Alanine 2,3-aminomutases and related polynucleotides

Abstract

The present disclosure relates generally to polypeptides having improved alanine 2,3-aminomutase (AAM) activity, the polynucleotides encoding the AAM polypeptides, and expression vectors and host cells for expressing the AAM polypeptides.

What is claimed is: 
     
         1 . An engineered alanine-2,3-aminomutase polypeptide with improved enzyme activity as compared to enzyme activity of the polypeptide of SEQ ID NO:4 and having at least about 80% sequence identity with SEQ ID NO:4. 
     
     
         2 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , in which the engineered alanine-2,3-aminomutase polypeptide has at least about 90% sequence identity with SEQ ID NO:4. 
     
     
         3 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , in which the enzyme activity is improved at least about 1.5 times to about 30 times or more the enzyme activity of the polypeptide of SEQ ID NO:4. 
     
     
         4 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , in which the enzyme activity is improved at least about 2 time to about 30 times or more the enzyme activity of the polypeptide of SEQ ID NO:4. 
     
     
         5 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , in which the enzyme activity is improved at least about 10 times to about 30 times or more enzyme activity of the polypeptide of SEQ ID NO:4. 
     
     
         6 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:4 and which includes one or more substitutions selected from: Y8H; D20N; L26I; 33P; K36E; E51G; N74K; Q85R or K; Q86R; A91S; Q95M; L99V; T111P; C130S; R1355; 138Y; A139T; Q141R; K142R; E149V; D156E; T163A; 1921; 5202T; T204A; E244V; E247G; N251Y or S; R275G; M283I or V; V292A; L297M or F; G300R; I313T; N315S; G331H; I339V; V341L or I; T342A; H353R; V355I; V362A; I363T; N371D; E379V; A383V; G384D; K385E; K387R; A392T; G395E; L399M; I401L; S404P; K410E; K412R; and F413L or S. 
     
     
         7 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 97% identity to a polypeptide selected from SEQ ID NO:295, 2068 and 2150. 
     
     
         8 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 98% identity to a polypeptide selected from SEQ ID NO: 258, 316, 350, 356, 790, 1006, and 1250. 
     
     
         9 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 99% identify to a polypeptide selected from SEQ ID NO:14, 24, 58, 86, 100, 312, 336, 380, 318, 404, 444, 490, 550 or 774. 
     
     
         10 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:4 and which includes one or more substitutions selected from: S4R; K7E; D15N; N19C; D20N; L26I or T; D33P or G; T40A; L41P; E44G; E47G; K50Q; S51G; Y63H; Y64C; S66C; T68L or M; D69N; N74K or H; A81T; Q85R or K; A91S; Q95M or V; P98S; L99V; R113C; C130S; H132R; R135S; R137H; Q141R; I155V; T163A; S178G or C; Y184C; I185T; K187R or E; E190C; I197T or V; S202T; V206I or A; I212M; D218G; V226A; H230A; E244G or V; E247G; R248G, K, or M; M249I; A250T or V; A252T; D268G; H278Y; M283I or V; L297M or F; I299M; R303H; E311G; I313N, V, S, or T; S321L; Y323F; G336A; V341L, I, or A; V355I; V362A or I; I363T; T367A; E368G; Y372C; K385E; Q397R; Q398R; I400V; P403S, G, or T; and P413L, G, or S. 
     
     
         11 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which has an amino acid sequence corresponding to the amino acid sequence of SEQ ID NO:4 and which includes one or more substitutions selected from: A2S or T; S4C or N; Y8H; Y9F; F10W; E16K; Q17K; Y19H; H22R; 129V; K30R; T31A or S; L32V; K36A; K37E; V49I; 168M; C75D; 177V; R78C; 182V; Q85K; Q86R; P106S; F120L; I122T or V, K142R, S145P, E149V, D156E, N160T, V164I, V177L, S178C, E180G, K187E, E191G, V192I, E196A or G, I197V, V198I, I200V, T204A, V206A, Q215H or R, V217A, K222R, Y223H, L228V, F232S, V238I, R248K or M, A250V, N251S, I254V, T260S, I266V, R275K, L279K, K282N, M2831, I290M or V, G300R or S, F302L, I310T or V, I313V, N315S, F328Y, P333A, V341A, V347I, Y359F, V362I, E370D, E374G, D377G, E379V, C381S, R382H, A383V, G384D, H386Q, K387A or R, E388I, V390I, A392E or T, S394L, G395S, G396S, I401M, E402V, P403G, I, L, M, or T, S404P, D405N, L406S, A407E, K409E or M, K410D or E, K412R, P413G or S, D414G or V, K415E or R, and N416A. 
     
     
         12 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 99% identity to a polypeptide selected from SEQ ID NOS: 2226, 2240, 2268, 2286, 2282, 2312, 2316, 2324, 2338, 2346, and 2348. 
     
     
         13 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 96% identity to a polypeptide selected from SEQ ID NOS: 2380 and 2378. 
     
     
         14 . The engineered alanine-2,3-aminomutase polypeptide of  claim 1 , which is a polypeptide having at least 95% identity to a polypeptide selected from SEQ ID NO: 2670, 2666, and 4586. 
     
     
         15 . A process for preparing beta-alanine, comprising contacting alpha-alanine with an engineered alanine-2,3-aminomutase of  claim 1 , under conditions suitable for conversion of alpha-alanine to beta-alanine. 
     
     
         16 . The process of  claim 15 , which is carried out with whole cells that express the engineered alanine-2,3-aminomutase, or an extract or lysate of such cells.