Isoprene synthase variants for improved microbial production of isoprene

Abstract

The present invention provides methods and compositions comprising at least one isoprene synthase enzyme with improved catalytic activity and/or solubility. In particular, the present invention provides variant plant isoprene synthases for increased isoprene production in microbial host cells. Biosynthetically produced isoprene of the present invention finds use in the manufacture of rubber and elastomers.

1 - 8 . (canceled) 
     
     
         9 . An isolated isoprene synthase variant, wherein the variant comprises one or more amino acid substitution(s) at one or more amino acid residue(s) of a wild type isoprene synthase, wherein said one or more amino acid residue(s) correspond to amino acid residues of  P. alba  isoprene synthase having the sequence of SEQ ID NO:120, wherein said amino acid substitution(s) are selected from the group consisting of V10M, F12S, T15A, E18G, V58I, V58F, L70Q, L70R, L70V, L70T, T71P, V79L, E89D, G94A, S119F, F120L, G127R, E175V, T212I, S257A, R262G, A266G, F280L, N297K, F305L, L319M, E323K, A328T, D342E, A359T, K366N, E368D, L374M, S396T, V418S, K438N, H440R, T442A, I449V, A469S, K500R, K505Q, G507S, S509N, F511Y, and N532K; and wherein the isoprene synthase variant has increased isoprene synthase activity compared to the wild type isoprene synthase. 
     
     
         10 . The isolated isoprene synthase variant of  claim 9 , wherein increased isoprene synthase activity is indicated by a host cell comprising the isoprene variant growing at a faster rate in the presence of dimethylallyl pyrophosphate (DMAPP) compared to a host cell comprising the wild type isoprene synthase. 
     
     
         11 . The isolated isoprene synthase variant of  claim 9 , wherein the isoprene synthase is a poplar isoprene synthase variant. 
     
     
         12 . The isolated poplar isoprene synthase variant of  claim 11 , wherein the wild type isoprene synthase is  P. alba  isoprene synthase of SEQ ID NO:120. 
     
     
         13 . The isolated poplar isoprene synthase variant of  claim 11 , comprising the L70R substitution. 
     
     
         14 . The isolated poplar isoprene synthase variant of  claim 11 , wherein the variant comprises two or more amino acid substitutions selected from the group consisting of G127R/F511Y, L70Q/G94A/R262G/F305L, F12S/T15A/E18G/N297K, S396T/T442I, V10M/E323K, F120L/A266G, K438N/K500R, V79L/S509N, E175V/S257A/E368D/A469S, T71P/L374M, F280L/H440R, E89D/H440R, V58F/A328T/N532K, S119F/D342E/I449V, and K366N/G507S. 
     
     
         15 - 53 . (canceled)