Engineered leucine decarboxylases

Abstract

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

We claim: 
     
         1 . An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92,%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to at least one of SEQ ID NO: 2, 4, 6, 8, 10, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766, wherein the amino acid positions of said amino acid sequence is numbered with reference to the amino acid sequence of SEQ ID NO: 2, 4, 6, 8, 10, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766. 
     
     
         2 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 2. 
     
     
         3 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 4. 
     
     
         4 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 6. 
     
     
         5 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 8. 
     
     
         6 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 10. 
     
     
         7 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 14. 
     
     
         8 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 48/64/164/324/343/353/357/364, 48/64/164/324/343/364, 48/64/164/353/357/364, 48/64/357/364, 64/164/324/343/353/357/364, 64/164/324/343/357/364, 64/164/353/357, 64/318/324/357/364, 64/324/353/357/364, 132/255/339/379/395, 164/196/324/357/364, 164/318/324/343/353/357, 164/318/324/357/364, 164/324/343/353/357/364, 164/324/357/364, 164/353/357/364, 164/364, 196/318/324/353/357/364, 318/343/357, 324/343/357/364, 324/353/357/364, 324/357/364, 339/379/389/394/395, 339/389/395, 339/391, 339/394/395/405, 357/364, 379/386, 379/394/395/397/404/405, 379/394/395/397/405, 389/394/395/397/405, and 394/397, wherein the amino acid positions are numbered with reference to SEQ ID NO: 38. 
     
     
         9 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 2, 3, 33, 48/64/255, 48/255/339, 48/255/379, 64, 64/255, 69, 161, 193, 255, 255/318/379, 259, 263, 318/339/379, 324, 324/389/394, 324/389/394/395, 324/389/394/397, 324/394, 324/394/395, 324/394/395/397, 324/395, 339, 340, 380, 382, 389, 389/394, 389/394/395, 389/394/395/397, 389/394/397, 389/395, 389/397, 390, 394, 394/395, 394/395/397, 395, 395/397, 397, 401, and 405, wherein the amino acid positions are numbered with reference to SEQ ID NO: 234. 
     
     
         10 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 2/64/69/324/380/382/388/389, 3/64/69/263/339/380/388, 3/64/69/389, 3/64/69/390, 3/64/379/380/390, 3/69/263/380, 3/69/324, 3/69/324/380/382/389/390, 12/135/259/263, 12/135/263/382, 12/259/263/304, 48/64/255, 64/69, 64/69/189/259/263/304, 64/69/189/259/263/304/339/340/379, 64/69/223/388, 64/69/223/388/389/390, 64/69/304/379/382, 64/69/324, 64/69/324/339/380/389/390, 64/69/339, 64/69/339/382/388/389, 64/69/339/389/390, 64/69/379/380, 64/69/380/388/390, 64/69/389, 64/69/390, 64/255/263, 64/263, 64/324/339/389/390, 69/223/263/324/382/388/390, 69/223/324/379/380/382/388/390, 69/263, 69/263/324, 69/263/339, 69/263/388, 69/263/389/390, 69/324/379/380/388, 69/324/380, 69/339/390, 69/382/390, 259/263/304, 259/263/304/339/340/379, 263/339/389/390, 263/390, and 304/340/379/380/382, wherein the amino acid positions are numbered with reference to SEQ ID NO: 284. 
     
     
         11 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 484, and 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 position 3/194/304, 3/259/263/304, 3/259/304, 3/259/304/324/339, 3/259/304/324/382, 3/259/304/382, 3/263/304/324, 3/263/304/324/339, 3/263/304/324/382, 3/304, 3/304/324, 16, 63, 77, 80, 87/270, 87/270/365, 87/328/365, 91, 92, 126, 140, 156, 168/270/328/338, 181, 194, 201, 256, 259, 259/263, 259/263/304, 259/263/304/324, 259/263/304/324/382, 259/263/304/379, 259/263/304/382, 259/304, 259/304/324, 259/304/324/339, 259/304/324/339/382, 259/304/382, 262, 263/304, 263/304/324, 263/304/324/339, 263/304/324/382, 263/324, 270, 270/319, 270/328/338, 270/328/338/365, 304, 304/324, 324, 328, 352, 365, 366, and 382, wherein the amino acid positions are numbered with reference to SEQ ID NO: 484. 
     
     
         12 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 16/63/80/126/168/366, 16/63/80/126/181/194/259/324/328/366, 16/63/126/168/270/328/366, 16/80/126/324/366, 16/80/126/366, 16/80/168, 16/80/168/270/366, 16/80/168/324, 16/80/168/366, 16/80/324, 16/91/126/168/324/366, 16/126/168/366, 16/168/259/366, 16/168/270/324/366, 16/168/324/328/366, 16/168/324/366, 16/168/366, 16/259/263/328, 16/324/328/366, 16/328/366, 80/126/168/270/366, 80/126/181/270/324/366, 80/168/270/366, and 168/366, wherein the amino acid positions are numbered with reference to SEQ ID NO: 594. 
     
     
         13 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 66/76/118/141/201/300, 66/76/198/200/296/303, 66/76/198/200/300, 66/118/200/296/303/317, 66/118/296, 66/118/296/300, 66/200, 76/118/141/200/296, 76/141/198/200/201/300, 80/201/270, 80/270, 80/270/324, 89/118/200, 106/270/324/352, 118/141/200, 126, 126/201/270/324, 126/270, 141/144/198/200/300, 156/270, 156/270/324, 201/270, 201/270/352, 270, and 270/324, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. 
     
     
         14 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said 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 position 19, 109, 123, 134, 170, 173, 187, 211, and 312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 686. 
     
     
         15 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 688, and 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 position 19/109/123/141/170/198/200/211/270/312, 19/109/123/141/170/198/211, 19/109/123/141/170/198/211/270/312, 19/109/123/170/211/270/312, 19/109/123/198/200/211/270/312, 19/109/170/173/211/270/312, 19/109/211/270/312, 109/170/211/270/312, and 109/211/270/312, wherein the amino acid positions are numbered with reference to SEQ ID NO: 688. 
     
     
         16 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from position 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. 
     
     
         17 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said polypeptide sequence has at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more sequence identity to SEQ ID NO: 766, and wherein the polypeptide sequence of said engineered leucine decarboxylase polypeptide comprises at least one substitution or substitution set at one or more amino acid positions selected from position 66, 66/118, 66/118/296, 66/118/296/300, 66/118/300, 66/296, 66/296/300, 66/300, 118, 118/296, 118/296/300, 118/300, 296, 296/300, and 300, wherein the amino acid positions are numbered with reference to SEQ ID NO: 766. 
     
     
         18 . An engineered leucine decarboxylase polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 2, 4, 6, 8, 10, 14, 38, 234, 284, 484, 594, 686, 688, and/or 766. 
     
     
         19 . An engineered leucine decarboxylase polypeptide, wherein said engineered leucine decarboxylase polypeptide comprises an engineered polypeptide provided in  claim 1 . 
     
     
         20 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said engineered leucine decarboxylase polypeptide is a Planctomycetaceae variant enzyme. 
     
     
         21 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said engineered leucine decarboxylase polypeptide exhibits more activity on leucine than the wild-type Planctomycetaceae species leucine decarboxylase. 
     
     
         22 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said engineered leucine decarboxylase more resistant to proteolysis than wild-type Planctomycetaceae species leucine decarboxylase. 
     
     
         23 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said engineered leucine decarboxylase polypeptide is more thermostable than wild-type Planctomycetaceae species leucine decarboxylase. 
     
     
         24 . The engineered leucine decarboxylase polypeptide of  claim 1 , wherein said engineered leucine decarboxylase polypeptide is purified. 
     
     
         25 . A composition comprising at least one engineered leucine decarboxylase polypeptide provided in  claim 1 . 
     
     
         26 . An engineered polynucleotide sequence encoding at least one engineered leucine decarboxylase polypeptide of  claim 1 . 
     
     
         27 . The engineered polynucleotide sequence of  claim 26 , wherein said polynucleotide sequence comprises a sequence at least 85%, 86%, 87%, 88%, 89%, 90%, 91%, 92%, 93%, 94%, 95%, 96%, 97%, 98%, 99%, or more identical to any of the odd-numbered sequences of SEQ ID NOS: 15-851. 
     
     
         28 . The engineered polynucleotide sequence of  claim 27 , wherein said sequence is operably linked to a control sequence. 
     
     
         29 . The engineered polynucleotide sequence of  claim 27 , wherein said polynucleotide is codon-optimized. 
     
     
         30 . An expression vector comprising at least one engineered polynucleotide sequence of  claim 27 , and at least one control sequence. 
     
     
         31 . The expression vector of  claim 30 , wherein said control sequence comprises a promoter. 
     
     
         32 . A host cell transformed with at least one polynucleotide sequence of  claim 26 . 
     
     
         33 . A host cell comprising the expression vector of  claim 30 . 
     
     
         34 . A method of producing an engineered leucine decarboxylase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered leucine decarboxylase polypeptide of  claim 1 , under suitable culture conditions, such that at least one engineered leucine decarboxylase polypeptide is produced. 
     
     
         35 . A composition comprising at least one engineered polynucleotide of  claim 26 . 
     
     
         36 . The composition of  claim 35 , wherein said composition is a pharmaceutical composition. 
     
     
         37 . The composition of  claim 36 , further comprising at least one pharmaceutically acceptable excipient and/or carrier. 
     
     
         38 . The composition of  claim 36 , wherein said composition is suitable for oral administration to a human. 
     
     
         39 . The composition of  claim 36 , wherein said composition is suitable for parenteral injection into a human. 
     
     
         40 . A method 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 of  claim 36 , to said subject.