Engineered phenylalanine ammonia lyase polypeptides

Abstract

The present invention provides engineered phenylalanine ammonia lyase (PAL) polypeptides and compositions thereof, as well as polynucleotides encoding the engineered phenylalanine ammonia lyase (PAL) polypeptides. In some embodiments, the engineered PAL polypeptides are optimized to provide enhanced catalytic activity, as well as reduced sensitivity to proteolysis and increased tolerance to storage at elevated temperatures. In some embodiments, the engineered PAL polypeptides contain fewer phenylalanine residues than wild-type PAL polypeptides. The present invention also provides methods for the use of the compositions comprising the engineered PAL polypeptides for therapeutic and industrial purposes.

We claim: 
     
         1 . An engineered phenylalanine ammonia lyase polypeptide comprising an amino acid sequence having at least 90% sequence identity to SEQ ID NO: 8, wherein said amino acid sequence comprises an amino acid residue difference at position 364, and further comprising at least one substitution or substitution set at one or more amino acid positions selected from 44/47/204/209/285, 44/47/364/470/495, 44/54/561204/239/285/364/495, 44/54/561204/239/470/495, 44/54/285/470, 44/102/285/364, 44/204/209/285, 44/204/285/364/470/495, 44/209/285/460/495, 44/285/364, 47/54/209, 47/204/209/239/285/495, 47/204/285/364/495, 47/209/239/364, 47/239/285/364, 47/470, 54, 54/56, 54/56/204/209/470, 54/561204/209/495, 54/561204/495, 54/561209/562, 54/56/285/364/470, 54/56/285/470, 54/165/204/209/239/285/470/495, 54/239/495, 54/285/470, 54/470, 56, 165, 204, 204/209/239/285/470/495, 204/209/364, 204/209/364/495, 204/239, 204/239/285, 204/364, 204/470, 209/285/364/470, 209/285/364/470/495, 209/364, 209/364/495, 209/470, 239/364, 285/364, 285/364/495, 364, 364/470, 470, and 495, wherein the amino acid positions of said amino acid sequences are numbered with reference to the amino acid sequence of SEQ ID NO:2. 
     
     
         2 . An engineered phenylalanine ammonia lyase polypeptide comprising an amino acid sequence having at least 95% sequence identity to the engineered phenylalanine ammonia lyase polypeptide of  claim 1 . 
     
     
         3 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide is an  Anabaena variabilis  variant enzyme. 
     
     
         4 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide is more thermostable than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         5 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide more resistant to proteolysis than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         6 . The engineered phenylalanine ammonia lyase polypeptide of  claim 5 , wherein said engineered phenylalanine ammonia lyase polypeptide is resistant to proteolysis by at least one digestive tract enzyme, than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         7 . The engineered phenylalanine ammonia lyase polypeptide of  claim 6 , wherein said engineered phenylalanine ammonia lyase polypeptide is more resistant to proteolysis by chymotrypsin, trypsin, carboxypeptidases, and/or elastases than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         8 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said engineered polypeptide is more acid stable than wild-type  Anabaena variabilis  phenylalanine ammonia lyase. 
     
     
         9 . The engineered phenylalanine ammonia lyase polypeptide of  claim 1 , wherein said polypeptide is purified. 
     
     
         10 . A method of producing an engineered phenylalanine ammonia lyase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered polypeptide and/or phenylalanine ammonia lyase polypeptide of  claim 1 , under suitable culture conditions, such that at least one engineered phenylalanine ammonia lyase polypeptide is produced. 
     
     
         11 . The method of  claim 10 , further comprising recovering at least one engineered phenylalanine ammonia lyase polypeptide from the culture and/or host cells. 
     
     
         12 . The method of  claim 10 , further comprising the step of purifying said at least one engineered phenylalanine ammonia lyase polypeptide. 
     
     
         13 . An engineered phenylalanine ammonia lyase polypeptide produced using a method of producing an engineered phenylalanine ammonia lyase polypeptide in a host cell comprising culturing a host cell comprising at least one polynucleotide encoding at least one engineered polypeptide and/or phenylalanine ammonia lyase polypeptide of  claim 1 , under suitable culture conditions, such that at least one engineered phenylalanine ammonia lyase polypeptide is produced. 
     
     
         14 . A composition comprising at least one engineered phenylalanine ammonia lyase polypeptide of  claim 1 . 
     
     
         15 . The composition of  claim 14 , wherein said composition is a pharmaceutical composition. 
     
     
         16 . The composition of  claim 15 , further comprising at least one pharmaceutically acceptable excipient and/or carrier. 
     
     
         17 . The composition of  claim 15 , wherein said composition is suitable for the treatment of phenylketonuria. 
     
     
         18 . The composition of  claim 15 , wherein said composition is suitable for the treatment of elevated blood phenylalanine. 
     
     
         19 . The composition of  claim 15 , wherein said composition is suitable for the treatment of tyrosinemia. 
     
     
         20 . The composition of  claim 15 , wherein said composition is suitable for the treatment of elevated blood tyrosine. 
     
     
         21 . The composition of  claim 15 , wherein said composition is suitable for oral administration to a human. 
     
     
         22 . The composition of  claim 21 , wherein said composition is in the form of a pill, tablet, capsule, gelcap, liquid, or emulsion. 
     
     
         23 . The composition of  claim 22 , wherein said pill, tablet, capsule, or gelcap further comprises an enteric coating. 
     
     
         24 . The composition of  claim 15 , wherein said composition is suitable for parenteral injection into a human. 
     
     
         25 . The composition of  claim 15 , wherein said composition is coadministered with at least one additional therapeutically effective compound. 
     
     
         26 . The composition of  claim 25 , wherein said composition comprises at least one additional therapeutically effective compound. 
     
     
         27 . A method for treating and/or preventing the symptoms of phenylketonuria in a subject, comprising providing a subject having phenylketonuria, and providing the composition of  claim 15 , to said subject. 
     
     
         28 . The method of  claim 27 , wherein the phenylalanine concentration in the blood of said subject is reduced upon providing said composition to said subject. 
     
     
         29 . The method of  claim 27 , wherein the cinnamic acid concentration in the blood of said subject is increased upon providing said composition to said subject. 
     
     
         30 . The method of  claim 27 , wherein said symptoms of phenylketonuria are ameliorated. 
     
     
         31 . A method for treating and/or preventing the symptoms of phenylketonuria in a subject, comprising providing a subject having phenylketonuria, and providing the composition of  claim 15 , wherein said subject is able to eat a diet that is less restricted in its phenylalanine content than diets required by subjects who have not been provided said composition. 
     
     
         32 . The method of  claim 31 , wherein said subject is an infant, child, young adult, or adult.