Improved methane monooxygenase enzymes

Abstract

Improved soluble methane monooxygenases and soluble methane monooxygenase systems are provided.

1 . A mutant soluble diiron monooxygenase system comprising at least one mutant subunit, wherein the at least one mutant subunit comprises one or more mutations, such that the activity of the mutant soluble diiron monooxygenase system is greater than the activity of a wild-type soluble diiron monooxygenase system. 
     
     
         2 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant hydroxylase alpha subunit wherein the mutant hydroxylase alpha subunit comprises one or more mutations at one or more residue position(s): 3, 14, 25, 26, 32, 33, 35, 42, 44, 53, 54, 55, 56, 57, 61, 130, 135, 154, 162, 163, 168, 216, 251, 262, 263, 264, 277, 316, 322, 330, 347, 356, 374, 375, 381, 387, 393, 399, 405, 419, 420, 422, 431, 432, 433, 434, 434, 444, 453, 456, 459, 469, 470, 475, 480, 485, 493, 504, 505, 510, 513, 515, 525, and/or 526, wherein the residue position(s) are numbered with respect to SEQ ID NO: 3. 
     
     
         3 . The mutant soluble diiron monooxygenase hydroxylase alpha subunit according to  claim 2 , wherein the one or more mutations comprises substitutions at one or more of residue position(s): L3P, L14P, A25S, Q26G, L32P, Q33K, Q33R, F35L, N42R, T44A, T44S, A53G, N54K, N54L, E55A, E55S, T56A, T56G, K57G, K57S, K57T, K61A, K61R, A130C, A130G, A130H, A130T, N135S, V154L, G162C, Q163F, H168G, L216M, Y251M, Y251W, A262E, S263Q, A264Q, T277G, I316S, G322N, R330T, Y347G, T356C, T356G, T356M, A374E, N375H, D381R, D381S, Y387I, Y387L, R393C, R399D, L405G, R419L, R419S, V420C, Q422E, K431E, G432A, A433G, A433Q, A433T, S434G, S434N, Q444E, G453V, M456V, A459E, I469V, F470I, G475*, E480G, L485E, K493E, K493V, D504E, K505E, D510E, K513R, K513T, L515G, A525Q, F526C, F526G, F526L, and/or F526S, wherein the residue position(s) are numbered with respect to SEQ ID NO: 3. 
     
     
         4 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant accessory protein subunit B wherein the mutant accessory protein subunit B comprises one or more mutations at one or more residue position(s): 3, 15, 16, 38, 58, 69, 81, 101, 102, 103, 106, 110, 112, 116, 118, 119, 120, 124, 126, 130, 131, 134, 136, 138, and/or 139, wherein the residue position(s) are numbered with respect to SEQ ID NO: 6. 
     
     
         5 . The mutant soluble diiron monooxygenase accessory protein subunit B of  claim 4 , wherein the one or more mutations comprises substitutions at one or more residue position(s): V3G, L15M, K16G, V38G, T58E, N69K, A81G, N101G, V102A, V102L, Y103H, L106V, S110A, T112G, T112V, A116S, A116T, T118E, T118G, L119E, G120A, G120E, T124C, T124G, T126S, M130D, M130V, G131S, R134A, R134G, R134W, L136A, D138S, Y139G, Y139S, and/or Y139V, wherein the residue position(s) are numbered with respect to SEQ ID NO: 6. 
     
     
         6 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant reductase subunit C wherein the mutant reductase subunit C comprises one or more mutations at one or more residue position(s): 17, 28, 38, 44, 45, 49, 58, 62, 64, 69, 74, 87, 90, 97, 98, 100, 110, 111, 116, 119, 125, 133, 135, 138, 143, 144, 152, 153, 155, 157, 174, 180, 184, 191, 196, 204, 208, 209, 211, 213, 216, 222, 235, 236, 238, 240, 242, 243, 244, 245, 246, 279, 281, 286, 288, 308, 324, 329, and/or 330, wherein the residue position(s) are numbered with respect to SEQ ID NO: 7. 
     
     
         7 . The mutant soluble diiron monooxygenase reductase subunit C of  claim 6 , wherein the one or more mutations comprises substitutions at one or more residue position(s): L17R, L17Q, I28G, L38G, E44G, E44R, G45R, T49L, D58K, K62L, C64A, C64Q, L69A, E74R, K87L, L90Q, T97V, H98G, R100G, E110R, A111V, L116V, V119C, Q125L, D133V, C135T, R138Q, E143K, P144C, I152S, P153H, T155A, V157R, E174A, L180S, R184G, N191D, G196V, P204S, F208Q, G209W, K211V, R213G, A216V, A222G, R235G, Q236M, Q238G, W240E, A242V, P243G, N244G, E245D, E245M, T246M, C279S, W281R, D286T, E288V, N308V, C324G, S329G, and/or R330W, wherein the residue position(s) are numbered with respect to SEQ ID NO: 7. 
     
     
         8 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant hydroxylase beta subunit wherein the mutant hydroxylase beta subunit comprises one or more mutations at one or more residue position(s): 32, 36, 46, 47, 58, 64, 87, 93, 109, 133, 143, 146, 151, 155, 157, 160, 162, 202, 167, 182, 183, 184, 186, 201, 202, 203, 204, 206, 208, 209, 211, 212, 213, 214, 216, 217, 220, 222, 224, 225, 226, 230, 233, 236, 239, 242, 244, 250, 256, 260, 266, 269, 270, 272, 273, 276, 281, 291, 303, 304, 305, 306, 308, 310, 311, 314, 318, 322, 325, 328, 329, 330, 333, 335, 336, 356, 364, and/or 372, wherein the residue position(s) are numbered with respect to SEQ ID NO: 4. 
     
     
         9 . The mutant soluble diiron monooxygenase hydroxylase beta subunit of  claim 8 , wherein the one or more mutations comprises substitutions at one or more residue position(s): N32V, G36L, L46V, T47H, T47V, P58N, A64G, T87V, V93G, Y109A, Y109E, Y109F, Y109V, I133S, E143T, N146R, A151G, N155G, Y157W, F160K, F160M, F160T, F160V, A162T, 202, A167Q, W182M, G183A, G183S, F184S, K186N, A201G, K202A, K202Q, K202R, K202S, I203V, V204S, G206E, D208A, E209R, T211I, A212G, V213G, V213W, P214V, A216G, E217L, N220V, E222R, Y224M, K225G, S226G, A230G, G233L, G233R, G233W, Q236G, Q236V, Q236W, F239L, N242G, N242P, N242V, S244G, A250C, F256W, V260C, Q266V, A269G, P270R, F272K, G273T, G273V, L276V, I281G, A291T, L303G, G304A, D305A, D305G, D305L, D305R, D305W, D306E, D306Q, D306R, E308G, S310R, S310W, D311A, D311G, R314G, R314H, R314Q, R318G, R3181, G322R, L325G, T328G, 1329W, A330R, R333V, F335G, M336G, L356G, I364G, and/or D372W, wherein the residue position(s) are numbered with respect to SEQ ID NO: 4. 
     
     
         10 . The mutant soluble diiron monooxygenase system, according to  claim 1 , comprising a mutant hydroxylase gamma subunit wherein the mutant hydroxylase gamma subunit comprises one or more mutations at one or more residue position(s): 10, 17, 18, 20, 21, 23, 24, 27, 29, 31, 33, 34, 41, 43, 45, 46, 47, 50, 51, 52, 53, 56, 63, 66, 67, 70, 79, 80, 103, 112, 116, 117, 127, 129, 136, 144, 147, 153, 154, 156, 165, and/or 166, wherein the residue position(s) are numbered with respect to SEQ ID NO: 5. 
     
     
         11 . The mutant soluble diiron monooxygenase hydroxylase gamma subunit of  claim 10 , wherein the one or more mutations comprises substitutions at one or more residue position(s): D10R, D10V, N17F, N17W, K18A, K18E, A20G, Q21H, N23A, N23D, N23E, N23G, N23L, N23V, T24E, T24V, K27G, K27Q, K27R, A29G, M31W, K33V, Q34A, Q34K, T41G, F43K, F43S, F43V, N45G, S46G, S46I, Y47S, Y47T, D50G, N51G, N51V, D52G, Y53C, I56L, K63G, K63T, V66D, L67E, R70E, R70G, R70V, H79E, K80M, D103G, I112V, Q116S, 1117L, Y127F, L129G, G136Q, G136S, N144D, N144G, N144V, Y147D, E153G, E154G, R156A, H165G, L166E, and/or L166G, wherein the residue position(s) are numbered with respect to SEQ ID NO: 5. 
     
     
         12 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant subunit D wherein the mutant subunit D comprises one or more mutations at one or more residue position(s): 4, 5, 7, 8, 9, 10, 13, 14, 17, 18, 20, 21, 22, 24, 27, 40, 60, 74, 79, 80, 89, 90, 92, 93, and/or 96, wherein the residue position(s) are numbered with respect to SEQ ID NO: 8. 
     
     
         13 . The mutant soluble diiron monooxygenase subunit D of  claim 12 , wherein the one or more mutations comprises substitutions at one or more residue position(s): S4R, A5S, Q7G, P8G, F9G, F9G, S10R, A13E, D14R, F17G, F17G, E18R, P20G, R21L, P22R, A24G, F27V, T40R, E60Q, S74A, T79G, T79S, H80G, E89D, Q90S, A92P, P93A, and/or D96R, wherein the residue position(s) are numbered with respect to SEQ ID NO: 8. 
     
     
         14 . The mutant soluble diiron monooxygenase system of  claim 1 , comprising a mutant protein folding chaperone groEL-2 wherein the mutant protein folding chaperone groEL-2 comprises one or more mutations at one or more residue position(s): 109, 160, 168, 169, 171, 182, 187, 396, 405, 409, 438, 444, 461, 466, 478, 484, 485, 495, 522, 524, and/or 529, wherein the residue position(s) are numbered with respect to SEQ ID NO: 9. 
     
     
         15 . The mutant protein folding chaperone groEL-2 of  claim 14 , wherein the one or more mutations comprises substitutions at one or more residue position(s): A109E, A109P, G160A, D168V, A169S, G171P, Q182W, L187V, T396L, V405G, V405D, N409G, A438K, D444S, P461V, G466L, A478L, A478R, A484E, A484G, N485T, G495H, V522L, G524T, and/or T529G, wherein the residue position(s) are numbered with respect to SEQ ID NO: 9. 
     
     
         16 . One or more isolated nucleic acids encoding one or more polypeptides comprising or consisting of the mutant soluble diiron monooxygenase system of  claim 1 . 
     
     
         17 . One or more vectors comprising or consisting of the one or more isolated nucleic acids of  claim 16 . 
     
     
         18 . One or more host cells comprising or consisting of the one or more vectors of  claim 17 . 
     
     
         19 . The one or more host cells of  claim 18 , wherein the one or more host cells comprises a prokaryotic cell. 
     
     
         20 . The one or more host cells of  claim 19 , wherein the prokaryotic cell comprises one or more of  Escherichia coli, Corynebacterium glutamicum , and/or  Bacillus methanolicus.