037] In some embodiments, the subject is an infant, child, young adult, or adult.
  • the subject is an infant. In some embodiments, the subject is a child. [0038] In some embodiments, the subject is a young adult. In some embodiments, the subject is an adult.
  • the present invention also provides for the use of the compositions provided herein.
  • the compositions comprise at least one engineered leucine decarboxylase polypeptide and/or polynucleotide provided herein. In some embodiments, the compositions comprise at least one engineered leucine decarboxylase polypeptide provided herein. In some embodiments, the compositions comprise an engineered leucine decarboxylase polypeptide provided herein.
  • the engineered LDC polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis, and/or increased tolerance to low pH environments. In some embodiments, the engineered LDC polypeptides are optimized to provide improved storage stability.
  • the present invention also provides methods for the use of the compositions comprising the engineered LDC polypeptides for therapeutic and industrial purposes.
  • the present invention provides engineered LDC polypeptides, mutants, biologically active fragments and analogues thereof, and pharmaceutical and industrial compositions comprising the same.
  • 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. [0044]
  • 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.
  • PBP pyridoxial 5’-phosphate
  • 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 (Gln or Q), histidine (His or H), isoleucine (Ile or I), leucine (Leu or L), lysine (Lys or K), methionine (Met or M), phenylalanine (Phe or F), proline (Pro or P), serine (Ser or S), threonine (Thr or T), tryptophan (Trp or W), tyrosine (Tyr or Y), and valine (Val or V).
  • 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.
  • 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 polypeptide sequence in the comparison window may comprise additions or deletions (i.e., gaps) as compared to the reference sequence for optimal alignment of the two sequences.
  • the percentage 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.
  • HSPs high scoring sequence pairs
  • the word hits are then extended in both directions along each sequence for as far as the cumulative alignment score can be increased. Cumulative scores are calculated using, for nucleotide sequences, the parameters “M” (reward score for a pair of matching residues; always >0) and “N” (penalty score for mismatching residues; always ⁇ 0). For amino acid sequences, a scoring matrix is used to calculate the cumulative score. Extension of the word hits in each direction are halted when: the cumulative alignment score falls off by the quantity “X” from its maximum achieved value; the cumulative score goes to zero or below, due to the accumulation of one or more negative-scoring residue alignments; or the end of either sequence is reached.
  • the BLAST algorithm parameters W, T, and X determine the sensitivity and speed of the alignment.
  • the 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 89:10915 [1989]).
  • 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 polypeptide 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 LDC, can be aligned to a reference sequence by introducing gaps to optimize residue matches between the two sequences.
  • amino acid difference and “residue difference” refer to a difference in the amino acid residue at a position of a polypeptide sequence relative to the amino acid residue at a corresponding position in a reference sequence.
  • 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., 109G).
  • 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 is the single letter identifier of the residue in the reference sequence
  • n is the number of the residue position in the reference sequence
  • 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.
  • amino acid substitution set and “substitution set” refers to a group of amino acid substitutions within a polypeptide sequence. In some embodiments, 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 LDC polypeptides listed in any of the Tables in the Examples (i.e., Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and/or 11-2).
  • “Conservative amino acid substitution” refers to a substitution of a residue with a different residue having a similar side chain, and thus typically involves substitution of the amino acid in the polypeptide with amino acids within the same or similar defined class of amino acids.
  • 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 amino acid with an acidic side chain (e.g., aspartic acid or glutamic acid); and a hydrophobic or hydrophilic amino acid is replaced with another hydrophobic or hydrophilic amino acid, respectively.
  • 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.
  • the terms “functional fragment” and “biologically active fragment” are used interchangeably herein, to refer to a polypeptide that has an amino-terminal and/or carboxy-terminal deletion(s) and/or internal 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.
  • “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 LDC 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 LDC 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 LDC 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.
  • “Improved enzyme property” refers to an engineered LDC polypeptide that exhibits an improvement in any enzyme property as compared to a reference LDC polypeptide, such as a wild-type LDC polypeptide (e.g., wild-type LDC having SEQ ID NO: 2) or another engineered LDC polypeptide.
  • a reference LDC polypeptide such as a wild-type LDC polypeptide (e.g., wild-type LDC having SEQ ID NO: 2) or another engineered LDC polypeptide.
  • “Increased enzymatic activity” and “enhanced catalytic activity” refer to an improved property of the engineered LDC 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 LDC) as compared to the reference LDC enzyme (e.g., wild-type LDC and/or another engineered LDC). 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.
  • the engineered LDC polypeptides have a specific activity of at least 0.01 ⁇ mol/min-mg, at least 0.02/ ⁇ mol/min-mg, at least 0.03/ ⁇ mol/min-mg, at least 0.05/ ⁇ mol/min-mg, at least 1.0/ ⁇ mol/min-mg, and in some preferred embodiments greater than 2.0/ ⁇ mol/min-mg.
  • the Km is in the range of about 1 ⁇ m to about 5mM; in the range of about 5 ⁇ m to about 2mM; in the range of about 10 ⁇ m to about 2mM; or in the range of about 10 ⁇ m to about 1mM.
  • the phrases “increased storage stability” means that an engineered LDC polypeptide according to the invention will retain more activity compared to a reference LDC 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.).
  • “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. Thus, the “enzymatic activity” or “activity” of a LDC 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.
  • moderately stringent 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, 5 ⁇ Denhart's solution, 5 ⁇ SSPE, 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, 5 ⁇ Denhart's solution, 5 ⁇ SSPE, 0.2% SDS at 42°C, followed by washing in 0.1 ⁇ SSPE, 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 0.1x 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.
  • “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.
  • the polynucleotides encoding the LDC enzymes are codon optimized for optimal production from the host organism selected for expression.
  • 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.
  • 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 LDC polypeptide.
  • “Product” in the context of an enzymatic conversion process refers to the compound or molecule resulting from the action of the LDC polypeptide on the substrate.
  • the term “culturing” refers to the growing of a population of microbial cells under suitable conditions using any suitable medium (e.g., liquid, gel, or solid).
  • Recombinant polypeptides e.g., LDC enzyme variants
  • LDC enzyme variants 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).
  • 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.
  • 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 heterologous 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.
  • the term “therapeutic” refers to a compound administered to a subject who shows signs or symptoms of pathology having beneficial or desirable medical effects.
  • pharmaceutical composition refers to a composition suitable for pharmaceutical use in a mammalian subject (e.g., human) comprising a pharmaceutically effective amount of an engineered LDC polypeptide encompassed by the invention and an acceptable carrier.
  • gene therapy is used in reference to the use of genes (i.e., genetic material) to treat and/or prevent disease in a mammalian subject (e.g., human). In some embodiments, the genetic material is introduced directly into at least some cells of the mammalian subject.
  • 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.
  • effective amount means an amount sufficient to produce the desired result. One of general skill in the art may determine what the effective amount by using routine experimentation.
  • 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 lagamorphs). 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 born 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.
  • 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.
  • composition and “formulation” encompass products comprising at least one engineered LDC of the present invention, intended for any suitable use (e.g., pharmaceutical compositions, dietary/nutritional supplements, feed, etc.).
  • compositions of the present invention 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.
  • the term “excipient” refers to any pharmaceutically acceptable additive, carrier, diluent, adjuvant, or other ingredient, other than the active pharmaceutical ingredient (API; e.g., the engineered LDC polypeptides of the present invention). Excipients are typically included for formulation and/or administration purposes.
  • the term “therapeutically 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).
  • 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.
  • the term “therapeutically 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.
  • the terms “treating,” “treat” and “treatment” 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.
  • engineered LDC polypeptides are produced by cultivating a microorganism comprising at least one polynucleotide sequence encoding at least one engineered LDC polypeptide under conditions which are conducive for producing the engineered LDC polypeptide.
  • the engineered LDC polypeptide is subsequently recovered from the resulting culture medium and/or cells.
  • the present invention provides exemplary engineered LDC polypeptides having LDC activity.
  • the Examples provide Tables (i.e., Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and/or 11-2) showing sequence structural information correlating specific amino acid sequence features with the functional activity of the engineered LDC polypeptides. This structure-function correlation information is provided in the form of specific amino acid residue differences relative to the reference engineered polypeptide of SEQ ID NO: 2, as well as associated experimentally determined activity data for the exemplary engineered LDC polypeptides.
  • the engineered LDC polypeptides of the present invention having LDC activity comprise a) an amino acid sequence having at least 85% sequence identity to reference sequence SEQ ID NO: 2; b) an amino acid residue difference as compared to SEQ ID NO: 2 at one or more amino acid positions; and c) which exhibits an improved property selected from i) enhanced catalytic activity, ii) reduced proteolytic sensitivity, iii) reduced aggregation, iv) increased stability as a lyophilized preparation to elevated temperatures, v) reduced immunogenicity, or a combination of any of i), ii), iii), iv), or v), as compared to the reference sequence.
  • 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.
  • the present invention provides functional fragments of engineered LDC polypeptides.
  • functional fragments comprise at least about 95%, 96%, 97%, 98%, or 99% of the activity of the engineered LDC polypeptide from which it was derived (i.e., the parent engineered LDC).
  • functional fragments comprise at least 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, or 99% of the parent sequence of the engineered LDC.
  • 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 engineered LDC polypeptide comprises an amino acid sequence having at least about 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99% or more identity to a reference sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, or the amino acid sequence of any variant (e.g., those provided in the Examples).
  • the reference sequence is selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • the engineered LDC polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to reference sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, and one or more residue differences as compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • the present invention provides methods and compositions for the production of each and every possible variation of LDC polynucleotides that could be made that encode the LDC polypeptides described herein by selecting combinations based on the possible codon choices, and all such variations are to be considered specifically disclosed for any polypeptide described herein, including the amino acid sequences presented in the Examples (e.g., in Tables 1-2, 2-1, 3-2, 4-1, 5-1, 6-1, 7-1, 8-1, 8-2, 10-1, 11-1, and/or 11- 2).
  • the codons are preferably optimized for utilization by the chosen host cell for protein production. For example, preferred codons used in bacteria are typically used for expression in bacteria.
  • the reference sequence is selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • the LDC polynucleotide encodes an engineered polypeptide having LDC activity with the properties disclosed herein, wherein the polypeptide comprises an amino acid sequence having at least 80%, 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to reference sequence SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, and one or more residue differences as compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • the polynucleotides are capable of hybridizing under highly stringent conditions to a reference polynucleotide sequence selected from SEQ ID NOS: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, or a complement thereof, or a polynucleotide sequence encoding any of the variant LDC polypeptides provided herein.
  • the polynucleotide capable of hybridizing under highly stringent conditions encodes a LDC polypeptide comprising an amino acid sequence that has one or more residue differences as compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766.
  • an isolated polynucleotide encoding any of the engineered LDC polypeptides herein is manipulated in a variety of ways to facilitate expression of the LDC polypeptide.
  • 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. coli lac operon, Streptomyces coelicolor agarase gene (dagA), Bacillus subtilis levansucrase gene (sacB), Bacillus licheniformis alpha-amylase gene (amyL), Bacillus stearothermophilus maltogenic amylase gene (amyM), Bacillus amyloliquefaciens alpha-amylase gene (amyQ), Bacillus licheniformis penicillinase gene (penP), Bacillus subtilis xylA and xylB genes, and prokaryotic beta-lactamase gene (See e.g., Villa-Kamaroff et al., Proc.
  • Exemplary transcription terminators for filamentous fungal host cells can be obtained from the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Aspergillus niger alpha-glucosidase, and Fusarium oxysporum trypsin- like protease.
  • Exemplary terminators for yeast host cells can be obtained from the genes for Saccharomyces cerevisiae enolase, Saccharomyces cerevisiae cytochrome C (CYC1), and Saccharomyces cerevisiae glyceraldehyde-3-phosphate dehydrogenase.
  • control sequence is also a suitable leader sequence (i.e., a non- translated 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 LDC 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
  • Exemplary polyadenylation sequences for filamentous fungal host cells include, but are not limited to the genes for Aspergillus oryzae TAKA amylase, Aspergillus niger glucoamylase, Aspergillus nidulans anthranilate synthase, Fusarium oxysporum trypsin-like protease, and Aspergillus niger alpha- glucosidase.
  • Useful polyadenylation sequences for yeast host cells are known (See e.g., Guo and Sherman, Mol. Cell. Bio., 15:5983-5990 [1995]).
  • 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.
  • 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.
  • 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 GAL1 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 LDC 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 LDC 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 LDC 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.
  • 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.
  • argB ornithine carbamoyltransferases
  • bar phosphinothricin acetyltransferase; e.g., from S. hygroscopicus), hph (hygromycin phosphotransferase), niaD (nitrate reductase), pyrG (orotidine-5'- phosphate decarboxylase; e.g., from A. nidulans or A. orzyae), sC (sulfate adenyltransferase), and trpC (anthranilate synthase), as well as equivalents thereof.
  • the present invention provides a host cell comprising at least one polynucleotide encoding at least one engineered LDC polypeptide of the present invention, the polynucleotide(s) being operatively linked to one or more control sequences for expression of the engineered LDC 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 ( ⁇ fhuA) and BL21).
  • the present invention provides methods of producing the engineered LDC polypeptides, where the methods comprise culturing a host cell capable of expressing a polynucleotide encoding the engineered LDC polypeptide under conditions suitable for expression of the polypeptide. In some embodiments, the methods further comprise the steps of isolating and/or purifying the LDC polypeptides, as described herein. [0136] Appropriate 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 LDC polypeptides into cells will find use in the present invention.
  • Engineered LDC polypeptides with the properties disclosed herein can be obtained by subjecting the polynucleotide encoding the naturally occurring or engineered LDC 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.
  • Mutagenesis and directed evolution methods can be readily applied to LDC-encoding polynucleotides to generate variant libraries that can be expressed, screened, and assayed.
  • 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 LDC 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 HPLC 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 LDC 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 LDC 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.
  • any of the engineered LDC 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 LDC polypeptides include, among others, reverse phase chromatography, high-performance liquid chromatography, ion-exchange chromatography, hydrophobic-interaction chromatography, size-exclusion chromatography, gel electrophoresis, and affinity chromatography.
  • affinity techniques may be used to isolate the improved LDC enzymes.
  • any antibody that specifically binds a LDC polypeptide of interest may find use.
  • various host animals including but not limited to rabbits, mice, rats, etc., are immunized by injection with a LDC polypeptide, or a fragment thereof.
  • the LDC 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 LDC 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 LDC polypeptide as described herein under conditions conducive to the production of the engineered LDC polypeptide and recovering the engineered LDC polypeptide from the cells and/or culture medium.
  • the host cell produces more than one engineered LDC polypeptide.
  • the present invention provides a method of producing an engineered LDC polypeptide comprising culturing a recombinant bacterial cell comprising a polynucleotide sequence encoding an engineered LDC polypeptide having at least 85%, 90%, 95%, 96%, 97%, 98%, or 99% sequence identity to reference sequences SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, and one or more amino acid residue differences as compared to SEQ ID NO: 2, 4, 6, 8, 10, 12, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, as provided herein, under suitable culture conditions to allow the production of the engineered LDC polypeptide and optionally recovering the engineered LDC polypeptide from the culture and/or cultured bacterial cells.
  • the present invention also provides engineered LDC polypeptides suitable for use in 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).
  • Pharmaceutical and Other Compositions [0151] The present invention provides engineered LDC polypeptides suitable for use in pharmaceutical and other compositions, such as dietary/nutritional supplements. [0152] Depending on the mode of administration, these compositions comprising a therapeutically effective amount of an engineered LDC according to the invention are in the form of a solid, semi- solid, or liquid.
  • Any suitable format for use in delivering the engineered LDC 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 LDC polypeptides are provided in a format suitable for injection (i.e., in an injectable formulation).
  • the engineered LDC polypeptides are provided in biocompatible matrices such as sol- gels, including silica-based (e.g., oxysilane) sol-gels.
  • the engineered LDC polypeptides are encapsulated.
  • the engineered LDC polypeptides are encapsulated in nanostructures (e.g., nanotubes, nanotubules, nanocapsules, or microcapsules, microspheres, liposomes, etc.).
  • the present invention be limited to any particular delivery formulation and/or means of delivery. It is intended that the engineered LDC 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. [0154] In some embodiments, the engineered LDC polypeptides are chemically modified by glycosylation, pegylation (i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.) or other compounds (See e.g., Ikeda, Amino Acids 29:283-287 [2005]; US Pat.
  • pegylation i.e., modified with polyethylene glycol [PEG] or activated PEG, etc.
  • the engineered LDC polypeptides are provided in formulations comprising matrix-stabilized enzyme crystals.
  • the formulation comprises a cross- linked crystalline engineered LDC enzyme and a polymer with a reactive moiety that adheres to the enzyme crystals.
  • the present invention also provides engineered LDC polypeptides in polymers.
  • 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 LDC 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. [0157] In some embodiments, the engineered LDC 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. In some embodiments, the engineered LDC 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 LDC polypeptides suitable for use in decreasing, ameliorating, or eliminating the signs and/or symptoms of MSUD.
  • the dosage of engineered LDC 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 LDC 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 LDC polypeptides are administered in combination with other pharmaceutical and/or dietary compositions.
  • the engineered LDC polypeptides of the present invention will find use in industrial compositions, including such areas as food flavorings (e.g., cheese).
  • the engineered LDC polypeptides are formulated for use in the food and/or feed industries.
  • the engineered LDC 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).
  • the engineered LDC polypeptides are used in liquid animal feed compositions (e.g., aqueous or oil-based slurries).
  • the engineered LDC variants of the present invention are sufficiently thermotolerant and thermostable to withstand the treatment used to produce pellets and other processed feed/foods.
  • ppm parts per million
  • M molar
  • mM millimolar
  • uM and ⁇ M micromolar
  • nM nanomolar
  • mol molecular weight
  • gm and g gram
  • mg milligrams
  • ug and ⁇ g micrograms
  • L and l liter
  • ml and mL milliliter
  • cm centimeters
  • mm millimeters
  • um and ⁇ m 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 ⁇ -D-1-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); innovative Research (Innovative Research, Novi, MI); Microfluidics (Microfluidics Corp., Newton, MA); Thermotron (Thermotron, Holland, MI); Waters (Waters Corp., Mil
  • EXAMPLE 1 Synthesis and Assaying of Amino Acid Decarboxylase Enzymes with Leucine Decarboxylase Activity
  • LDC leucine decarboxylase
  • Method used in the synthesis and assaying of amino acid decarboxylase enzymes for leucine decarboxylase (LDC) activity are described.
  • Amino Acid Decarboxylase Gene Acquisition and Construction of Expression Vectors [0165] Polynucleotide sequences encoding amino acid decarboxylases from Streptomyces sp. GP55 (SEQ ID NO: 2; Acc. No. WP_101384472.1), Saccharothrix sp. ST-888 (SEQ ID NO: 4; Acc. No.
  • Kitasatospora sp. MBT63 SEQ ID NO: 6; Acc. No. WP_051812394.1
  • Kitasatospora sp. MMS61-BH015 SEQ ID NO: 8; Acc. No. WP_104818078.1
  • Streptomyces sp. NRRL F-6131 SEQ ID NO: 10; Acc. No. WP_051769113.1
  • Planctomycetaceae bacterium SEQ ID NO: 12; Acc. No. RPI63066.1
  • Larkinella arboricola SEQ ID NO: 14; Acc. No.
  • A0A327WPB0 were synthesized as the genes of SEQ ID NOS: 1, 3, 5, 7, 9, 11, and 13, respectively. These synthetic genes were cloned into a pCK110900 vector system (See e.g., US Pat. No.7,629,157 and US Pat. Appln. Publn. No.2016/0244787, both of which are hereby incorporated by reference), and subsequently expressed in an E. coli strain derived from W3110. In some embodiments, expression vectors lacking antimicrobial resistance markers find use. Production of Shake Flask Powders (SFP) [0166] E.
  • coli cultures transformed with plasmids containing amino acid decarboxylases were plated onto Luria Broth-agar plates with 1% glucose and in some instances, 30 ⁇ g/mL chloramphenicol, and grown overnight at 37oC.
  • a single colony from each culture was transferred to 5 mL of Luria Broth (LB) with 1% glucose and 30 ⁇ g/mL chloramphenicol, where appropriate.
  • the cultures were grown for 18 h at 30 ⁇ C, 250 rpm, and subcultured approximately 1:50 into 250 ml of Terrific Broth (TB) with 30 ⁇ g/mL of chloramphenicol.
  • the cultures were grown for approximately 3-4 h at 30 ⁇ C, 250 rpm, to an OD 600 of 0.6-0.8 and induced with 1 mM of IPTG.
  • the cultures were grown for 20 h at 30 ⁇ C, 250 rpm.
  • Cells were harvested by centrifugation (7000 rpm x 10 min, 4°C), and the supernatant was discarded.
  • the pellets were resuspended in 30 mL of 50 mM sodium phosphate, pH 7.0, with 1 mM PLP and lysed using a single pass through a microfluidizer (Microfluidics) at 110 psi.
  • thermostability is a valuable trait useful in manufacture and storage of enzyme therapeutics and often occurs as a byproduct of other stabilization efforts.
  • thermostability of the enzymes was assessed as follows: 100 ⁇ L of amino acid decarboxylase SFP at 10 g/L were incubated for 1.5 h at 30-700C in a thermocycler. After incubation, samples were briefly centrifuged, and 10 ⁇ L of the heat-treated SFP was added to 90 ⁇ L of reaction mix for a final concentration of 5 mM leucine in 50 mM sodium phosphate, pH 7.0. Reactions were incubated for 2 h at 37°C at 250 rpm in a THERMOTRON ® titre-plate and processed and analyzed by LC-MS/MS as described above.
  • results for SEQ ID NOS: 2-14 are shown in Table 1-2.
  • SFP Characterization Assay for Resistance to Proteases [0170] To evaluate the relative stability of enzymes to representative intestinal proteases, a mix of porcine trypsin (Sigma Aldrich) and bovine chymotrypsin (Sigma Aldrich) was dissolved in 50 mM sodium phosphate, pH 7.0, to a concentration of 3 g/L each and serially diluted 2-fold. Then, 10 g/L amino acid decarboxylase SFP were incubated with 0-1.5 g/L trypsin/chymotrypsin at 37°C for 1 h at 250 rpm in a THERMOTRON ® titre-plate shaker.
  • EXAMPLE 2 LDC Variants of SEQ ID NO: 12 [0171] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 12, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 11, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. High-Throughput (HTP) Growth of Cultures Expressing LDC Enzymes [0172] Transformed E. coli cells were selected by plating onto LB agar plates containing 1% glucose.
  • HTP High-Throughput
  • reaction mix resulting in a final concentration of 0.6-3 mM of all twenty amino acids, with isoleucine-d10 (Cambridge Isotope Laboratories) in place of isoleucine, and 10 ⁇ M PLP in 50 mM sodium phosphate, pH 7.0, was used.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer as described in Example 2, with the following conditions: heat-treated clarified lysate was preincubated 1:1 with McIlvaine buffer, pH 3.3. The resulting acidic buffer-treated lysate was then preincubated 1:1 with a final concentration of 0.5 g/L trypsin and chymotrypsin (1:1) for 1 h at 37°C with shaking. After incubation, samples were centrifuged, and 40 ⁇ L of sample was added to 60 ⁇ L of reaction mix for a final concentration of 3 mM leucine and 10 ⁇ M PLP in 50 mM sodium phosphate, pH 7.0.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin to simulate the gastric environment.
  • heat-treated clarified lysate was preincubated 1:1 with McIlvaine buffer, pH 3.2, containing 0.05 g/L pepsin from porcine gastric mucosa (Sigma), in a COSTAR ® 96-well round bottom plates (Corning). The plates were sealed and incubated for 1 h at 37°C in a THERMOTRON ® titre-plate shaker (250 rpm).
  • the samples were centrifuged briefly, and the resulting supernatant was preincubated 1:1 with a final concentration of 0.5 g/L trypsin and chymotrypsin (1:1) for 1 h at 37°C with shaking. After incubation, 40 ⁇ L of sample was added to 60 ⁇ L of reaction mix for a final concentration of 3 mM leucine and 10 ⁇ M PLP in 50 mM sodium phosphate, pH 7.0.
  • LDC Variants of SEQ ID NO: 484 [0181] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 484, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 484, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer and Protease [0182] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, with the exception that the lysis buffer contained 40 ⁇ M PLP.
  • EXAMPLE 7 LDC Variants of SEQ ID NO: 594 [0183] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 594, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 594, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer and Protease [0184] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, with the exception that the lysis buffer contained 40 ⁇ M PLP.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin and subsequently challenged with proteases as described in Example 5. Specifically, heat-treated clarified lysate was preincubated 1:1 with a final concentration of 0.2 g/L pepsin in McIlvaine buffer, pH 2.8, for 1 h at 37°C, and the resulting supernatant was preincubated 1:1 with a final concentration of 1.5 g/L trypsin and chymotrypsin (1:1) dissolved in 400 mM sodium phosphate, pH 8, for 1 h at 37°C.
  • EXAMPLE 8 LDC Variants of SEQ ID NO: 686 [0185] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 686, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 686, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer and Protease [0186] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, with the exception that the lysis buffer contained 40 ⁇ M PLP.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin and subsequently challenged with proteases as described in Example 5. Specifically, heat-treated clarified lysate was preincubated 1:1 with a final concentration of 0.4 g/L pepsin in McIlvaine buffer, pH 2.8, for 1-1.5 h at 37°C, and the resulting supernatant was preincubated 1:1 with a final concentration of 2-4 g/L trypsin and 1.5 g/L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, for 1-2 h at 37°C.
  • reaction mix resulting in a final concentration of 2.5 mM leucine, isoleucine-d 10 , valine, asparagine, methionine, and cysteine in 50 mM sodium phosphate, pH 7.0, was used. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. The results of these assays are provided in Tables 8-1 and 8-2.
  • the lysate was pelleted (10,000 x rpm, 30 min, 4°C), and the resulting supernatant was heat-treated at 60°C for 1 h in a water bath. Samples were then centrifuged (10,000 x rpm, 1 h, 4°C) before the resulting supernatant was frozen and lyophilized to generate a powder containing the expressed enzyme.
  • EXAMPLE 10 LDC Variants of SEQ ID NO: 688 [0190] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 688, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 688, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer and Protease [0191] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 2, with the exception that the lysis buffer contained 20 mM sodium phosphate and 40 ⁇ M PLP.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin and subsequently challenged with proteases as described in Example 5. Specifically, heat-treated clarified lysate was preincubated 1:1 with a final concentration of 0.4 g/L pepsin in McIlvaine buffer, pH 3, for 1.5 h at 37°C, and the resulting supernatant was preincubated 1:1 with a final concentration of 4 g/L trypsin and 1.5 g/L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, for 2 h at 37°C.
  • EXAMPLE 11 LDC Variants of SEQ ID NO: 766 [0192] In this Example, experiments for evolution and screening of LDC variants derived from SEQ ID NO: 766, for improved leucine activity, low pH tolerance, and protease resistance are described. Directed evolution of the LDC encoded by SEQ ID NO: 766, was carried out by constructing libraries of variant genes. These libraries were then plated, grown, and screened using the methods described below. HTP Activity Analysis of Clarified Lysates Pretreated with Acidic Buffer and Protease [0193] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 10.
  • Heat-treated lysates containing LDC variants were challenged with acidic buffer containing pepsin and subsequently challenged with proteases as described in Example 5. Specifically, heat-treated clarified lysate was preincubated 1:1 with a final concentration of 0.8 g/L pepsin in McIlvaine buffer, pH 2.6-2.8, for 2 h at 37°C, and the resulting supernatant was preincubated 1:1 with a final concentration of 4 g/L trypsin and 1.5 g/L chymotrypsin dissolved in 400 mM sodium phosphate, pH 8, for 2 h at 37°C.
  • HTP Activity Analysis of Clarified Lysates on Leucine [0194] HTP growth and lysis of E. coli cells expressing LDC variants were performed as described in Example 10.
  • Heat-treated lysates were diluted 20-fold in water, and 20 ⁇ L of sample was added to 80 ⁇ L of reaction mix, resulting in a final concentration of 3 mM leucine in 50 mM sodium phosphate, pH 7. Reactions were incubated, quenched, diluted, and analyzed as described in Example 6. Activities on leucine were normalized by the concentration of leucine decarboxylase in heat-treated clarified lysates, which was determined by SDS-PAGE and size exclusion chromatography. The results of these assays are provided in Tables 11-2.
  • Plasma samples were obtained at 90 minutes and 30 minutes pre-prandial, and at 5, 15, 30 minutes, as well as 1, 2, 4, 8, 12, and 24 hours post-prandial (the 24 hour time point was collected the following day, prior to feeding). Samples were transferred into tubes containing the anticoagulant, K2EDTA, placed on wet ice pending processing, and centrifuged at 2500 rpm for 10 minutes at approximately 4 ⁇ C. The resulting plasma was recovered and stored frozen ( ⁇ -60°C) until analysis. Plasma leucine, phenylalanine, tyrosine, and methionine were quantified using LC-MS/MS to evaluate efficacy. A significant increase in plasma leucine was observed with meal challenge alone, compared to pre-prandial baseline.
  • iAUC was reported by first subtracting pre-prandial leucine values (fasted background) from each post-prandial time point value for each animal, and then performing AUC calculations. Statistical calculations and significance were determined using GraphPad Prism 7 (GraphPad Software).
  • mice Prior to dosing and starting at weaning, iMSUD mice were maintained on leucine free mouse chow with leucine supplemented in the drinking water (5.75 g leucine/L), in order to support growth and extend survival. Animals at least 2 months old and 20 g BW were used for experiments. Animals were fasted overnight ( ⁇ 15 hours) prior to dosing. On each dosing day 45 mg whey protein powder (BN Labs Grass Fed Whey Protein; 9.14% leucine), formulated with water for a total volume of 100 mL/animal, was provided by oral gavage.
  • whey protein powder BN Labs Grass Fed Whey Protein; 9.14% leucine

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KR1020227030236A KR20220137057A (ko) 2020-02-04 2021-02-03 조작된 류신 데카르복실라제
BR112022015139A BR112022015139A2 (pt) 2020-02-04 2021-02-03 Polipeptídeo da leucina descarboxilase engenheirada, composição, sequência de polinucleotídeo engenheirado, vetor de expressão, célula hospedeira, métodos para produzir um polipeptídeo da leucina descarboxilase engenheirada e para tratar e/ou prevenir os sintomas de doença da urina de xarope de bordo, composição, e, uso
PH1/2022/551776A PH12022551776A1 (en) 2020-02-04 2021-02-03 Engineered leucine decarboxylases
AU2021216389A AU2021216389A1 (en) 2020-02-04 2021-02-03 Engineered leucine decarboxylases
MX2022009531A MX2022009531A (es) 2020-02-04 2021-02-03 Leucina descarboxilasas modificadas geneticamente.
JP2025114444A JP2025143444A (ja) 2020-02-04 2025-07-07 操作されたロイシンデカルボキシラーゼ

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