Malonate decarboxylases for industrial applications

Abstract

The present invention relates to a method for the enzymatic decarboxylation of malonic acid (propanedioic acid) derivatives catalyzed by enzymes structurally and/or functionally related to arylmalonate decarboxylase (AMDase) as isolated from microorganisms of the genus Bordetella . The present invention also relates to novel enzymes with a decarboxylase activity, useful for performing the claimed method, mutants thereof, corresponding coding sequences and expression systems, methods of preparing said novel enzymes, and screening methods for obtaining further suitable enzymes also having said decarboxylase activity.

1 . A protein having decarboxylase activity comprising an amino acid sequence having sequence identity of less than 99% to the  Bordetella bronchiseptica  polypeptide of SEQ ID NO: 2 and comprising at least one functional mutation in one of the amino acid sequence positions 14, 15, 159 and 190 of the polypeptide of SEQ ID NO: 2. 
     
     
         2 . The protein of  claim 1 , wherein said protein has at least 95% sequence identity to residues 1 to 240 of the  Bordetella bronchiseptica  polypeptide of SEQ ID NO: 2 and comprises a functional mutation in at least one amino acid position selected from the group consisting of positions 13, 14, 21, 40, 43, 48, 104, 126, 156, 159, 188, 189, 190, 191 and 212 of the polypeptide of SEQ ID NO: 2. 
     
     
         3 . The protein of  claim 1 , wherein said protein has at least 95% sequence identity to residues 1 to 240 of the  Bordetella bronchiseptica  polypeptide of SEQ ID NO: 2 and comprises a functional mutation in one of the amino acid sequence positions 14, 189 and 190 of the polypeptide of SEQ ID NO: 2. 
     
     
         4 . The protein of  claim 1 , wherein said protein has at least 95% sequence identity to residues 1 to 240 of the  Bordetella bronchiseptica  polypeptide of SEQ ID NO: 2 and comprises mutations in a combination of amino acid positions of the polypeptide of SEQ ID NO: 2 selected from the group consisting of positions:
 13 and 14,   13 and 15,   14 and 15,   13, 14 and 15,   14, 15 and 189,   14, 15 and 190,   14, 15, 189 and 190,   13, 14, 15 and 189,   13, 14, 15 and 190,   13, 14, 15, 189 and 190   13, 14 and 191,   13, 15 and 191;   14, 15 and 191,   13, 14, 15 and 191,   14, 15, 189 and 191   14, 15, 190 and 191,   14, 15, 189, 190 and 191,   13, 14, 15, 189 and 191,   13, 14, 15, 190 and 191,   and   13, 14, 15, 189, 190 and 191.   
     
     
         5 . The protein of  claim 1 , wherein said decarboxylase activity is a stereoselective mono-decarboxylase activity. 
     
     
         6 . A nucleic acid encoding the protein of  claim 1 . 
     
     
         7 . An expression cassette comprising the nucleic add of  claim 6  operably linked to at least one regulatory nucleic acid sequence. 
     
     
         8 . A recombinant expression vector comprising at least one expression cassette of  claim 7 . 
     
     
         9 . A recombinant microorganism comprising:
 a) the nucleic acid of  claim 6 ;   b) an expression cassette comprising the nucleic acid of a) operably linked to at least one regulatory nucleic acid sequence; or   c) a recombinant expression vector comprising at least one expression cassette of b).   
     
     
         10 . A bioreactor comprising at least one protein having decarboxylase activity as defined in  claim 1 , optionally in immobilized form. 
     
     
         11 . A method of preparing an enzyme having decarboxylase activity, comprising cultivating the recombinant microorganism of  claim 9 , and optionally isolating said enzyme having decarboxylase activity from the culture. 
     
     
         12 . A mutant of a protein having decarboxylase activity selected from the group consisting of:
 a) a protein comprising amino acid residues 2 to 241 of SEQ ID NO: 4 and originating from  Mesorhizobium  sp. BNCI;   b) a protein comprising amino acid residues 2 to 240 of SEQ ID NO: 6 and originating from  Pyrcoccus horikoshii  OT3;   c) a protein comprising amino acid residues 2 to 267 of SEQ ID NO: 8 and originating from  Bordetella Bronchiseptica  RB50; and   d) a protein comprising amino acid residues 2 to 246 of SEQ ID NO: 10 and originating from  Aklaliphilus metalliredigenes  QYMF;   wherein said mutant has sequence identity less than 100% to 60% to the corresponding parent protein and comprises at least one functional mutation in one of the amino acid sequence positions 14, 15, 159 and 190 said positions being numbered according to the alignment of  FIG. 3  ( b ).   
     
     
         13 . A nucleic acid encoding the mutant of  claim 12 . 
     
     
         14 . An expression cassette comprising the nucleic acid of  claim 13  operably linked to at least one regulatory nucleic acid sequence. 
     
     
         15 . A recombinant microorganism comprising:
 a) the nucleic acid of  claim 13 ; or   b) an expression cassette comprising the nucleic acid of a) operably linked to at least one regulatory nucleic acid sequence.   
     
     
         16 . A mutant of a protein having decarboxylase activity, wherein said protein comprises amino acid residues 2 to 256 of SEQ ID NO: 12 and is originated from  Aquifex pyrophilus , and wherein said mutant has sequence identity less than 100% to 60% to the corresponding parent protein. 
     
     
         17 . The mutant of  claim 16 , comprising:
 a) a Cystein containing catalytic site, the Cystein residue of which is able to interact with the substrate to, in particular by transferring a proton to the enolate intermediate of the decarboxylated substrate; and   b) an oxy-anion hole, comprising a plurality of polar amino acid side chains interacting with the carboxylic group of the substrate not to be cleaved off.   
     
     
         18 . A nucleic acid encoding the mutant of  claim 16 . 
     
     
         19 . An expression cassette comprising the nucleic acid of  claim 18  operably linked to at least one regulatory nucleic acid sequence. 
     
     
         20 . A recombinant microorganism comprising:
 a) the nucleic acid of  claim 18 ; or   b) an expression cassette comprising the nucleic acid of a) operably linked to at least one regulatory nucleic acid sequence.