Thermostable peroxide-driven cytochrome P450 oxygenase variants and methods of use

Abstract

The invention relates to novel variants of cytochrome P450 oxygenases. These variants have at least one mutation improving their ability to use peroxide as an oxygen donor as compared to the corresponding wild-type enzyme. The variants also have at least one mutation improving thermostability as compared to the parent enzyme or corresponding wild-type enzyme. Preferred variants include cytochrome P450 BM-3 heme domain variants having L52I, I58V, F87A, H100R, S106R, F107L, A135S, M145A/V, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and/or V446I amino acid substitutions.

1 . An isolated variant of a cytochrome P450 BM-3 comprising the amino acid sequence of SEQ ID NO:3, the variant comprising at least a first mutation in an amino acid residue selected from K9, I58, F87, E93, H100, F107, K113, A135, M145, 145A, A184, N186, D217, M237, E244, S274, L324, I366, K434, E442, and V446 of SEQ ID NO:3, and at least a second mutation in an amino acid residue selected from L52, S106, N239, and V340. 
     
     
         2 . The isolated variant of  claim 1 , comprising mutations in at least three amino acid residue selected from K9, I58, F87, E93, H100, F107, K113, A135, M145, 145A, A184, N186, D217, M237, E244, S274, L324, 1366, K434, E442, and V446 of SEQ ID NO:3, and mutations in at least three amino acid residues selected from L52, S106, N239, and V340. 
     
     
         3 . The isolated variant of  claim 1 , comprising a mutation in L52, I58, F87, H100, S106, F107, A135, A184, N239, S274, L324, V340, I366, K434, E442, and V446. 
     
     
         4 . The isolated variant of  claim 1 , wherein the cytochrome P450 BM-3 does not include a reductase domain. 
     
     
         5 . The isolated variant of  claim 1 , wherein the variant has a higher thermostability than P450 BM-3. 
     
     
         6 . The isolated variant of  claim 1 , wherein the variant has a higher thermostability than the cythchrome P450 BM-3 heme domain. 
     
     
         7 . The isolated variant of  claim 1 , wherein the first mutation is selected from K9I, I58V, F87A, F87S, E93G, H100R, F107L, K113E, A135S, M145A, M145V, A184V, N186S, D217V, M237L, E244G, S274T, L324I, I366V, K434E, E442K, and V446I. 
     
     
         8 . The isolated variant of  claim 1 , wherein the second mutation is selected from L52I, S106R, N239H, and V340M. 
     
     
         9 . The isolated variant of  claim 1 , wherein the variant comprises at least 6 mutations selected from K9I, L52I, I58V, F87A, F87S, E93G, H100R, S106R, F107L, K113E, A135S, M145A, M145V, A184V, N186S, D217V, M237L, N239H, E244G, S274T, L324I, V340M, I366V, K434E, E442K, and V446I. 
     
     
         10 . The isolated variant of  claim 1 , wherein the variant comprises the mutations L52I, I58V, F87A, H100R, S106R, F107L, A135S, A184V, N239H, S274T, L324I, V340M, I366V, K434E, E442K, and V446I. 
     
     
         11 . A method of thermostabilizing a parent cytochrome P450 oxygenase heme domain having at least a first mutation in a wild-type cytochrome P450 oxygenase heme domain, the method comprising:
 (a) preparing a protein library of variants of the parent having at least a second mutation, which second mutation is located no more than 10 Ångströms from the first mutation; and   (b) selecting any variant having a higher thermostability than the parent.   
     
     
         12 . The method of  claim 11 , wherein selecting any variant comprises selecting any variants having a T50 higher than that of the parent cytochrome P450 oxygenase heme domain. 
     
     
         13 . The method of  claim 11 , wherein selecting any variant comprises selecting any variants having a T50 higher than that of the wild-type cytochrome P450 oxygenase heme domain. 
     
     
         14 . The method of  claim 11 , wherein the parent cytochrome P450 oxygenase heme domain comprises an amino acid sequence at least 95% identical to SEQ ID NO:3. 
     
     
         15 . The method of  claim 11 , wherein the first mutation is in at least one amino acid selected of the group consisting of I58, H100, F107, A135, M145, N239, S274, K434, and V446. 
     
     
         16 . The method of  claim 15 , wherein the first mutation is at least one amino acid substitution selected from the group consisting of I58V, H100R, F107L, A135S, M145V, M145A, N239H, S274T, K434E, and V446I. 
     
     
         17 . The method of  claim 11 , wherein the second mutation is in at least one amino acid selected from the group consisting of L52, S106, M145, L324, I366, and E442. 
     
     
         18 . The method of  claim 17 , wherein the second mutation is at least one amino acid substitution selected from the group consisting of L52I, S106R, M145A, L324I, I366V, E442K, and a reversal of a mutation in M145. 
     
     
         19 . The method of  claim 18 , wherein the second mutation is at least one amino acid substitution selected from L52I, S106R, and E442K. 
     
     
         20 . An isolated variant of a parent cytochrome P450 oxygenase heme domain, the parent comprising at least a first mutation in a wild-type cytochrome P450 oxygenase heme domain and having at least 90% sequence identity to SEQ ID NO:3; and the variant comprising at least a second mutation no more than 10 Ångströms from the first mutation, the second mutation promoting a higher thermostability in the variant. 
     
     
         21 . The isolated variant of  claim 20 , wherein the parent has a higher capability of using peroxide as an oxygen donor than the corresponding wild-type cytochrome P450 oxygenase heme domain. 
     
     
         22 . The isolated variant of  claim 20 , having a T 50  higher than the wild-type cytochrome P450 domain.